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Repeated dose toxicity: oral

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008

Materials and methods

Test guidelineopen allclose all
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

Test material

Constituent 1
Reference substance name:
-
EC Number:
482-220-0
EC Name:
-
Cas Number:
848301-69-9
IUPAC Name:
482-220-0

Test animals

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.

Administration / exposure

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.
Doses / concentrations
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.

Examinations

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.

Results and discussion

Results of examinations

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.

Effect levels

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'

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.