Hydrocarbons, C18-C24, iso-alkanes <2% aromatics

Endpoint summary

Description of key information

Based on weight of evidence from subchronic toxicity studies on related substances covering the relevant carbon number range, it can be concluded that Hydrocarbons, C18-24, isoalkanes, <2% aromatics, does not produce systemic toxicity effects relevant for hazard assessment at dose levels up to 750 or 1000 mg/kg bw/day. The most significant findings in the studies conducted were light hydrocarbon induced nephropathy in the kidney of male rats and forestomach lesions. Neither of these effects is relevant to humans (Baetcke, 1991).  

Baetcke, KP, GC Hard, IS Rodgers, RE McGaughy, LM Tahan, 1991. Alpha 2micro-globulin: association with chemically induced renal toxicity and neoplasia in the male rat. EPA/625/3-91/019F, September 1991.

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:

comparable to guideline study

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 consecutive 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:

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 238 g, the females weighed 143 to 170 g, 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.

Reference 3

Endpoint:

sub-chronic toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Justification for type of information:
1. Hypothesis for the analogue approach:
The hypothesis for the analogue approach is that the test substance, GTL Base Oil Distillates (C18-C50 branched, cyclic and linear hydrocarbons – Distillates / 848301-69-9 / 482-220-0 is produced in the same Fischer-Tropsch process as GTL Gasoil which is the starting material from which the registration substance is produced by fractional distillation. The source substance contains constituents of the target substance, Hydrocarbons, C18-C24, iso-alkanes, <2% aromatics, although it covers a wider carbon number distribution. The substances therefore have qualitatively similar properties (RAAF Scenario 2 applies). See Endpoint Summary (CSR Section 5.6.3 for additional information).
2. Source and target chemical(s)
The source substance GTL Base Oil Distillates (Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear) is composed of linear, branched and cyclic hydrocarbons of chain length C18-C50.
The target substance, Hydrocarbons, C18-C24, iso-alkanes, <2% aromatics, is composed of predominantly branched hydrocarbons of chain length C18-24.
3. Analogue approach justification
The constituents of the source and target substances are all hydrocarbons. Identical constituents have identical toxicity profiles. There is no evidence of toxicity in the studies on the source substance, and no evidence of toxicity in studies on hydrocarbons with shorter carbon chain length and a narrower range of carbon number, therefore there is no evidence for mixture effects or for increased toxicity with increased concentration..

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Dose descriptor:

NOEL

Effect level:

50 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: effects observed at 1000 and 200 mg/kg/day

Critical effects observed:

no

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

750 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

10 000 mg/m³

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

10 000 mg/m³

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

No repeated dose toxicity data are available for Hydrocarbons, C18-24, isoalkanes, <2% aromatics itself. However, good quality repeated-dose toxicity studies for the oral route are available for related substances covering the relevant carbon number range. These are used as read-across to demonstrate the lack of toxicity of hydrocarbons in the relevant carbon number range.

GTL GASOIL (C8-C26)

A two-generation reproductive toxicity study (oral, gavage) has been carried out using GTL Gasoil following OECD Test Guideline 416 and conducted according to GLP (RTI, 2011a). The test material was administered by gavage to four groups of male and female Crl:CD (SD) rats, each of 25 and 28 animals of each sex for the F₀ and F₁ generations, respectively. Test concentrations were 0 (control), 50, 200 and 750 mg/kg bw/day for the F₀and F₁generations. Dosing was performed for 70 consecutive days prior to mating. Males continued to be dosed during the 14-day mating period and post-mating holding (up to Day 107). Females were dosed during the mating period, gestation and lactation (up to Day 128). Additional endpoints (including detailed histopathology) were included in the test protocol such that the study can be used to assess general systemic toxicity in addition to reproductive effects.

Overall, there were 19 unscheduled deaths of parental animals (4 F0 males, 6 F0 females, 5 F1 males, and 4 F1 females). None of the deaths were directly attributed to the test item. Based on macroscopic (and in some cases microscopic) findings, 11 of the deaths were likely due to gavage error and/or aspiration of the dose formulation into the lungs. With the low viscosity nature of the test item and the use of corn oil as the vehicle, aspiration events were not unexpected. Two F0 females were injured by their mating partner and euthanized for humane reasons, and 1 F0 male was euthanized for humane reasons due to lesions on the neck, likely caused by scratching at the ear tag. One F0 female given 750 mg/kg/day was euthanized due to dystocia, and 1 F1 female given 750 mg/kg/day was found dead the day after dystocia was observed. The cause of death for 3 animals (1 F1 male given 50 mg/kg/day, 1 F1 male given 750 mg/kg/day, and 1 F1 female given 750 mg/kg/day) could not be definitively determined at necropsy.

There were no adverse test item-related effects on F0 and F1 parental body weights, body weight changes, feed consumption, and food efficiency. Test item-related histopathological lesions were identified in the lungs of both males and females of the F0 and F1 generations and the kidneys (males only) of the F1 generation. No test-item related histopathological changes were found in any of the organs that were evaluated beyond those of the standard OECD 416 protocol. There was an increased incidence and severity of chronic interstitial/alveolus inflammation in the F0 and F1 males and females given 750 mg/kg/day; this microscopic finding correlated with macroscopic observations in F0 and F1 males and F1 females, as well as increased absolute and relative lung weights in F0 and F1 males and females. As these lung lesions were considered to be secondary to aspiration of the dose formulation, and the chronic interstitial/alveolus inflammation finding was not unanticipated based on a previous 90-day, repeated-dose study with a similar test item, the lungs from the low and mid-dose animals were not evaluated. In the F1 males, test item-related slight increases in renal tubule degeneration/necrosis and renal tubule hyaline droplets, suggestive of hydrocarbon-induced alpha-2-microglobulin male rat nephropathy, were seen in the males given 750 mg/kg/day. Special staining of kidneys from males in the control and high-dose group confirmed this lesion to be hydrocarbon-induced alpha-2-microglobulin male rat nephropathy, an anticipated outcome of this study; therefore, the kidneys of the low- and mid-dose animals were not evaluated. In the F0 males, renal tubule mineralization was noted. Since this finding may be an artefactual change resulting from tissue fixation and/or processing, and since mineralized tubules were only observed in the F0 males given 750 mg/kg/day, with no similar mineralized tubules seen in the F0 control males, F0 females given 750 mg/kg/day, and F1 males and females given vehicle or 750 mg/kg/day, this finding was considered equivocal and non-adverse.

In conclusion, test item-related histopathological lesions were identified in the lungs (chronic interstitial/alveolus inflammation) of both males and females of the F0 and F1 generations with corresponding macroscopic findings and increased lung weights and the kidneys (renal tubule degeneration/necrosis and renal tubule hyaline droplets confirmed to be alpha-2-microglobulin) of F1 males only. The lung lesions were most likely secondary to aspiration of the test material and therefore not relevant for human risk assessment. The renal effects are a well known male rat specific effect which is induced by hydrocarbons and has no relevance for humans. Additional equivocal, non-adverse findings included renal tubule mineralization in the kidneys of F0 males given 750 mg/kg/day and slightly decreased spleen weights in F1 and F2 pups. Based on the absence of adverse, test item-related findings directly attributable to the test item in non-reproductive tissues, a dosage level of 750 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for reproductive and systemic toxicity.

Based on the available repeated dose oral studies to assess systemic, reproductive and developmental toxicity on GTL Gasoil (C8-C26) and considering the relative gut absorption discussed in Section 5.1 of the CSR, it is concluded that Hydrocarbons, C18-C24, isoalkanes, < 2% aromatics (GS310) will have a similar toxicological profile after repeated oral exposure. Additional information on systemic toxicity of related GTL substances can be found in the literature (Boogaard et al., 2017)

Therefore, for repeated dose exposure, for GS310 the following is established NOAEL (systemic toxicity) = 750 mg/kg body weight/day. 

GTL BASE OIL (C18-C50, branched, cyclic and linear)

A 90-day oral (gavage) toxicity study has been carried out using GTL Base Oil (C18-C50, CAS 858301-69-9), following OECD Test Guideline 408 and conducted according to GLP (Harlan, 2008). 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 ninety consecutive days, at dose levels of 50, 200 and 1000 mg/kg/day in Arachis oil BP. Two recovery groups, each of ten males and ten females, were treated with the high dose or the vehicle alone for 90 consecutive days and then maintained without treatment for a further 28 days.

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. Such observations of this nature are often reported following oral administration of an unpalatable test material and, in isolation, are not indicative of systemic toxicity. 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. No toxicologically significant effects were detected in the haematological and clinical chemistry parameters measured, nor were any toxicologically significant macroscopic abnormalities detected in terminal kill animals.

Microscopic histopathology, however, revealed some findings. 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 and at 200 mg/kg/day for males. The cytoplasm of macrophages seen in the lungs of animals of either sex treated with 1000 mg/kg/day and at 200 mg/kg/day for females was generally more vacuolated than that seen in alveolar macrophages in control animals. Vacuolated histiocytes were observed in relation to treatment for females treated with 1000 and 200 mg/kg/day but a similar effect was not seen for males treated with 200 mg/kg/day, or animals of either sex treated with 50 mg/kg/day.

Although the relevance to human health has not been established, the increased vacuolization of the macrophages seems adaptive in nature and did not have any health consequences in the animals. Gopinath (1987) refers to similar events when animals are exposed to various chemical forms such as phospholipids. Because contact to the material is via the gut and secondarily through aspiration of small quantities of test material in the lungs, such material is phagocytosed by the macrophage-like histiocytes. With some test materials, the cellular breakdown of such material may be slow and hence the accumulation of material may lead to a “foamy” appearance of the macrophage. Recovery may therefore exceed the normal twenty-eight day period as described in the test guideline but does not necessarily represent an irreversible change. The process can be regarded as neither proliferative or degenerative but merely part of a normal body process response to exposure to high levels of certain test materials which are inherently inert in their activity. As such the effects detected in the lungs and mesenteric lymph nodes in this study were not considered to be an adverse effect of treatment. The NOEL was considered to be 50 mg/kg/day. The effects detected at 1000 and 200 mg/kg/day in the lungs and mesenteric lymph nodes were not considered to be an adverse effect of treatment. The accumulation of petroleum hydrocarbons in mesenteric lymph nodes is a well known effect in the rat with little relevance to humans. In addition, it is related to the carbon chain length and not expected to occur to a significant extent with GTL Base Oil. The overall NOAEL considered relevant for human health hazard assessment of GTL Base Oil was considered to be 1000 mg/kg/day.

GTL BASE OIL 3 (C18 -C26)

A 28-day dietary study in rats has been conducted with GTL Base Oil 3 in accordance with OECD test guideline 407 and in compliance with GLP (Harlan, 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. Treatment related microscopic findings characterised 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.

On the basis of these findings the No Observed Effect Level was determined to be 15000 ppm for females and 3750 ppm in males, equivalent to achieved doses of 1267 and 308 mg/kg bw/day, respectively.

C9-C14 isoparaffins

The toxicity of C9-C14 isoakanes by inhalation route has been recently reviewed (Carrillo et al., 2013).

Results from a 13-week inhalation study in rats on a C10–C12 isoparaffinic solvent at nearly vapour saturated concentrations are compared to the results of repeated inhalation and oral toxicity studies of four other isoparaffinic hydrocarbon solvents. Statistically significant findings which were consistent across all studies included: nephropathy and small but significant changes in hematological parameters in male rats and liver enlargement in both male and female rats. The male rat kidney changes were due to an alpha-2u- globulin process and not relevant for human health or risk assessment. The liver enlargement without pathologic changes or elevations in liver enzyme markers was considered to be an adaptive response. The reason for the reductions in hematological parameters that were observed in males only is not clear, but it is suggested that these were either due to normal variation or a secondary consequence of the nephropathy. Thus, the overall No Observed Adverse Effect Concentration (NOAEC) was the highest concentration tested in the C10-C12 isoalkane study, >10,000 mg/m³. Because of the overall pattern of response, and the test being conducted at nearly saturated concentrations due to the low volatility this solvent, it is considered that the NOAEC determined for C10–C12 isoalkane solvents brackets the low end of the Hydrocarbons, C18-C24, isoalkanes, <2% aromatics. Such high exposure concentrations would not be technically obtained in an inhalation study using Hydrocarbons, C18-C24, isoalkanes, <2% aromatics, indicating that the NOAEC of , >10,000 mg/m³ is a reasonable worse-case read across.

Conclusion

The available data indicate that repeated exposure to hydrocarbons in the range relevant for Hydrocarbons, C18-C24, isoalkanes, <2% aromatics (C18 to C24) do not cause serious adverse effects relevant to human health via either the oral or inhalation routes. The most significant findings in the studies conducted were light hydrocarbon induced nephropathy in the kidney of male rats and forestomach lesions. Neither of these effects is relevant to humans (Baetcke, 1991).

Repeated dose inhalation studies are not available for constituents present in Hydrocarbons, C18-C24, isoalkanes, <2% aromatics. This is due to the low volatility of hydrocarbons in the C18-C24 range. However, it is considered that the NOAEC determined for C10–C12 isoalkane solvents brackets the low end of Hydrocarbons, C18-C24, isoalkanes, <2% Aromatics. Such high exposure concentrations would not be technically obtained in an inhalation study using Hydrocarbons, C18-C24, isoalkanes, <2% aromatics, indicating that the NOAEC of , >10,000 mg/m³ is a reasonable worse-case read across.

The key study is an extended 2-generation reproductive toxicity study (oral, gavage) with GTL Gasoil (C8-C26, branched and linear) reported a NOAEL of 750 mg/kg bw/day for systemic effects (RTI, 2011a). Dosing in this study was performed for up to 107 consecutive days in males and up to 128 consecutive days in females.

In a 90-day oral (gavage) study with GTL Base Oil (C18-C50, branched, linear and cyclic), the NOAEL was 1000 mg/kg bw/day (Harlan, 2008).

In all cases the NOAEL was the highest dose tested.

References:

Baetcke, KP, GC Hard, IS Rodgers, RE McGaughy, LM Tahan, 1991. Alpha 2micro-globulin: association with chemically induced renal toxicity and neoplasia in the male rat. EPA/625/3-91/019F, September 1991.

Carrillo JC, Adenuga MD, McKee RH, Roth RN, Steup D, Simpson BJ. The sub-chronic toxicity in rats of isoparaffinic solvents. Regul Toxicol Pharmacol. 2013 (3):446-55.

Gopinath C., Prentice D.E. and Lewis D.J., eds., The Respiratory System in: Atlas of Experimental Toxicological Pathology, Lancaster, UK, MTP Press Limited, 1987

Justification for classification or non-classification

Based on the absence of significant effects for human health in available sub-chronic toxicity studies for analogue substances there is no requirement to classify Hydrocarbons, C18-C14, isoalkanes, <2% aromatics for specific target organ toxicity following repeated exposures, according to the criteria of Regulation (EC) No. 1272/2008.