Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2009

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
Principles of method if other than guideline:
A rat 2-generation study was extended to provide information on repeated dose toxicity in the F0 generation (design equivalent OECD 408). Functional observation battery, haematology and clinical chemistry assessments were performed following 11 wk treatment. Necropsy and collection of tissues for histopathological assessment was conducted after 18 wk treatment.
GLP compliance:
yes (incl. QA statement)
Remarks:
UK Department of Health, 4 March 2009

Test material

Constituent 1
Reference substance name:
NEXBTL renewable diesel
IUPAC Name:
NEXBTL renewable diesel
Details on test material:
- Name of test material (as cited in study report): NExBTL Renewable diesel
- Description: clear colourless liquid
- Date received: 18 December 2007

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Strain: HsdRccHan:WIST
- Source: Harlan Laboratories UK Ltd
- Group mean weight at start of treatment: males 244-265 g; females 148-174 g
- Housing: group, 4/sex /cage
- Diet (e.g. ad libitum): yes
- Water (e.g. ad libitum): yes
- Acclimation period: 12 d


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23
- Humidity (%): 40-70
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12 (lights on 0600-1800 hr)


IN-LIFE DATES : Dose range finder
- Protocol signed 29 January 2008
- First treatment: 12 February 2008
- Necropsy: 4 March 2008

IN-LIFE DATES : Main study
- Protocol signed 29 January 2008
- First treatment: 24 March 2008 (males) or 25 March 2008 (females)
- Necropsy: 29-30 July 2009 (males) or 4-29 July 2008 (females)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

- Justification for vehicle used: the test substance was insufficiently soluble in water but readily soluble in arachis oil
- Concentration in vehicle: 0, 12.5, 62.5 and 250 mg/ml
- Dosing volume: 4 ml/kg bwt

Dosing volume adjusted based on most recent bodyweight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Sample were analysed by GC-FID. Results of stability and homogeneity studies showed that formulated dosing solutions were stable for at least 16 days and that the test substance was evenly distributed. Analyses for achieved concentration were within + or - 10% of nominal.

Duration of treatment / exposure:
Dose range finder: at least 21 consecutive days
Main study: at least 11 weeks treatment prior to haematological, clinical chemistry and neurobehavioural assessments; at leat 18 weeks treatment prior to necropsy.
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
200, 600, 1000 mg/kg bwt/d (range-finder)
Basis:
other: nominal in vehicle (range finder)
Remarks:
Doses / Concentrations:
50, 250, 1000 mg/kg bwt/d (main study)
Basis:
other: nominal in vehicle (main study)
No. of animals per sex per dose:
Range-finder: 3/sex/dose level
Main study: 10/sex/dose level
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection:
- based on results of dose range-finder investigation
- 1000 mg/kg bwt/d is a limit dose according to AnnexV to Directive 67/548/EEC
- treatments in excess of 1000 mg/kg bwt/d were considered unnecessary and undesirable

Animals used in the main study formed part of a larger group used for reproductive toxicity testing of the test substance.

Examinations

Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS:
- before dosing; immediately after dosing; 1 and 5 hr post-dosing

BODY WEIGHT:
- before dosing; weekly thereafter

FOOD CONSUMPTION:
- weekly intervals

WATER CONSUMPTION:
- weekly intervals

OPTHALMOSCOPIC EXAMINATION
- performed pre-treatment and during wk 10

HAEMATOLOGY:
- performed on all animals/treatment levels during wk 11
- blood (lateral tail vein) collected into EDTA
- animals not fasted or anaesthesitised
- parameters examined: haemoglobin, haematocrit, erythrocyte count, erythrocyte indices (mean corpuscular haemoglobin, mean corpuscular volume, mean corpuscular haemoglobin concentration); total leucocyte (white cell)count; differential leucocyte count (neutrophils, lymphocytes monocytes, eosinophils, basophils), platelet count, prothrombin time, activated partial thromboplastin time

CLINICAL CHEMISTRY:
- performed on all animals/treatment levels during wk 11
- blood (lateral tail vein) collected into lithium heparin, determinations run on plasma
- animals not fasted or anaesthesitised
- parameters examined: urea, glucose, total protein, albumin, albumin/globulin ratio, sodium, potassium, chloride, calcium, inorganic phosphorus, aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT), alkaline phosphatase (AP), creatinine, total cholesterol, total bilirubin
NEUROBEHAVIOURAL EXAMINATION:
- performed on all animals/treatment levels during wk 11
- functional observation tests: standard behavioural assessment battery
- functional performance tests: motor activity, forelimb/hindlimb grip strength, sensory reactivity
Sacrifice and pathology:
GROSS PATHOLOGY:
The following organs were sampled from all animals and weighed before fixation: adrenals, brain, epididymides, heart, kidneys, liver, ovaries, prostate, seminal vesicles (with coagulating gland), spleen, testes, thymus, uterus (with cervix)

HISTOPATHOLOGY:
Samples of the following tissues were preserved:
adrenals, aorta (thoracic), bone and bone marrow (femur, sternum), brain (including cerebrum, cerebellum and pons), caecum, colon, duodenum, epididymides, eyes, gross lesions, heart, ileum (inlcuding Peyer's patches), jejunum, kidneys, liver, lungs (with bronchi), lymph nodes (cervical, mesenteric), mammary glands, muscle, oesophagus, ovaries, pancreas, pituitary, prostate, rectum, salivary glands (submaxillary), sciatic nerve, seminal vesicles (coagulating gland), skin (hind limb), spinal cord (cervical, mid-thoracic, lumbar), spleen, stomach, testes, thymus, thyroid/parathyroid, tongue, trachea, urinary bladder, uterus (with cervix)
-tissues from all control and 1000 mg/kg/day dose group animals processed and 5 µm sections stained (haematoxylin and eosin) and examined by light microscopy
- liver and kidney examination were was subsequently extended to include all animals from all treatment groups
Other examinations:
KIDNEY:
- renal tissue from all male rats (F0 and F1 generations) was stained with Mallory-Heidenhain stain and examined by light microscopy
Statistics:
Haematological, blood chemical, organ weights, bodyweight gain and quantitative functional performance and sensory reactivity data were assessed for dose response relationships by linear regression analysis, followed by ANOVA incorporating Levene’s test for homogeneity of variance. Where variances were shown to be homogenous, pairwise comparisons were conducted using Dunnett’s test. Where Levene’s test showed unequal variances the data were analysed using non-parametric methods: Kruskal-Wallis ANOVA and Mann-Whitney ‘U’ test.

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):
effects observed, treatment-related
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS
Increased salivation were detected following treatment at 1000 mg/kg bwt/d. The study director considered this to be due to the unpleasant taste of the test substance rather than a toxicological response.

FOOD CONSUMPTION
Food intake was increased slightly in animals from the 1000 mg/kg/d groups versus controls. This achieved statistical significance (elevated 10-14%) in males during weeks 7, 8 and 10. There was no effect in high dose females or animals of either sex treated with 250 or 50 mg/kg/day. Feed efficiency was unaffected. This observation was considered due to the unpleasant taste of the test substance and of no biological or toxicological significance.

WATER CONSUMPTION
Water intake by high dose males exhibited variable, ocassionally significant, increases of 25-50% on several occasions between days 43 to 75 and in intermediate dose males on days 61, 69 and 74 (intake increased 28-31%). No differences in water intake were detected for low dose males or any of the female treatment groups. The findings in intermediate and high dose males were not considered to be biologically or toxicologically significant.

HAEMATOLOGY
Males from the 1000 mg/kg/d group showed a small (-2%) but statistically significant reduction in mean cell haemoglobin concentration relative to controls. Males from the 1000 and 250 mg/kg/d groups exhibited statistically significant (70-80%) increases in neutrophil counts, however a number of individual values were outside the normal expected range for this parameter (0.09 – 1.29 x 10^9/l). No comparable alterations were present in low dose males or any treated females while all other haematological parameters assessed were unaltered. These findings for intermediate and high dose male groups were not considered to be biologically or toxicologically significant.

CLINICAL CHEMISTRY
Females from the 1000 mg/kg/d treatment group showed a statistically significant (approx. -50%) reduction in alkaline phosphatase activity, with a slight (-20%) but non-significantly reduction present also in high dose males. All other clinical chemistry parameters were unaffected by treatment. These isolated findings were not to be biologically or toxicologically significant.

ORGAN WEIGHTS
Males treated with 1000 mg/kg/d showed a statistically significant increase (+13%) in relative liver weight. No such effect was detected for females, or animals of either sex given 250 or 50 mg/kg/d. The finding was not considered to be biologically or toxicologically significant.

HISTOPATHOLOGY: NON-NEOPLASTIC
The following treatment-related changes were observed:

LIVER: The occurrence of generalised hepatocyte enlargement was significantly increased in females treated with 1000 and 250 mg/kg/d but not 50 mg/kg/day. There was no convincing evidence of an effect on hepatocyte size for male rats at any treatment level. Generalised hepatocyte enlargement is seen occasionally among untreated rats although such was not the case in this investigation. The occurrence of this finding in female rats from the intermediate and top treatment groups was therefore not considered to be biologically or toxicologically significance.

KIDNEY: Globular accumulations of eosinophilic material were present at significantly elevated incidence in the tubular epithelium of males treated with 1000 mg/kg/d, with isolated instances present in males from the 250 and 50 mg/kg/d groups. However the presence of one such instance among control males from this study confirms the condition to be present occasionally in untreated male rats indicating that an effect of treatment at the low and intermediate dose levels was not convincing. The finding in the high dose animals appeared consistent with hydrocarbon nephropathy, which results from the excessive accumulation of a2-microglobulin in renal proximal tubular epithelial cells. This was subsequently confirmed by the additional staining of kidney tissue with Mallory-Heidenhain stain. Since a2-microglobulin occurs only in the proximal tubular epithelium of adult male rats this finding is of no biological or toxicological significance.

OTHER: all other tissues were unremarkable

Effect levels

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: absence of biologically or toxicologically relevant findings up to a limit dose of 1000 mg/kg bwt/d

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Generalised hepatocyte enlargement was present in females treated for up to 18 wk with 1000 or 250 mg/kg bwt/d. Findings by dose level (0, 50, 250 or 1000 mg/kg bwt/d) were as follows: Number examined: 10, 10, 10, 10 Finding absent: 9, 3, 4, 4 Minimal severity: 1, 7, 3, 0 Slight severity: 0, 0, 3**, 6** ** = P<0.01 (Chi-square) Globular accumulations of eosinophilic material were observed in the tubular epithelium of male rats treated with 1000 mg/kg bwt/d for 18 wk. Findings by dose level (0, 50, 250 or 1000 mg/kg bwt/d) for Mallory-Heidenhain staining of kidney tissue from F0 and F1 males were as follows: F0 males: Number examined: 10, 10, 10, 10 Finding absent: 6, 6, 5, 0 Minimal severity: 4, 4, 5, 2 Slight severity: 0, 0, 0, 4 Moderate severity: 0, 0, 0, 4 Overall incidence for high dose group: P<0.01 (Chi-square) F1 males: Number examined: 10, 10, 10, 10 Finding absent: 6, 6, 2, 0 Minimal severity: 3, 3, 4, 7 Slight severity: 1, 1, 4, 3 Overall incidence for high dose group: P<0.01 (Chi-square)

Applicant's summary and conclusion

Conclusions:
No adverse systemic, neuro-functional or neuro-behavioural toxicity present in rats following treatment with 1000 mg/kg/d for up to 18 wk. A NOAEL of 1000 mg/kg bwt/d was obtained for sub-chronic toxicity.
Executive summary:

The repeated dose oral toxicity of NExBTL renewable diesel was investigated in a GLP-compliant rat 2-generation study modified to provide information on repeated dose effects. Enhancements included the assessment of clinical signs, effects on bodyweight and dietary intake, determination of neuro-functional and neuro-behavioural parameters and an assessment of clinical chemistry and haematological endpoints. At study end all animals were subject to full necropsy and tissues processed for histological examination. Following a preliminary range finding investigation, the test substance was administered by oral gavage to male and female Wistar rats at doses of 0, 50, 250 or 1000 mg/kg bwt/d for up to 18 wk; higher exposures were considered unnecessary and unrealistic since 1000 mg/kg bwt/d is an upper limit dose according to Annex V of Directive 67/548/EEC. Treatment-related clinical signs were limited to increased salivation following treatment, an observation presumed to reflect the unpleasant taste of the test substance. Observations for functional and behavioural toxicity together with determinations of haematological and clinical chemistry parameters were unremarkable. Histopathological findings were limited to an increased occurrence of generalised hepatocyte enlargement (a spontaneous finding, seen occasionally among untreated rats) in females from the high and intermediate dose groups while kidney tissue from high dose males exhibited globular accumulations of eosinophilic material in the tubular epithelium, shown to contain a2-microglobulin (Mallory-Heidenhain staining). The microscopic appearance of a wide range of other tissues was unremarkable. In conclusion, oral administration of NExBTL renewable diesel to rats for a period of up to 18 wk resulted in minor treatment-related changes at 1000 and 250 mg/kg/day, however these were considered not to represent an adverse health effect giving a No Observed Adverse Effect Level for systemic and functional/behavioural toxicity of 1000 mg/kg/day