[No public or meaningful name is available]

Endpoint summary

Description of key information

'Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear' 90 day repeated dose oral toxicity study in the rat:

- treatment-related effects in animals of either sex treated with 1000 and 200 mg/kg/day

- no such effects were detected in animals of either sex treated with 50 mg/kg/day and the “No Observed Effect Level” (NOEL) was, therefore, considered to be 50 mg/kg/day

- the effects detected at 200 and 1000 mg/kg/day in the lungs and mesenteric lymph nodes were considered to be secondary to aspiration following the oral gavage and a normal response of the lymph nodes clearing the material, respectively, and were therefore not considered to be an adverse effect of treatment; the “No Observed Adverse Effect Level” (NOAEL) was therefore considered to be 1000 mg/kg/day.

Supporting information:

A publication / study is available, which demonstrates that the toxicological response to dietary mineral hydrocarbons seen in F-33 rats is different than the response in other rat strains and other species. As in previous studies, dietary treatment with the related substance LMPW ("Paraffin waxes and Hydrocarbon waxes", CAS 8002-74-2) resulted in elevated MHC levels in selected organs and histopathological / immunohistochemical evidence of an inflammatory response unique to the livers of F-344 rats. In contrast, only minimal changes in MLNs were observed at the highest dietary concentration tested in the S-D rats with no other significant effects observed. These results add to the evidence from other studies that the dietary effects of MHCs in the F-344 rat are of questionable relevance for human safety (Griffis, 2009).

A 28-day dietary study in rats has been conducted with GTL Base Oil 3 (C18-C30) in accordance with OECD test guideline 407 and in compliance with GLP (Harlan Laboratories Ltd, 2014). The test item was administered by continuous dietary admixture to three groups, each of five male and five female Wistar Han™:RccHan™:WIST strain rats, for twenty-eight consecutive days, at dietary concentrations of 750, 3750 and 15000 ppm (equivalent to mean achieved dosages for males and females combined of 63, 308 and 1267 mg/kg bw/day). A control group of five males and five females were treated with basal laboratory diet. Clinical signs, functional observations, body weight, dietary intake and water consumption were monitored during the study. Hematology and blood chemistry were evaluated for all animals at the end of the study.

All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues from high dose and control animals was performed.

There were no unscheduled deaths during the study. There were no clinical signs, no changes in behavioural assessment, functional observations or sensory reactivity, and no effects on body weight, food consumption or food efficiency at any dose level. During the third week of treatment, males given the test item at all dose levels showed generally dose-related increases in water consumption levels in comparison with controls. There were no changes in water consumption for the treated females during the same period.

There were no treatment-related changes in the haematology or blood chemistry parameters examined, no macroscopic abnormalities at necropsy and no organ weight changes at any dose level. Microscopic examination of tissues revealed changes in male rats at the high dose level of 15000 ppm. During the third week of treatment, males given the test item at all dose levels showed generally dose-related increases in water consumption levels in comparison with controls. There were no changes in water consumption for the treated females during the same period.

On the basis of these findings the No Observed Effect Level was determined to be 15000 ppm for females and 3750 ppm in male.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was performed between January 2008 and 08 August 2008.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: guideline study on Gas-to-liquids (GTL) substance covering the carbon range from C18 to C50

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Approximately 18 to 21 weeks.
- Housing: The animals were housed in groups of three or four by sex in polypropylene cages with stainless steel mesh lids and solid bases containing softwood chip bedding.
- Diet (e.g. ad libitum): Free access to food (pelleted diet).
- Water (e.g. ad libitum): Free access to mains drinking water.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): Temperature controls set to achieve target value of 21 ± 2ºC.
- Humidity (%): Relative humidity controls set to achieve target values of 55 ± 15%.
- Air changes (per hr): At least fifteen air changes per hour.
- Photoperiod (hrs dark / hrs light): Low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness.

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test material was prepared at the appropriate concentrations as a solution in Arachis oil BP.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test material formulations were shown to be stable for at least fourteen days. Formulations were therefore prepared weekly and stored at 40ºC in the dark.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of Distillates (Fischer-Tropsch) heavy, C18-50-branched, cyclic and linear in the test material formulations was determined by high performance liquid chromatography mass selective (HPLC/MS) using an external standard technique.

The test material formulations were sampled and analysed within three days of preparation.

The results indicate that the prepared formulations were within the acceptable limits of the nominal concentration.

Duration of treatment / exposure:

Up to ninety consectutive days.

Frequency of treatment:

The test material was administered daily.

Remarks:

Doses / Concentrations:
50, 200 and 1000 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

10 males and 10 females per dose group for 50, 200 and 1000 mg/kg/day.
10 males and 10 females in control group (0 mg/kg/day).
Two recovery groups, each of 10 males and 10 females, were treated with the high dose (1000 mg/kg/day) or the vehicle alone for ninety days and then maintained without treatment for a further twenty-eight days.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were chosen based on the results of the 21-day range-finding study (please refer to 21-day range finding study).
The range-finding study used doses of 0, 100, 300 and 1000 mg/kg/day.

Results:
Clinical observations:
An isolated incident of increased salivation was detected for one male treated with 300 mg/kg/day on Day 11 only. No such effects were detected in females treated with 300 mg/kg/day or animals of either sex treated with 1000 or 100 mg/kg/day. One control male had scab formation between Days 15 and 21, this was unrelated to treatment.

Bodyweight:
No adverse effect on bodyweight gain was detected for animals of either sex treated with 1000, 300 or 100 mg/kg/day.

Organ Weights:
No toxicologically significant effects were detected in the organ weights measured.

Necropsy:
No macroscopic abnormalities were detected.

CONCLUSION
The dose levels for the ninety day phase of the study were chosen, following consultation with the Sponsor, as:
High dose: 1000 mg/kg/day
Intermediate dose: 200 mg/kg/day
Low dose: 50 mg/kg/day
- plus a control group treated with vehicle alone.

Positive control:

No.

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS:
All animals were examined for overt signs of toxicity, ill-health or behavioural change immediately before dosing, immediately post dosing and one and five hours after dosing during the working week. Animals were observed immediately before and after dosing and one hour after dosing at weekends and public holidays. During the treatment-free period, animals were observed twice daily, morning and afternoon (once daily at weekends). All observations were recorded.

FUNCTIONAL OBSERVATIONS:
Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioural toxicity. During Week 12 functional performances tests were also performed on all animals together with an assessment of sensory reactivity to different stimuli.

BEHAVIOURAL ASSESSMENTS:
Detailed individual clinical observations were performed for each animal using a purpose built
arena. The following parameters were observed:
Gait, Hyper/Hypothermia, Tremors, Skin colour, Twitches, Respiration, Convulsions, Palpebral closure, Bizarre/Abnormal/Stereotypic behaviour, Urination, Salivation, Defecation, Pilo-erection, Transfer arousal, Exophthalmia, Tail elevation, Lachrymation

FUNCTIONAL PERFORMANCE TESTS:
MOTOR ACTIVITY:
Twenty purpose built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals of one sex were tested at each occasion and were randomly allocated to the activity monitors. The tests were performed at approximately the same time each day, under similar laboratory conditions. The evaluation period was one hour for each animal. The time in seconds each animal was active and mobile was recorded for the overall one hour period and also during the final 20% of the period (considered to be the asymptotic period).

FORELIMB/HINDLIMB GRIP STRENGTH:
An automated grip strength meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. The animal was pulled by the base of the tail until its grip was broken. A record of the force required to break the grip for each animal
was made. Three consecutive trials were performed for each animal.

SENSORY REACTIVITY:
Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli.
The following parameters were observed:
Grasp response, Touch escape, Vocalisation, Pupil reflex, Toe pinch, Blink reflex, Tail pinch, Startle reflex, Finger approach

BODYWEIGHT:
Individual bodyweights were recorded on Day 1 and at weekly intervals thereafter. Bodyweights were also recorded at terminal kill.

FOOD CONSUMPTION:
Food consumption was recorded for each cage group at weekly intervals throughout the study.

WATER CONSUMPTION:
Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes.

OPHTHALMOSCOPIC EXAMINATION:
The eyes of all control and high dose animals were examined pre-treatment and before termination of treatment (during Week 12). Examinations included observation of the anterior structures of the eye, pupillary and corneal blink reflex. Following pupil dilation with 0.5% “Tropicamide” solution, detailed examination of the internal structure of the eye using a direct ophthalmoscope was performed.

LABORATORY INVESTIGATIONS:
Haematological and blood chemical investigations were performed on all surviving animals from each test and control group at the end of the study (Day 90) and on all recovery group animals at the end of the treatment-free period (Day 118). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Day 91 or 119. Animals were not fasted prior to sampling.

HAEMATOLOGY:
The following parameters were checked:
Haemoglobin (Hb)
Erythrocyte count (RBC)
Haematocrit (Hct)
Erythrocyte indices - mean corpuscular haemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular haemoglobin concentration (MCHC)
Total leucocyte count (WBC)
Differential leucocyte count - neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count (PLT)
Reticulocyte count (Retic) - Cresyl blue stained slides were prepared but reticulocytes were not assessed

Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/l).

BLOOD CHEMISTRY:
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:
Urea, Inorganic phosphorus (P), Glucose, Aspartate aminotransferase (ASAT), Total protein (Tot.Prot.), Alanine aminotransferase (ALAT), Albumin, Alkaline phosphatase (AP), Albumin/Globulin (A/G) ratio (by calculation), Creatinine (Creat), Sodium (Na+) Total cholesterol (Chol), Potassium (K+), Total bilirubin (Bili), Chloride (Cl-), Calcium (Ca++)

Sacrifice and pathology:

PATHOLOGY:
On completion of the dosing period, or in the case of recovery group animals, at the end of the treatment-free period, all surviving animals were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination.
All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

ORGAN WEIGHTS:
The following organs, removed from animals that were killed at the end of the study, were dissected free from fat and weighed before fixation:
Adrenals, Ovaries, Brain, Spleen, Epididymides, Testes, Heart, Thymus, Kidneys, Uterus, Liver.

HISTOPATHOLGY:
Samples of the following tissues were removed from all animals and preserved in buffered 10% formalin:
Adrenals, Ovaries, Aorta (thoracic), Pancreas, Bone & bone marrow (femur including stifle joint), Pituitary, Bone & bone marrow (sternum), Prostate , Brain (including cerebrum, cerebellum and pons), Rectum, Caecum Salivary glands (submaxillary), Colon, Sciatic nerve, Duodenum, Seminal vesicles (including coagulating gland),Epididymides, Skin (hind limb), Eyes, Spinal cord (cervical, mid-thoracic and lumbar), Gross lesions, Heart, Spleen , Ileum (including Peyer’s patches), Stomach, Jejunum, Testes, Kidneys, Thymus, Liver, Thyroid/parathyroid, Lungs (with bronchi), Tongue, Lymph nodes (cervical and mesenteric), Trachea, Mammary glands, Urinary bladder, Muscle (skeletal), Uterus, Oesophagus.

All tissues from control and 1000 mg/kg/day dose group animals were prepared as paraffin blocks, sectioned at nominal thickness of 5 μm and stained with haematoxylin and eosin for subsequent microscopic examination. Any macroscopically observed lesion were also processed.
Since there were indications of treatment-related lungs and mesenteric lymph node changes, examination was subsequently extended to include similarly prepared sections from all animals in the other treatment groups.

Other examinations:

None.

Statistics:

Data were processed to give group mean values and standard deviations where appropriate.

All data were summarised in tabular form. Where appropriate, quantitative data were analysed by the Provantis™ Tables and Statistics Module. For each variable, the most suitable transformation of the data was found, the use of possible covariates checked and the homogeneity of means assessed using ANOVA or ANCOVA and Bartlett’s test. The transformed data were analysed to find the lowest treatment level that showed a significant effect, using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found, but the data showed non-homogeneity of means, the data were analysed by a stepwise Dunnett (parametric) or Steel (non-parametric) test to determine significant differences from the control group. Finally, if required, pair-wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).

Prbability values (p) are presented as follows:
p <0.01**
p <0.05*
p >0.05 (not significant).

Histopathology data were analysed using the following methods to determine significant differences between control and treatment groups for the individual sexes.

Chi squared analysis for differences in the incidence of lesions occurring with an overall frequency of 1 or greater.

1. Kruskal-Wallis one way non-parametric analysis of variance for the comparison of severity grades for the more frequently observed graded conditions.

p<0.001 +++ --- ***
p<0.01 ++ -- **
p<0.05 + - *
p<0.1 (+) (-) (*)
p>0.1 N.S. (not significant)

With plus signs indicating positive differences from the control group and minus signs indicating negative differences. Asterisks refer to overall between group variation which is non-directional.

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

no effects observed

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

MORTALITY:
One male treated with 200 mg/kg/day was killed in extremis on Day 88. Histopathological examination of the tissues for this animal revealed peripheral inflammatory cell infiltrations and pleural adhesions indicating that the animal had been mal-dosed into the thorax. There were no further unscheduled deaths.

CLINICAL OBSERVATIONS:
Animals of either sex treated with 1000 mg/kg/day and males treated with 200 mg/kg/day showed episodes of increased salivation throughout the treatment period.
The male that was killed in extremis showed hunched posture, lethargy and respiratory pattern changes prior to termination.
No toxicologically significant signs were detected in females treated with 200 mg/kg/day, animals of either sex treated with 50 mg/kg/day or in recovery animals following twenty-eight days treatment free period.

FUNCTION OBSERVATIONS:-
BEHAVIORAL ASSESSMENTS:
There were no treatment-related changes in behaviour detected during the weekly open field arena assessments. All inter and intra group differences in behavioural scores were considered to be a result of normal variation for rats of the strain and age used and were, of no toxicological importance.

FUNCTIONAL PERFORMANCE TESTS:
There were no treatment-related changes in the functional performance parameters measured.
Statistical analysis revealed no significant intergroup differences.

SENSORY REACTIVITY ASSESSMENTS:
There were no treatment-related changes in sensory reactivity.
All inter and intra group differences in sensory reactivity scores were considered to be a result of normal variation for rats of the strain and age used, and was of no toxicological importance.

BODYWEIGHT:
No toxicologically significant effects on bodyweight development were detected.
The statistically significant intergroup differences detected during Weeks 3, 15, 16 and 17 were considered to be of no toxicological importance.

FOOD CONSUMPTION:
There was no adverse effect on food consumption during the study period. Food efficiency (the ratio of bodyweight gain to dietary intake) was similar to that of controls.

WATER CONSUMPTION:
Daily visual inspection of water bottles revealed no intergroup differences.

OPHTHALMOSCOPY EXAMINATION:
There were no treatment-related ocular effects. The incidental finding recorded was that normally encountered in laboratory maintained rats of this age and strain.

LABORATORY INVESTIGATIONS:
HAEMATOLOGY:
There were no toxicologically significant changes in the haematological parameters measured.
The intergroup differences detected in mean cell haemoglobin concentration, platelet count, eosinophils and clotting time were considered to be of no toxicological importance.

BLOOD CHEMISTRY:
There were no toxicologically significant changes in the blood chemical parameters measured.
The intergroup differences detected in total protein, albumin, alanine aminotransferase, calcium, chloride and potassium were considered to be of no toxicological importance.

PATHOLOGY:-
ORGAN WEIGHTS:
There were no toxicologically significant changes in the organ weights measured.
The intergroup differences detected in the adrenals and kidneys were considered to be of no toxicological importance.

NECROPSY:
The decedent male showed pale lungs, duodenum, ileum and jejunum, gaseous distension in the stomach and dark patches on the liver.
The remaining findings recorded for terminal kill animals at necropsy were considered to be of no toxicological importance.

HISTOPATHOLOGY:
The following treatment-related changes were detected:

LUNGS:
A greater incidence of higher grades of severity of alveolar macrophage accumulations was observed for animals of either sex treated with 1000 mg/kg/day (p<0.01 for males and for females) or at 200 mg/kg/day for males (p<0.05). The cytoplasm of macrophages seen in the lungs of animals of either sex treated with 1000 mg/kg/day (p<0.001) or at 200 mg/kg/day (p<0.001 for males and p<0.01 for females) was generally more vacuolated than that seen in alveolar macrophages in control animals.

There were no associated changes in the airways of affected animals indicating that this condition probably did not arise as a consequence of accidental installation of the test material into the respiratory tract during gavage dosing and thus was a systemic effect.

There was no convincing evidence of similar effects in either sex at the 50 mg/kg/day treatment level and no indication that the condition had regressed to any extent among Recovery 1000 mg/kg/day rats of either sex following an additional twenty-eight days without treatment.

MESENTERIC LYMPH NODES:
Vacuolated histiocytes were observed in relation to treatment for females treated with 1000 mg/kg/day (p<0.001) or at 200 mg/kg/day (not
significant). A similar effect was not seen for males treated with 1000 or 200 mg/kg/day or animals of either sex treated with 50 mg/kg/day.

There was no evidence to suggest that the effect had regressed among Recovery 1000 mg/kg/day animals following completion of an additional twenty-eight days without treatment.

All remaining morphological changes were those commonly observed in laboratory maintained rats of the age and strain employed, and there were no differences in incidence or severity between control and treatment groups that were considered to be of toxicological significance.

Please see attached background material for summary forms and justification of No Observed Effect Level.

Dose descriptor:

NOEL

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

not specified

Discussion:

The administration of 'Distillates (Fischer-Tropsch), heavy, C18-50-branched, cyclic and linear' by oral gavage, at dose levels of 1000, 200 and 50 mg/kg/day for a period of up to ninety consecutive days resulted in treatment related effects in animals of either sex treated with 1000 and 200 mg/kg/day.

There was no apparent adverse effect on the physical condition of the animals. Microscopic examinations revealed a greater incidence of higher grades of severity of alveolar macrophage accumulations in the lungs for animals of either sex treated with 1000 mg/kg/day and in males treated with 200 mg/kg/day. The cytoplasm of macrophages seen in the lungs of animals of either sex treated with 1000 mg/kg/day or at 200 mg/kg/day was also generally more vacuolated than that seen in alveolar macrophages in control animals. The lack of effect in control animals plus no changes consistent with dosing trauma suggest that the effect seen amongst treated animals at 200 and 1000 mg/kg/day are not a result of the process of gavage administration.

The remaining histopathological change was evident in the mesenteric lymph nodes. Vacuolated histiocytes were observed in relation to treatment for females dosed at 1000 and 200 mg/kg/day.

There were no indication that the conditions seen in the lungs and mesenteric lymph nodes had regressed to any extent among recovery 1000 mg/kg/day animals of either sex following an additional twenty-eight days without treatment.

Please see attached background material for summary forms and justification of No Observed Effect Level.

Conclusions:

- treatment-related effects in animals of either sex treated with 1000 and 200 mg/kg/day
- no such effects were detected in animals of either sex treated with 50 mg/kg/day and the “No Observed Effect Level” (NOEL) was, therefore, considered to be 50 mg/kg/day
- the effects detected at 200 and 1000 mg/kg/day in the lungs and mesenteric lymph nodes were considered to be secondary to aspiration following the oral gavage and a normal response of the lymph nodes clearing the material, respectively, and were therefore not considered to be an adverse effect of treatment; the “No Observed Adverse Effect Level” (NOAEL) was therefore considered to be 1000 mg/kg/day.

Executive summary:

Introduction.

The study was designed to investigate the systemic toxicity of the test material ‘Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear’ and complies with the following regulatory guidelines:

i) The OECD Guidelines for Testing of Chemicals No. 408 "Subchronic Oral Toxicity - Rodent: 90 Day Study” (Adopted 21 September 1998).

ii) The EU Annex B Method B26 - “Subchronic Oral Toxicity Test - Repeated Dose 90-Day Oral Toxicity Study in Rodents” - updated 21 August 2001.

iii) The United States Environmental Protection Agency (EPA), Health Effects Test Guidelines, OPPTS 870.3100 - 90 Day Oral Toxicity in Rodents.

Methods.

The test material was administered by gavage to three groups, each of ten male and ten female Sprague-Dawley Crl:CD® (SD) IGS BR strain rats, for up to ninety consecutive days, at dose levels of 50, 200 and 1000 mg/kg/day. A control group of ten males and ten females was dosed with vehicle alone (Arachis oil BP). Two recovery groups, each of ten males and ten females, were treated with the high dose (1000 mg/kg/day) or the vehicle alone for ninety consecutive days and then maintained without treatment for a further twenty-eight days.

Clinical signs, functional observations, bodyweight development and food and water consumption were monitored during the study. Haematology and blood chemistry were evaluated for all nonrecovery animals at the end of the treatment period and for all recovery group animals at the end of the treatment-free period. Ophthalmoscopic examination was also performed on control group and high dose animals.

All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues was performed.

Results.

Mortality.

One male treated with 200 mg/kg/day was killed in extremis on Day 88.

Histopathological examination of the respiratory tissues for this animal revealed peripheral inflammatory cell infiltrations and pleural adhesions indicating that the animal had been maldosed into the thorax. There were no further unscheduled deaths.

Clinical Observations.

Animals of either sex treated with 1000 mg/kg/day and males treated with 200 mg/kg/day showed episodes of increased salivation throughout the treatment period. The male that was killed in extremis showed hunched posture, lethargy and respiratory pattern

changes prior to termination.

No clinically observable signs of toxicity were detected in females treated with 200 mg/kg/day, animals of either sex treated with 50 mg/kg/day or in recovery animals following twenty-eight day treatment free period.

Behavioural Assessment.

There were no toxicologically significant changes in the behavioural parameters measured.

Functional Performance Tests.

There were no toxicologically significant changes in the functional performance parameters measured.

Sensory Reactivity Assessments.

There were no treatment-related changes in sensory reactivity.

Bodyweight.

No toxicologically significant effects on bodyweight development were detected.

Food Consumption.

No adverse effect on dietary intake or food efficiency was detected.

Water Consumption.

No intergroup differences were detected.

Ophthalmoscopy.

No treatment-related ocular effects were observed.

Haematology.

No toxicologically significant effects were detected in the haematological parameters measured.

Blood Chemistry.

No toxicologically significant effects were detected in the blood chemical parameters measured.

Organ Weights.

No toxicologically significant effects were detected.

Necropsy.

No toxicologically significant macroscopic abnormalities were detected in terminal kill animals. The decedent male showed pale lungs, duodenum, ileum and jejunum, gaseous distension in the stomach and dark patches on the liver.

Histopathology.

The following treatment-related changes were detected:

LUNGS:

A greater incidence of higher grades of severity of alveolar macrophage accumulations was observed for animals of either sex treated with 1000 mg/kg/day (p<0.01 for males and for females) or at 200 mg/kg/day for males (p<0.05). The cytoplasm of macrophages seen in the lungs of animals of either sex treated with 1000 mg/kg/day (p<0.001) and at 200 mg/kg/day (p<0.001 for males and 0.01 for females) was generally more vacuolated than that seen in alveolar macrophages in control animals.

There were no associated changes in the airways of affected animals indicating that this condition probably did not arise as a consequence of accidental installation of the test material into the respiratory tract during gavage dosing and thus was a systemic effect.

There was no convincing evidence of similar effects in either sex at the 50 mg/kg/day treatment level and no indication that the condition had regressed to any extent among Recovery 1000 mg/kg/day rats of either sex following an additional twenty-eight days without treatment.

MESENTERIC LYMPH NODES:

Vacuolated histiocytes were observed in relation to treatment for females treated with 1000 mg/kg/day (p<0.001) and at 200 mg/kg/day (not significant). A similar effect was not seen for males treated with 1000 or 200 mg/kg/day or animals of either sex treated with 50 mg/kg/day.

There was no evidence to suggest that the effect had regressed among Recovery 1000 mg/kg/day animals following completion of an additional twenty-eight days without treatment.

Conclusion.

The oral administration of 'Distillates (Fischer-Tropsch), heavy, C18-50-branched, cyclic and linear' to rats for a period of ninety consecutive days at dose levels of 50, 200 and 1000 mg/kg/day resulted in treatment-related effects in animals of either sex treated with 1000 and 200 mg/kg/day. No such effects were detected in animals of either sex treated with 50 mg/kg/day and the “No Observed Effect Level” (NOEL) was, therefore, considered to be 50 mg/kg/day.

However, the effects detected at 200 and 1000 mg/kg/day in the lungs and mesenteric lymph nodes were considered to be secondary to aspiration following the oral gavage and a normal response of the lymph nodes clearing the material, respectively, and were therefore not considered to be an adverse effect of treatment. The “No Observed Adverse Effect Level” (NOAEL) was therefore considered to be 1000 mg/kg/day.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Qualifier:

according to guideline

Guideline:

other: US federal guidelines for the care and use of laboratory animals

GLP compliance:

not specified

Limit test:

no

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: in a closed container at ambient temperature
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: not applicable
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test material was incorporated into the diet at two concentrations, 0.2 or 2.0% (w/w). The LMPW pellets were first converted to a fine powder by generating a fine spray of molten LMPW with an atomizing nozzle operated with heated nitrogen and then cooling the sprayed material. Fresh batches of test diet were prepared weekly and stored in sealed containers under refrigeration until use.
- Preliminary purification step (if any): not specified

Species:

rat

Strain:

other:

Remarks:

Fisher-344 and Sprague-Dawley

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, NC, USA
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 4 weeks
- Weight at study initiation:
- Fasting period before study:
- Housing: For the first week of acclimation period two rats of the same strain were housed in the same cage. Thereafter, the rats were individually housed.
- Diet: Certified rodent diet, ad libitum
- Water: tap water ad libitum
- Acclimation period: 14 days

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-23 °C
- Humidity (%): 34-76%
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test material was incorporated into the diet at two concentrations, 0.2 or 2.0% (w/w). The LMPW pellets were first converted to a fine powder by generating a fine spray of molten LMPW with an atomizing nozzle operated with heated nitrogen and then cooling the sprayed material. Fresh batches of test diet were prepared weekly and stored in sealed containers under refrigeration until use.

DIET PREPARATION
- Rate of preparation of diet (frequency): Fresh batches of test diet were prepared weekly.
- Mixing appropriate amounts with (Type of food): Certified rodent diet
- Storage temperature of food: Fresh batches of test diet were stored in sealed containers under refrigeration until use.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dietary concentration, homogeneity and stability were verified using the hydrocarbon analysis methodology. Dietary concentration was determined at study weeks 1, 2, 3, 4, 9 and 13 and all samples were 90-101% of the nominal concentration. In the homogeneity determination the mean recoveries and standard deviation at the 0.2% and 2.0% treatment levels were 98. and 99.1 respectively.
The ability of the rats to differentiate between the powdered test material and Certified Rodent Diet (CRD) was assessed by determining the test material concentration of samples taken from the feeders of five rats per strain from the high dose groups at the end of a 7-day feeding period. No increase in test material concentration was observed in the samples, confirming that the rats had not selectively consumed CRD.

Duration of treatment / exposure:

Animals were treated up to 90 days. Ten control and high-dose rats of each strain were euthanized and necropsied after 30 and 60 days of treatment. Ten control, low and high-dose rats of each strain were necropsied after 90 days. Additional rats were used for determination of mineral hydrocarbon in target tissues after 30, 60 or 90 days of treatment.

Frequency of treatment:

The test material in CRD was available ad libitum

Dose / conc.:

157 mg/kg bw/day (nominal)

Remarks:

0.2 % (w/w) for F-344 rats

Dose / conc.:

160 mg/kg bw/day (nominal)

Remarks:

0.2 % (w/w) for S-D rats

Dose / conc.:

1 609 mg/kg bw/day (nominal)

Remarks:

2.0 % (w/w) for F-344 rats

Dose / conc.:

1 644 mg/kg bw/day (nominal)

Remarks:

2.0 % (w/w) for S-D rats

No. of animals per sex per dose:

Control group: 30 rats (10 rats sacrificed at 30, 60 and 90 days)
Low dose group: 10 rats (10 rats sacrificed at 90 days)
High dose group: 30 rats (10 rats sacrificed at 30, 60 and 90 days)
Additional rats were used for determination of mineral hydrocarbon in target tissues after 30, 60 or 90 days of treatment.
Control group: 5 rats (sacrificed at 90 days)
Low dose group: 5 rats (sacrificed at 90 days)
High dose group: 15 rats (5 rats sacrificed at 30, 60 and 90 days)

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale:
- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: No satellite groups were selected.
- Post-exposure recovery period in satellite groups: not applicable
- Section schedule rationale (if not random): random

Positive control:

Not used.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
- Cage side observations checked included: clinical signs of toxicity

DETAILED CLINICAL OBSERVATIONS: Yes / No / Not specified
- Time schedule:

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

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: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not applicable

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were collected from the abdominal aorta at necropsy.
- Anaesthetic used for blood collection: Yes (barbiturate anesthesia)
- Animals fasted: Yes
- How many animals: 10
- Parameters checked in table [No.1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Serum from the blood sample collected at necropsy.
- Animals fasted: Yes
- How many animals: 10
- Parameters checked in table [No.1] were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table No 2). The heart, liver, kidney, ovaries and spleen from all subchronic study animals and MLNs from half the rats in each group were weighed at necropsy and organ weight/terminal body weight ratios were calculated.

HISTOPATHOLOGY: Yes (see table No 2). Sections of the heart, liver and MLN from subchronic study rats in control and high-dose groups were evaluated histologically at 30 and 60 days and in all groups at 90 days. In addition, sections of kidney, cervical lymph nodes, ovary, spleen and gross lesions were examined from all subchronic study animals.

IMMUNOHISTOCHEMICAL ANALYSIS OF LIVER AND MLNS; EM EVALUATION OF LIVER: Replicate sections of paraffin-embeded samples of liver, MLN and cardiac mineral valves from five randomly selected rats in each group were stained immunohistochemically to demonstrate the presence of cells expressing CD3 (T-cells), CD45RA (B-cells), CD8 (CD8a, suppresser/cytotoxic T-cells), CD4 (helper T-cells), ED2 (resident macrophages), and lysozyme (activated microphages). Liver and MLN sections of selected animals were stained with a lipid stain and sections of all animals were evaluated microscopically under polarized light for the presence of birefringent material in attempts to observe the presence of test material droplets. Liver sections were also evaluated by EM to determine Kupffer cell activation and the nature of the lipid-like mineral observed in some samples.

Other examinations:

MINERAL HYDROCARBON ANALYSES (MHC)
- Dietary concentration: The LMPW content of the test diets was determined by use of a gas chromatograph equipped with a flame ionization detector and a capillary column. The LMPW was extracted from the feed with hexanes and then analysed against known test standards;
- Tissue levels: The MHC content in selected tissues (liver, kidney, heart, spleen and MLNs) was determined. The tissues were saponified and the mineral and the mineral hydrocarbon was extracted into carbon tetrachloride. The extracts were then analysed by infrared spectroscopy. The samples were quantified against analytical standards of LMPW prepared in carbon tetrachloride. The limit of quantitation of the method was 0.5 mg/g for liver, kidney, heart and spleen and 2.5 mg/g for MLNs. In addition, the mineral hydrocarbon profile in the liver of F-344 rats was determined by gas chromatography-mass spectrometry (GC/MS). The major peaks were identified on the chromatogram.

Statistics:

Statistical evaluation of the data was conducted where appropriate. The data were analysed at each time point (30, 60 and 90 days) by a standard two-way analysis of variance. Differences between corresponding dose groups both across and within strains were tested using contrast statements. Pair-wise comparisons between groups were performed using the Tukey-Kramer method. Residuals from the model on untransformed data were tested for normality by the Shapiro-Wilk test. If the residuals failed this test at the 1% level, the dependent variable was converted to a function of its percentile rank, and then transformed by the inverse normal distribution Blom's transformation. The two-way analyses were then performed on the transformed data.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no adverse effects due to treatment on physical appearance or behaviour of the rats.

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths during the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weight gain was not affected by LMPW treatment in either rat strain. As expected the S-D rats were heavier than the F-344 rats at the start of the study and gained more weight on a week-to-week basis throughout the study.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Feed consumption was not affected by the treatment.

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

A number of haematological parameters were affected by LMPW treatment in F-344 rats. Increase in number of WBC and neutrophils in low and high dose group were observed at 30, 60 and 90 days of the study. The increase in total WBC was statistically significant only in the 2% group at 60 days while neutrophils were significanly increased at 0.2% and 2.0% treatment levels at 90 days. These WBC changes were considered treatment-related. Modest decrease in RBC counts and haemoglobin concentration along with accompanying reductions in hematocrit, mean corpuscular volume, mean corpuscular haemoglobin and mean corpuscular haemoglobin concentration. The decrease in RBC was statistically significant only in the low dose treatment group. Similar changes in RBC count and haemoglobin concentration were observed in the 2% LMPW-treated rats at 60 but not at 30 days. Platelet count were also decreased in F-344 rats but this decrease was statistically significant only at 0.2% treatment group at 90 days.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Elevated levels of a number of enzymes (AST, ALT, GGT and ALKP) were observed in F-344 rats at both the 0.2% and 2.0% doses at all treatment intervals. Statistically significant increases in ALT and GGT were observed in F-344 rats at 30, 60 and 90 days of the study at both doses while significant increases were seen only at 60 and 90 days for AST and only in the 2% treatment group at 90 days for ALKP. Serum albumin levels were consistently lower and cholesterol levels consistently higher than constrols for F-344 rats at 30, 60 and 90 days. At 90 days albumin levels were significantly lower than controls. No treatment-related haematological changes were observed in the S-D rats.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant increases in hepatic, splenic and MLN weights were seen in F-344 rats at all dose levels and treatment intervals. The only effect of LMPW treatment on organ weight in the S-D rats was increased MLN weight but this was not statistically significant at 90 days. Statistical significance was observed at the 30 and 60 day treatment intervals. There was also a dose-related, statistically significant increase in ovary weight F-344 rats at 90 days. This change was not seen in the S-D rats.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopic changes related to LMPW treatment were observed in liver, lymph nodes, spleen and heart of the F-344 rats. In the S-D rats treatment-related changes were limited to the MLNs.
The salient change in the livers of the F-344 rats was microgranuloma formation. In the 2% LMPW group, microgranulomas were observed in the livers of three rats at 30 days and in all rats at 60 and 90 days. Microgranulomas were also seen in all 0.2% F-344 rats at 90 days. The severity of microgranuloma formation was similar in the 0.2% and 2.0% dose groups and in the 60 abd 90-day animals. Microgranulomas were observed in one S-D rat from the 0.2% group at 60 days but this was considered an incidential finding since microgranulomas were not seen in other S-D rats.
After 60 and 90 days of treatment, lymphoid cell infiltrates/aggregates were observed at the periphery of the hepatic microgranulomas in all F-344 rats from the 2.0% group. Greater severity was seen at 90 days than 60. Scattered foci of necrosis were associated with the microgranulomas and were observed only in F-344 rats from the 2% group at 60 and 90 days. Both incidence and severity of the necrosis increased with duration of treatment.
Vacuolated hepatocytes were observed in rats from both strains and groups including control.
Ultrastructural examination by EM confirmed the presence of widespread granulomatous inflammation in the livers from LMPW-treated F-344 rats. The size and number of Kupffer cells with features of activation were increased. EMs also showed large numbers of variably-sized polygonal crystaloid structures within the lysisimes of Kupffer cells. These crystalloid structures were also common in hepatocytes. There was no evidence of inflammation with microgranuloma formation by EM evaluation in the S-D rat livers.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

- CD3 immunohistochemical (T-cell) marker: In S-D rats, F-344 controls and treated F-344 rats at day 30, CD3-positive cells were infrequent. The number of CD3-positive cells increased in F-344 rats with increasing dose and treatment duration. In small granulomas, approximately half the cells of the infiltrate were positive. Most of the other cells were histocytic and epitheliolid macrophages. Larger microgranulomas were surrounded by a rim of CD3-positive lymphocytes of variable width. There were no significant changes in S-D rats.
- CD8 (CD8a) (suppresser/cytotoxic T-cells) and CD4 (helper T-cells) markers: CD8a-positive cells were scattered throughout the sinusoids in control animals. In treated F-344 rats, increased number of small aggregates of CD8a-positive cells were observed in centrilobular and midzonal regions. Increased number of CD8a-positive cells surrounded the granulomas and were part of a peripheral lymphoid rim. This change was increased with both dose and treatment duration. The distribution of CD4-positive cells in frozen sections was similar to that of CD8a-positive cells in paraffin sections. In S-D rats, treatment-related microgranulomas were not found and the number of CD8a-positive cells reflected normal distribution.
- CD45RA (B-cell) marker: CD45RA-positive cells were a minor component of background microgranulomas in both rat strains. The number of positive cells increased with dose and treatment duration in F-344 rats and large CD45RA-positive cells were seen in the microgranulomas. There was no treatment-related response in the S-D rats.
- ED2 (resident macrophage) marker and EM evaluation: ED2-positive cells lining hepatic sinusoids were seen in control and 30-day rats of both strains. S-D rats appeared to have a higher background number of positive cells compared to the F-344 rats. Using ED2-positive cells, a treatment-related increase in Kupffer cell numbers was not observed in S-D rats. EM evaluation, however, revealed Kupffer cell hypertrophy and hyperplasia in livers from treated F-344 rats. The majority of macrophages in microgranulomas in F-344 rats were ED2-negative.
-Lysozyme: Epithelioid macrophages, multinucleated giant cells and polymorphonuclear cells were lysozyme-positive.
-Mesenteric lymph nodes (MLN): Treatment-related effects in the MLNs consisted of microgranuloma formation and reticuloendothelial hyperplasia. In the F-344 rats, microgranulomas and reticuloendothelial hyperplasia wer4e observed in almost all rats at 30, 60 and 90 days. The severity of both findings tended to increase with increasing duration of treatment but there was no difference in severity of microgranuloma formation between the 60 and 90 day groups. microgranuloma formation and reticuloendothelial hyperplasia were observed in S-D rats with substentially lower severity. Microgranulomas and reticuloendothelial hyperplasia were also observed in a few control animals and was considered incidential.
- CD3, CD8 (CD8a) and CD4 markers: In MLNs from control rats, CD3-positive cells were seen in the parafollicular cortical zone and scattered throughout the medulla and follicular areas. In both F-344 and S-D rats, the number of CD3-positive cells decreased with increasing number and size of microgranulomas. The distribution of CD8a-positive cells in control MLNs was similar to CD3-positive cells and there was no treatment effect in either strain of the rat. The distribution of CD4-positive cells was comparable to CD8a-positive cells.
- CD45RA marker: CD45RA-positive cells occupied most of the nodal cortex in MLNs without microgranulomas. The number appeared to decrease with microgranuloma formation. The reduction in CD3-, CD8a-, CD4- and CD45RA-positive lymphocytes was considered to be the result of spatial displacement by macrophages.
- Lysozyme and ED2 markers: Epithelioid cells in MLN microgranulomas were variably lysozyme-positive, similar to the liver findings. There was a treatment-related increase in both strains. ED-positive macrophages were more prominent in S-D rats. Cervical lymph nodes and spleens were examined microscopically only from rats treated for 90 days. Microgranulomas were observed in the cervical lymph nodes in F-344 rats. Extramedullary haematopoisesis was seen in the spleen of all rats examined at 90 days. The severity of the observed changes in the spleen appeared to be increased in LMPW-treated F-344 rats at both doses but there was no apparents effect of LMPW treatment in S-D rats. Lymphoid cell infiltrates in the base of the mitral valve were observed at increased frequency, compared to the controls in tissue sections from F-344 rats in the 2% LMPW group after 60 and 90 days. A similar finding in the base of mitral valve was observed in the control F-344 and one control S-D rat but not in LMPW-treated S-D rats. Recut sections of the same samples were also examined. Many of the mitral valve lessions were not observed in these recut sections and treatment-related changes were not apparent in either strain of rat.
- Tissue MHC concentration: Quantifiable MHC levels were detected in the MLNs from both strains fed LMPW but only in the livers from F-344 rats. There was evidence of accumulation as the mean MHC level increased from 1.86 mg/g at 30 days to 19.8 mg/g at 90 days in the livers and from below 2.5 mg/g at 30 and 60 days to 6.2 mg/g at 90 days in MLNs. At 90 days the mean MHC level in the MLNs of the F-344 rats fed 2% LMPW was more than twice the value in the S-D rats fed the same concentration.
The GC-MS analysis of a cyclohexane extract of liver from the LMPW-treated F-344 rats identified peaks that were for the most part saturated linear and branched hydrocarbons. There were also some peaks that were identified as methyl esters of fatty acids. These esters may have been formed from free fatty acids reacting with methanol during the methanol/cyclohexane extraction.

Remarks on result:

other: No NOAEL was determined.

Critical effects observed:

not specified

Conclusions:

The present study further demonstrates that the toxicological response to dietary mineral hydrocarbons seen in F-33 rats is different than the response in other rat strains and other species. As in previous studies, dietary treatment with LMPW resulted in elevated MHC levels in selected organs and histopathological/immunohistochemical evidence of an inflammatory response unique to the livers of F-344 rats. In contrast, only minimal changes in MLNs were observed at the highest dietary concentration tested in the S-D rats with no other significant effects observed. These results add to the evidence from other studies that the dietary effects of MHCs in the F-344 rat are of questionable relevance for human safety.

Executive summary:

The study was conducted with the most biologically active MHC material evaluated in earlier studies. The results with LMPW extend the observations of a biological response to MHC materials that appears to be unique to F-344 rats. There were no treatment-related effects in the present study on survival, clinical signs, body weight gain or feed consumption with LMPW in either rat strain after 90 day treatment. The treatment-related changes that were observed either occurred only in F-344 rats or were substantially more severe in F-344 rats than in S-D rats. The haematological changes, elevated serum enzyme levels, and increased hepatic and splenic weights were observed only in F-344 rats. The statistically significant effects noted on haematology and serum enzyme levels were not well correlated with actuall dose. The changes at the high dose were equivalent or marginally less than that at the low dose suggesting that liver toxicity was not strongly dose-related. Histopathological changes, however, were more severe at the high dose than at the low dose.

In the present study, MLN weights were increased with 3 -4 fold in F-344 rats after 90 days compared to controls, while MLN weights were increased only about 1.5 -fold. Moreover, liver weights were increased only in F-344 strain at both doses. Hepatic microgranuloma formation was also observed only in F-344 rats at both doses. Similarly, the treatment-related inflammatory changes seen in the livers, spleens and cervical lymph nodes of F-344 rats were not evident in S-D rats. Microgranulomas and reticuloendothelial hyperplasia in the MLNs were observed in both rat strains but at a lower incidence and severity in S-D rats.

The severity of cellular vacuolations which resembled microvesicular steatosis increased with treatment duration. The increase of positive staining of cellular markers of CD3, CD8a, CD4 and CD45RA correlated with the increase in lymphoid cells in the livers of treated F-344 rats. Although, associated with lymphocytes, no determination could be made on the specificity of the microgranulomas as being the hypersensitive or non-imune type. Cytokines from macrophages can elicit lymphocyte attraction, accumulation and granuloma formation.

The population of Kupffer cells in the liver is maintained by both local proliferation and extrahepatic recruitment of mononuclear precursor cells. The magority of epitheliolid macrophages in the hepatic microgranulomas from LMPW-treated F-344 rats were ED2 -negative indicating a proable origin from non-resident macrophages. It is likely that resident Kupffer cells phagocytized hepatic paraffin wax residues leading to lipid-like pseudocysts, release of inflammatory cytokines, recruitment of extrahepatic macrophages and microgranuloma formation. In the study, lysozyme straining of epithelioid macrophages decreased with increasing microgranuloma size. EM evaluation revealed Kupffer cell hypertrophy and increased cell numbers in the livers of treated F-344 rats. It is possible that these Kupffer cells originated from recruited inflammatory cells not expressing DC2 antigen but not yet sufficiently activated to be lysozyme-positive.

Changes in the cardiac mitral valve were seen in this study although they were not confirmed in the immunohistochemical evaluation. No cardiac mitral valve changes were noted in any of the S-D rats treated.

One mechanism that may contribute to the observed effects in F-344 rats may be species- and strain-specific differences in absorption and metabolism of mineral hydrocarbons. In the present study , quantifiable MHC levels were detected in livers from F-344 rats but not S-D rats.

The GC/MS analysis of MHC residues in the livers of F-344 rats treated with LMPW indicated that most of the detected hydrocarbons were in the C22 -C31 range. Similarly, other studies demonstrated preferential retention of hydrocarbon compounds in the C20 -C35 range (de Rooji et al., 1993).

Along with the previously observed diffreneces in pharmacokinetics, differences in immune sensitivity may also play a central role in the greater response of F-344 rats to MHCs. The F-344 rats appear to be more sensitive immunologically to a number of agents than other rat strains.

The results from the present study provide further evidence of the unique response of F-344 rats to dietary MCHs. In numerous earlier studies, highly refined white oils and paraffin waxes such as LMPW have shown a relatively low order of toxicity in all species tested.

In conclusion, the present study further demonstrates that the toxicological response to dietary mineral hydrocarbons seen in F-33 rats is different than the response in other rat strains and other species. As in previous studies, dietary treatment with LMPW resulted in elevated MHC levels in selected organs and histopathological/immunohistochemical evidence of an inflammatory response unique to the livers of F-344 rats. In contrast, only minimal changes in MLNs were observed at the highest dietary concentration tested in the S-D rats with no other significant effects observed. These results add to the evidence from other studies that the dietary effects of MHCs in the F-344 rat are of questionable relevance for human safety.

Reference 3

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Experimental Starting Date: 30 September 2013; Experimental Completion Date: 11 August 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

yes

Remarks:

: Minor deviations which did not affect the study integrity or validity of study

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

: Minor deviations which did not affect the study integrity or validity of study

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

A sufficient number of male and female Wistar Han:RccHan:WIST strain rats were obtained from Harlan Laboratories U.K. Ltd. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatized for eight days during which time their health status was assessed. A total of forty animals (twenty males and twenty females) were accepted into the study. At the start of treatment the males weighed 207 to 238g, the females weighed 143 to 170g, and were approximately six to eight weeks old.

The animals were housed in groups of five by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding. The animals were allowed free access to food and water. A ground diet (Rat and Mouse SQC Ground Diet No. 1, Special Diet Services, Dietex International Ltd, Witham, Essex, UK) was used. Mains drinking water was supplied from polycarbonate bottles attached to the cage. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels. The diet, drinking water, bedding and environmental enrichment were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

The animals were housed in a single air-conditioned room within the Harlan Laboratories Ltd Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerized system, and print-outs of hourly temperatures and humidities are included in the study records. The Study Plan target ranges for temperature and relative humidity were 22 ± 3 °C and 30 to 70%. Short term variations from these targets were considered not to have affected the purpose or integrity of the study.

The animals were randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomized. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

Test Item Preparation:
The test item was incorporated into the diet at concentrations of 750, 3750 and 15000 ppm as follows:

A known amount of test item was mixed with a small amount of basal laboratory diet in a Robot Coupe Blixer 4 mixer set at a constant speed. This pre-mix was then transferred to a Hobart QE200 mixer and the required amount of basal laboratory diet was added and mixed for a further thirty minutes at a constant speed, setting 1.

The stability and uniformity of distribution of the test item in the diet were determined as part of this study. Results showed the dietary admixtures to be stable for fourteen days at room temperature. Dietary admixtures were prepared prior to the first treatment and weekly thereafter. The diet was stored in labeled, double plastic bags in labeled, covered plastic bins at room temperature. Samples were taken of each dietary admixture and analyzed for uniformity of distribution and concentration of GTL Base Oil 3. The results indicate that the prepared dietary admixture concentrations were within acceptable ranges for the purpose of this study.

Procedure:
The test item was administered continuously, for twenty-eight consecutive days, by dietary admixture. Control animals were treated in an identical manner with basal laboratory diet.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples were taken of each dietary admixture and analyzed for uniformity of distribution and concentration of GTL Base Oil 3.

The test item concentration in the test samples was determined by gas chromatography (GC) using an external standard technique. The test item gave a chromatographic profile consisting of a peak profile.

The admixtures investigated during the study were found to comprise test item in the range of 96% to 107% and, thus, the required content limit of ±20% with reference to the nominal content was met.

In addition, the test item was found to be stable in the admixtures when kept 14 days in the dark at room temperature due to results which met the variation limit of 10% from the time-zero mean.

In conclusion, the results inidcate the accurate use of the test item and Diet as vehicle during this study. The formulations were found to be homogeneously prepared and sufficient formulation stability under storage conditions was approved.

Duration of treatment / exposure:

Twenty-eight consecutive days

Frequency of treatment:

Administered continuously for twenty-eight days.

Remarks:

Doses / Concentrations:
Dietary concentration: 750, 3750, 15000 ppm. Mean acheieved dose level: 63, 308, 1267 mg/kg bw/day
Basis:
nominal in diet

No. of animals per sex per dose:

5 males and 5 females per dose group.

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The dietary concentrations were chosen in consultation with the Study Sponsor's Monitoring Scientists following review of the toxicity information provided by the Study Sponsor.

Positive control:

None.

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS:
All animals were examined for overt signs of toxicity, ill-health or behavioral change daily from the start of treatment. All observations were recorded.

FUNCTIONAL OBSERVATIONS:
Prior to the start of treatment and on Days 7, 14, 21 and 27, all animals were observed for signs of functional/behavioral toxicity. Functional performance tests were also performed on all animals during Week 4, together with an assessment of sensory reactivity to different stimuli.

BEHAVIORAL ASSESSMENT:
Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed:
Gait
Tremors
Twitches
Convulsions
Bizarre/Abnormal/Stereotypic behavior
Salivation
Pilo-erection
Exophthalmia
Lachrymation
Hyper/Hypothermia
Skin color
Respiration
Palpebral closure
Urination
Defecation
Transfer arousal
Tail elevation

FUNCTIONAL PERFORMANCE TESTS:
Motor Activity:
Twenty purpose built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals of one sex were tested at each occasion and were randomly allocated to the activity monitors. The tests were performed at approximately the same time each occasion, under similar laboratory conditions. The evaluation period was one hour for each animal. The time in seconds each animal was active and mobile was recorded for the overall one hour period and also during the final 20% of the period (considered to be the asymptotic period, Reiter and Macphail 1979).

Forelimb/Hindlimb Grip Strength:
An automated grip strength meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal. The assessment was developed from the method employed by Meyer et al (1979).

Sensory Reactivity:
Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. The following parameters were observed:
Grasp response
Vocalization
Toe pinch
Tail pinch
Finger approach
Touch escape
Pupil reflex
Blink reflex
Startle reflex

BODY WEIGHT:
Individual body weights were recorded on Day 1 (prior to the start of treatment) and at weekly intervals thereafter. Body weights were also performed prior to terminal kill.

FOOD CONSUMPTION:
Food consumption was recorded for each cage group at weekly intervals throughout the study. Food conversion efficiency and mean achieved dosage were calculated retrospectively.

WATER CONSUMPTION:
Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes except during Week 3 where water intake was measured gravimetrically.

LABORATORY INVESTIGATIONS:
Hematological and blood chemical investigations were performed on all animals from each test and control group at the end of the study (Day 28). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture at termination on Day 29. Animals were not fasted prior to sampling.

HEMATOLOGY:
The following parameters were investigated:
Hemoglobin (Hb)
Erythrocyte count (RBC)
Hematocrit (Hct)
Erythrocyte indices - mean corpuscular hemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular hemoglobin concentration (MCHC)
Total leukocyte count (WBC)
Differential leukocyte count - neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count (PLT)
Reticulocyte count (Retic)
Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/L).

BLOOD CHEMISTRY:
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:
Urea
Glucose
Total protein (Tot.Prot.)
Albumin
Albumin/Globulin (A/G) ratio (by calculation)
Sodium (Na+)
Potassium (K+)
Chloride (Cl-)
Calcium (Ca++)
Inorganic phosphorus (P)
Aspartate aminotransferase (ASAT)
Alanine aminotransferase (ALAT)
Alkaline phosphatase (AP)
Creatinine (Creat)
Total cholesterol (Chol)
Total bilirubin (Bili)
Bile acids
Gamma glutamyltranspeptidase
Triglycerides

Sacrifice and pathology:

NECROPSY:
On completion of the dosing period, all animals were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination.

All animals were subjected to a full external and internal examination.

At termination, following organ weighing, a small section (ca. 1 cm2) of the liver was removed, placed on ice until chilled and then stored frozen pending shipment to the study sponsor for future analysis.

THYROID HORMONE ASSESSMENT:
At termination, blood samples were taken from the exsanguination procedure and the serum from each animal was stored frozen at approximately -20°C. No treatment related effects on the pituitary-thyroid axis were identified.

ORGAN WEIGHTS:
The following organs, removed from animals that were killed at the end of the dosing period were dissected free from fat and weighed before fixation:
Adrenals
Brain
Epididymides
Heart
Kidneys
Pituitary (post-fixation)
Prostate and Seminal Vesicles (with coagulating glands and fluids)
Liver
Ovaries
Spleen
Testes
Thymus
Thyroid/Parathyroid (post fixation)
Uterus with Cervix

HISTOPATHOLOGY:
Samples of the following tissues were removed from all animals:
Adrenals
Aorta (thoracic)
Bone & bone marrow (femur including stifle joint)
Bone & bone marrow (sternum)
Brain (including cerebrum, cerebellum and pons)
Caecum
Colon
Duodenum
Epididymides
Esophagus
Eyes
Gross lesions
Heart
Ileum
Jejunum
Kidneys
Liver
Lungs (with bronchi)
Lymph nodes (mandibular and mesenteric)
Mammary gland
Muscle (skeletal)
Pituitary
Prostate
Rectum
Salivary glands (submaxillary)
Sciatic nerve
Seminal vesicles (with coagulating glands and fluids)
Skin (hind limb)
Spinal cord (cervical, mid thoracic and lumbar)
Spleen
Stomach
Testes
Thymus
Thyroid/Parathyroid
Trachea
Urinary bladder
Uterus & Cervix
Vagina

All tissues were dispatched to the histology processing Test Site for processing.

Since there were indications of treatment-related changes, examination was subsequently extended to include similarly prepared sections of spleen, mesenteric lymph nodes and small intestine (duodenum, jejunum and ileum) from males in the low and intermediate groups.

Statistics:

Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters:

Grip Strength, Motor Activity, Body Weight Change, Hematology, Blood Chemistry, Absolute Organ Weights, Body Weight-Relative Organ Weights.

Data were analyzed using the decision tree from the ProvantisTM Tables and Statistics Module as detailed as follows:

Where appropriate, data transformations were performed using the most suitable method. The homogeneity of variance from mean values was analyzed using Bartlett’s test. Intergroup variance were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covarities. Any transformed data were analyzed to find the lowest treatment level that showed a significant effect using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found but the data shows non-homogeneity of means, the data were analyzed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests was performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).

Probability values (p) are presented as follows:

p<0.01 **
p<0.05 *
p>0.05 (not significant)

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

effects observed, treatment-related

Description (incidence and severity):

males given the test item at all dose levels showed generally dose-related increases in water consumption levels in comparison with controls.

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Males at 15000 ppm.

Histopathological findings: neoplastic:

not examined

Details on results:

MORTALITY:
There were no unscheduled deaths during this study.

CLINICAL OBSERVATIONS:
No clinical signs were seen in any of the animals throughout the study.

FUNCTIONAL OBSERVATIONS:
Behavioral Assessments:
There was no effect of treatment with the test item on behavioral assessment or scores.

Functional Performance Tests:
There were no treatment-related changes in the functional performance parameters measured.

Sensory Reactivity Assessments:
There were no treatment-related changes in sensory reactivity.

All inter and intra group differences in sensory reactivity scores were considered to be a result of normal variation for rats of the strain and age used and the differences were of no toxicological importance.

BODY WEIGHT:
There were no detrimental treatment-related changes in body weight development detected throughout the study.

Males and females treated with 3750 ppm and males treated with 15000 ppm showed a statistically significant increase (p<0.05) in weight gain in comparison with controls after two weeks of treatment. A similar trend (p<0.05) was evident in all male test groups after three weeks of treatment. However, as an increase in weight is not seen as an adverse response in studies of this nature these findings were considered to be of no toxicological importance.

Females treated at 3750 ppm showed low weight gains in comparison with the controls and remaining test groups after one week of treatment but showed recovery thereafter.

FOOD CONSUMPTION:
No adverse effect on food consumption or for food efficiency (the ratio of body weight gain to dietary intake) was identified throughout the treatment period.

WATER CONSUMPTION:
Gravimetric measurement of water consumption performed during the third week of treatment revealed males from all test groups to have been consuming more drinking water (20% for 750 ppm, 36% for 3750 ppm and 50% for 15000 ppm) than the current control. However, there was no convincing difference between test and control females for water intake.

LABORATORY INVESTIGATIONS:
HEMATOLOGY:
There were no treatment related changes in the hematological parameters measured.

Females receiving 3750 or 15000 ppm diet showed statistically significantly lower (p<0.05) mean corpuscular hemoglobin values than that of the concurrent control. In addition females treated with 15000 ppm were also observed to have a statistically significantly lower (p<0.05) mean corpuscular volume in comparison with the respective controls. However, in the absence of convincing supporting evidence these findings were considered to have arisen fortuitously.

An increased platelet count was noted in all male test groups (750 and 15000 ppm: p<0.05 and 3750 ppm: p<0.01) and accompanied in males treated at 15000 ppm only by an increase (p<0.05) for activated partial thromboplastin time. However, there was no supporting evidence to indicate these coagulation factors were affected by treatment (the liver being the major site of synthesis).

BLOOD CHEMISTRY:
There were no treatment-related effects detected in the blood chemistry parameters measured.

Incidental findings were confined to a statistically significant reduction in plasma calcium levels that was detected for all female test groups (750 ppm, p<0.05, 3750 and 15000 ppm p<0.01) in comparison with the concurrent controls. However, all individual values were entirely within the normal ranges for rats of this strain and age and on this basis this isolated finding was considered not to be associated with test item toxicity.

THYROID HORMONE ASSESSMENT:
No treatment-related effects on the pituitary-thyroid axis were identified therefore no thyroid hormone parameters were measured.

PATHOLOGY:
NECROPSY:
There were no macroscopic abnormalities detected.

ORGAN WEIGHTS:
There were no treatment-related changes in organ weights.

Incidental findings included an increase in absolute and relative (to terminal body weight) heart weight in males treated at 15000 ppm while females from this test group showed a reduction (p<0.01) in absolute and relative brain weight when compared with controls. However, as the majority of individual values were within the normal historical background ranges and in the absence of microscopic changes in these organs these findings were considered to be incidental and not to be related to test item toxicity.

Males treated at 750 ppm showed a statistically significant reduction (p<0.01) for testes weights in comparison with controls. Similar changes were not evident among males treated at 15000 ppm and as such this intra group difference was considered fortuitous.

HISTOPATHOLOGY:
Treatment related microscopic findings were recorded in males treated at 15000 ppm these findings were detected in the duodenum, jejunum, ileum, Peyer’s patches, spleen and mesenteric lymph node and consisted of apoptosis/single cell necrosis in the lamina propria/crypt up to moderate severity in the duodenum and jejunum in all five males and in the ileum of four males.

Increased severity of lymphocytolysis above background minimal grade was noted in Peyer’s patches, spleen and mesenteric lymph node up to moderate degree in the majority of males (15000 ppm). These findings may be considered as an exacerbation of the normal processes of cell attrition which occurs in these organs.

No treatment-related histopathological findings were detected in males treated at 750 or 3750 ppm or for any of the female test groups.

Dose descriptor:

NOEL

Effect level:

3 750 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

other: See discussion and conclusion for details.

Dose descriptor:

NOEL

Effect level:

15 000 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

other: See discussion and conclusion for details.

Critical effects observed:

not specified

Discussion:

The oral administration of GTL Base Oil 3 to rats by dietary admixture for a period of twenty eight consecutive days at dietary concentrations of 750, 3750 and 15000 ppm did not produce evidence of test item toxicity.

There were no remarkable findings during the course of treatment in this study and histopathological changes were confined to the males treated at 15000 ppm. These findings involved evidence of moderate severities of apoptosis/single cell necrosis in the lamina propria crypt in all five males together with a slightly above normal severity lymphocytolysis in Peyer’s patches, spleen and mesenteric lymph node in the majority of males from this test group. These findings were considered as an exacerbation of the normal processes of cell attrition which occurs in these organs and for this reason excludes classification of a No Observed Adverse Effect Level (NOAEL) in the males treated at 15000 ppm.

Conclusions:

The oral administration of GTL Base Oil 3 to rats by dietary admixture for a period of twenty eight consecutive days at dietary concentrations of 750, 3750 and 15000 ppm resulted in treatment-related histopathological findings along the alimentary canal, mesenteric lymph nodes and spleen of males treated at 15000 ppm preventing classification of a "No Observed Adverse Effect Level" (NOAEL) in males at this dose level. However, as there was no other convincing evidence of adverse treatment related changes detected the No Observed Effect Level (NOEL) in males was considered to be 3750 ppm and in females 15000 ppm.

Executive summary:

Introduction:

This study was designed to investigate the systemic toxicity of the test item. It is compatible with the requirements for notification of a new chemical substance in the EC and follows the testing method described in Commission Directive 96/54/EC (Method B7) and OECD Guidelines for Testing of Chemicals No. 407 "Repeated Dose 28 Day Oral Toxicity Study in Rodents" (adopted 03 October 2008).

This study was also designed to be compatible with Commission Regulation (EC) No 440/2008 of 30 May 2008, laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Methods:

The test item was administered by continuous dietary admixture to three groups, each of five male and five female Wistar Han™:RccHan™:WIST strain rats, for twenty-eight consecutive days, at dietary concentrations of 750, 3750 and 15000 ppm (equivalent to mean achieved dosages for males and females combined of 63, 308 and 1267 mg/kg bw/day). A control group of five males and five females were treated with basal laboratory diet.

Clinical signs, functional observations, body weight, dietary intake and water consumption were monitored during the study. Hematology and blood chemistry were evaluated for all animals at the end of the study.

All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues from high dose and control animals was performed. Males receiving the high dose showed treatment-related microscopic changes in the spleen, mesenteric lymph nodes and small intestine (duodenum, jejunum, ileum) and the histopathological evaluation of these tissues was extended to males receiving the low and intermediate dose levels.

Results:

Mortality:

There were no unscheduled deaths during this study.

Clinical Observations:

There were no clinical signs in any of the animals throughout the treatment period.

Behavioral Assessment:

There were no changes detected in the behavioral parameters measured.

Functional Performance Tests:

There were no treatment-related changes in the functional performance parameters measured.

Sensory Reactivity Assessments:

There were no treatment-related changes in sensory reactivity.

 

Body Weight:

There were no adverse changes in body weight gain detected throughout the study.

 

Food Consumption:

No adverse effects on food consumption or food efficiency were detected in test animals in comparison with controls.

 

Water Consumption:

During the third week of treatment, males given the test item at all dose levels showed generally dose-related increases in water consumption levels in comparison with controls. There were no changes in water consumption for the treated females during the same period.

 

Hematology:

There were no treatment-related changes in the hematological parameters measured.

 

Blood Chemistry:

There were no treatment-related changes in the blood chemistry parameters measured.

Necropsy:

There were no macroscopic abnormalities detected.

 

Organ Weights:

There were no treatment-related changes in the measured organ weights.

Histopathology:

Treatment related microscopic findings characterized by apoptosis/single cell necrosis in the lamina propria/crypt were identified in the duodenum, jejunum, ileum, Peyer’s patches, spleen and mesenteric lymph node of males treated with 15000 ppm. 

 

Above normal severities of lymphocytolysis was noted in Peyer’s patches, spleen and mesenteric lymph node in the majority of males treated at 15000 ppm.

 

No histopathological changes were detected in males at 3750 or 750 ppm or in any of the female test groups.

Conclusion:

The oral administration of GTL Base Oil 3 to rats by dietary admixture for a period of twenty eight consecutive days at dietary concentrations of 750, 3750 and 15000 ppm resulted in treatment-related histopathological findings along the alimentary canal, mesenteric lymph nodes and spleen of males treated at 15000 ppm preventing classification of a "No Observed Adverse Effect Level" (NOAEL) in males at this dose level. However, as there was no other convincing evidence of adverse treatment related changes detected the No Observed Effect Level (NOEL) in males was considered to be 3750 ppm and in females 15000 ppm.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

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

No repeated dose toxicity data are available for 'Paraffin waxes (Fischer-Tropsch), full range, C15-C50, branched and linear' itself. However, good quality repeated-dose toxicity studies for the oral route are available for the closely related substance ‘Distillates (Fischer-Tropsch), heavy, C18-50-branched, cyclic and linear’:

The oral administration of the closely related substance ‘Distillates (Fischer-Tropsch), heavy, C18-50-branched, cyclic and linear’ to rats for a period of ninety consecutive days at dose levels of 50, 200 and 1000 mg/kg/day in an OECD 408 study resulted in treatment-related effects in animals of either sex treated with 1000 and 200 mg/kg/day. The “no observed effect level” (NOEL) was considered to be 50 mg/kg/day. However the effects detected at 200 and 1000 mg/kg/day in the lungs and mesenteric lymph nodes were considered to be secondary to aspiration following the oral gavage and a normal response of the lymph nodes clearing the material, respectively, and were therefore not considered to be an adverse effect of treatment. The “No Observed Adverse Effect Level” (NOAEL) was therefore considered to be 1000 mg/kg/day.

Additionally, the available two-generation reproductive toxicity studies (see IUCLID section 7.8) with GTL Diesel (C8-C26) and GTL Base oil distillates (C18-C50) covering the entire carbon number range C15-50, also demonstrated an absence of systemic effects in both F1 and F2 litters at the highest dose tested. Both studies were undertaken with additional histopathology on several organs not normally included in these studies to further characterise the systemic toxicity of the substance.

Supporting information:

In a study, the response of female F-344 and S-D rats was compared after 90 days dietary treatment with LMPW ("Paraffin waxes and Hydrocarbon waxes", CAS 8002-74-2).

The test material was incorporated into the diet at two concentrations, 0.2 or 2.0 % (w/w). Fresh batches of test diet were prepared weekly and stored in sealed containers under refrigeration until use. The test material preparations in diet were available daily ad libitum. Dietary concentration, homogeneity and stability were verified using the hydrocarbon analysis methodology.

Approximately 30 animals of each strain were used for high dose and control groups from which 10 were euthanized and necropsied after 30, 60 and 90 days of treatment. Ten low-dose rats of each strain were necropsied after 90 days. Additional rats were used for determination of mineral hydrocarbon in target tissues after 30, 60 or 90 days of treatment. Regular observations for mortality, clinical signs of toxicity and changes in body weight gain were performed. Haematological and clinical chemistry examinations were performed at necropsy along with macroscopic and histopathological examination of organ tissues.

The study was conducted with the most biologically active MHC material evaluated in earlier studies. The results with LMPW extend the observations of a biological response to MHC materials that appears to be unique to F-344 rats. There were no treatment-related effects in the present study on survival, clinical signs, body weight gain or feed consumption with LMPW in either rat strain after 90 day treatment. The treatment-related changes that were observed either occurred only in F-344 rats or were substantially more severe in F-344 rats than in S-D rats. The haematological changes, elevated serum enzyme levels, and increased hepatic and splenic weights were observed only in F-344 rats. The statistically significant effects noted on haematology and serum enzyme levels were not well correlated with actual dose. The changes at the high dose were equivalent or marginally less than that at the low dose suggesting that liver toxicity was not strongly dose-related. Histopathological changes, however, were more severe at the high dose than at the low dose.

In the present study, MLN weights were increased with 3-4 fold in F-344 rats after 90 days compared to controls, while MLN weights were increased only about 1.5 -fold. Moreover, liver weights were increased only in F-344 strain at both doses. Hepatic microgranuloma formation was also observed only in F-344 rats at both doses. Similarly, the treatment-related inflammatory changes seen in the livers, spleens and cervical lymph nodes of F-344 rats were not evident in S-D rats. Microgranulomas and reticuloendothelial hyperplasia in the MLNs were observed in both rat strains but at a lower incidence and severity in S-D rats.

The severity of cellular vacuolations which resembled microvesicular steatosis increased with treatment duration. The increase of positive staining of cellular markers of CD3, CD8a, CD4 and CD45RA correlated with the increase in lymphoid cells in the livers of treated F-344 rats. Although, associated with lymphocytes, no determination could be made on the specificity of the microgranulomas as being the hypersensitive or non-immune type. Cytokines from macrophages can elicit lymphocyte attraction, accumulation and granuloma formation.

The population of Kupffer cells in the liver is maintained by both local proliferation and extrahepatic recruitment of mononuclear precursor cells. The majority of epitheliolid macrophages in the hepatic microgranulomas from LMPW-treated F-344 rats were ED2 -negative indicating a probable origin from non-resident macrophages. It is likely that resident Kupffer cells phagocytized hepatic paraffin wax residues leading to lipid-like pseudocysts, release of inflammatory cytokines, recruitment of extrahepatic macrophages and microgranuloma formation. In the study, lysozyme straining of epithelioid macrophages decreased with increasing microgranuloma size. EM evaluation revealed Kupffer cell hypertrophy and increased cell numbers in the livers of treated F-344 rats. It is possible that these Kupffer cells originated from recruited inflammatory cells not expressing DC2 antigen but not yet sufficiently activated to be lysozyme-positive.

Changes in the cardiac mitral valve were seen in this study although they were not confirmed in the immunohistochemical evaluation. No cardiac mitral valve changes were noted in any of the S-D rats treated.

One mechanism that may contribute to the observed effects in F-344 rats may be species- and strain-specific differences in absorption and metabolism of mineral hydrocarbons. In the present study, quantifiable MHC levels were detected in livers from F-344 rats but not S-D rats.

The GC/MS analysis of MHC residues in the livers of F-344 rats treated with LMPW indicated that most of the detected hydrocarbons were in the C22 -C31 range. Similarly, other studies demonstrated preferential retention of hydrocarbon compounds in the C20 -C35 range (de Rooji et al., 1993).

Along with the previously observed differences in pharmacokinetics, differences in immune sensitivity may also play a central role in the greater response of F-344 rats to MHCs. The F-344 rats appear to be more sensitive immunologically to a number of agents than other rat strains.

The results from the present study provide further evidence of the unique response of F-344 rats to dietary MCHs. In numerous earlier studies, highly refined white oils and paraffin waxes such as LMPW have shown a relatively low order of toxicity in all species tested.

In conclusion, the present study further demonstrates that the toxicological response to dietary mineral hydrocarbons seen in F-33 rats is different than the response in other rat strains and other species. As in previous studies, dietary treatment with LMPW resulted in elevated MHC levels in selected organs and histopathological/immunohistochemical evidence of an inflammatory response unique to the livers of F-344 rats. In contrast, only minimal changes in MLNs were observed at the highest dietary concentration tested in the S-D rats with no other significant effects observed. These results add to the evidence from other studies that the dietary effects of MHCs in the F-344 rat are of questionable relevance for human safety.

Justification for classification or non-classification

Based on the information from the available two-generation reproductive toxicity studies where additional histopathology on several organs was undertaken to further characterise the systemic toxicity and the absence of significant effects for human health in the available study of the closely related substance 'Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear', there is no requirement to classify 'Paraffin waxes (Fischer-Tropsch), full range, C15-C50, branched and linear' for specific target organ toxicity following repeated exposures, according to the criteria of Regulation 1272/2008/EC.