Renewable hydrocarbons (diesel type fraction)

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

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

Adequacy of study:

key study

Study period:

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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP guideline study. Read-across is considered to be reliability 2.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

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

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

GTL base oil 3

IUPAC Name:

GTL base oil 3

Test material form:

other: liquid

Details on test material:

Identification : GTL Base Oil 3
Description : Clear colorless liquid
Chemical Name: Distillates (Fischer-Tropsch), heavy, C18-C50 - branched, cyclic and linear
Purity : Not supplied
Batch Number : Unknown
Label : GTL XHVI-3 (Ras Laffan)
Date Received : 23 August 2013
Storage Conditions: Ambient temperature and humidity, stored in darkness
Expiry Date : 01 September 2015

Test animals

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.

Administration / exposure

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.

Doses / concentrations

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.

Examinations

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)

Results and discussion

Results of examinations

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.

Effect levels

1

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.

2

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.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.