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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:
April 2007 - May 2008
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study on supporting substance (with carbon range C4-10) conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevant results.

Data source

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

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:
481-730-0
EC Name:
-
IUPAC Name:
481-730-0
Constituent 2
Reference substance name:
848301-65-5
Cas Number:
848301-65-5
IUPAC Name:
848301-65-5
Constituent 3
Reference substance name:
Naphtha (Fischer-Tropsch), light, C4-10 - branched and linear
IUPAC Name:
Naphtha (Fischer-Tropsch), light, C4-10 - branched and linear
Constituent 4
Reference substance name:
C4-C10 branched and linear hydrocarbons (light) – Naphtha
IUPAC Name:
C4-C10 branched and linear hydrocarbons (light) – Naphtha
Details on test material:
- Name of test material (as cited in study report): Naphtha (Fischer-Tropsch), light, C4-10-branched and linear
- Substance type: Organic
- Physical state: Clear colourless liquid
- Analytical purity: 100%
- Impurities (identity and concentrations): Information not available
- Composition of test material, percentage of components: Information not available
- Isomers composition: Information not available
- Purity test date: Information not available
- Lot/batch No.: Information not available
- Expiration date of the lot/batch: Information not available
- Stability under test conditions: formulations of test material were stable for at least fourteen days
- Storage condition of test material: approximately +4°C, in the dark

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: six to eight weeks
- Weight at study initiation:it averages for males 168,2 g and for females 149,8 (these means are calculated from data of first study day for all groups tested)
- Fasting period before study: no
- Housing: in groups of three or four by sex in polypropylene cages with stainless steel mesh lids and solid bases containing softwood chip bedding in a single air-conditioned room
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2°C
- Humidity (%): 55 ± 15%
- Air changes (per hr): at least fifteen air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on oral exposure:
The test material was administered daily by gavage using stainless steel cannula attached to a disposable plastic syringe. Conrol animals were treated only with vehicle in an identical manner.

PREPARATION OF DOSING SOLUTIONS: test material was prepared at the appropriated concentrations as solutions in Arachis oil BP, which was served as vehicle and as common control.
- Rate of preparation of dosing solution (frequency): weekly
- Mixing appropriate amounts with (Type of food): vehicle
- Storage temperature of food: at +4°C in the dark


VEHICLE
- Justification for use and choice of vehicle (if other than water): Arachis oil BP was chosen as a suitable diluter for hydrocarbons. Arachis oil is a monounsaturated fat, wide used as a most neutral food component in a variety of diets, also known for its high smoke point and stability. The oil ensures an optimal intake of test material in gastrointestinal tract.
- Concentration in vehicle: 0; 12,5; 50 and 188 mg/mL
- Amount of vehicle (if gavage): 4mL/kg/day
- Lot/batch no. (if required): data not avilable
- Purity: data not available
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of each test material formulation were taken and analysed for concentration of 'Naphtha (Fischer-Tropsch), light, C4—10-branched and
linear'. The concentration of test material in formulations was determined by gas chromatography (GC) using an external standard technique. The analytical method has been satisfactorily validated in terms of linearity, specificity and accuracy for the purposes of the study.The results indicate that the prepared formulations were within ± 10% of the nominal concentration and were stable for at least fourteen days.
Duration of treatment / exposure:
1.Non-recovery groups of animals: ninety consecutive days/oral gavage.
2.Two recovery groups 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/oral gavage.
Frequency of treatment:
daily at the same timepoint
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
50 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
200 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
750 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
- ten male and ten female Sprague-Dawley Crl:CD® (SD) IGS BR strain rats at dose levels of 50, 200 and 750 mg/kg/day
- control group: ten males and ten females with vehicle alone (Arachis oil BP)
- two recovery groups, each of ten males and ten females, were treated with the high dose (750 mg/kg/day) or the vehicle alone

Dose Level Animal Numbers
------------------------------------------------
(mg/kg/day) Male Female
------------------------------------------------
Control 0 10 10
Recovery Control 0 10 10
Low 50 10 10
Intermediate 200 10 10
High 750 10 10
Recovery High 750 10 10
Control animals:
yes, concurrent vehicle
Details on study design:
Animals were randomly assigned to the control and treatment groups according to OECD Guidline 408 "Repeated Dose 90-Day Oral Toxicity in
Rodents"

- Dose selection rationale: were ascertained from the preliminary fourteen day repeated dose oral (gavage) range-finder study in the rat: test material in the concentrations 300 mg/kg/day and 1000 mg/kg/day as well as control group treated with vehicle alone was administered daily. Increased salivation which was detected for males and females at the 1000 mg/kg/day and for males at the 300 mg/kg/day as well as individual incident of palish kidney and sloughing on the non-glandular region of stomach from the treatment group of 1000 mg/kg/day were the reason to determine the dose levels for the ninety day study as high dose 750 mg/kg/day, intermediate dose 200 mg/kg/day and low dose 50 mg/kg/day.

- Rationale for animal assignment (if not random): randomized

- Rationale for selecting satellite groups: randomized

A common control group, treated only with vehicle alone, is needed to exclude any effect of vehicle and make possible to interpret the effects of test material. The aim of recovery groups is to observe reversibility or persistence of any toxic effects.

- Post-exposure recovery period in satellite groups: 28 days

- Section schedule rationale (if not random): randomized
Positive control:
no

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: No
- Time schedule:
- Cage side observations checked in table [No.?] were included.


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


BODY WEIGHT: Yes
- Time schedule for examinations: on day 1 of study and at weekly intervals thereafter.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No feeding study but oral gavage.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

FOOD CONSUMPTION: was recorded for each cage group at weekly intervals throughout the study (calculated in g/animal/day).


FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No drinking water study but oral gavage.
- Time schedule for examinations:

WATER CONSUMPTION:daily by visual inspection.


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: the eyes were examined pretreatment and before termination of treatment (during Week 12).
- Dose groups that were examined: non-recovery control and non-recovery high dose animals.

Examinations included observation of the anterior structures of the eye, pupillary and corneal blink reflex. Following
pupil dilation with 1.0% “Tropicamide” solution, detailed examination of the internal structure of the eye using a direct
ophthalmoscope was performed.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the treatment period (Day 90) and on all recovery group animals at
the end of the treatment-free period (Day 118).
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: all animals from each test and control group as well as all recovery group animals.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the treatment period (Day 90) and on all recovery group animals at the end of the treatment-free period (Day 118).
- Animals fasted: No
- How many animals: all animals from each test and control group as well as all recovery group animals
- The parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant.

URINALYSIS: No
- Time schedule for collection of urine:
- Metabolism cages used for collection of urine: Yes / No / No data
- Animals fasted: Yes / No / No data



NEUROBEHAVIOURAL EXAMINATION: Yes


All animals were observed for signs of functional/behavioural toxicity. During Week 12 functional performances tests were also
performed on all non-recovery animals together with an assessment of sensory reactivity to different stimuli.


- Time schedule for examinations: Prior to the start of treatment and at weekly intervals thereafter.
- Dose groups that were examined: only non-recovery (without 28 days treatment-free period) animals.
- Battery of functions tested: sensory activity (Grasp response, Touch escape,Vocalisation, Pupil reflex, Toe pinch, Blink reflex,Tail pinch, Startle reflex
Finger approach) grip strength and motor activity.
- Parameters were examined using a purpose built arena.

OTHER:
Sacrifice and pathology:

GROSS PATHOLOGY: (Yes ) On completion of the dosing period, or in the case of recovery group animals, at the end of the
treatment-free period, all 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: (in order to determine general toxicological effects) Adrenals, Ovaries,Brain, Spleen,Epididymides, Testes, Heart, Thymus, Kidneys, Uterus and Liver

HISTOPATHOLOGY: Yes

 All tissues from control and 750 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 was also processed.

Since there were indications of treatment-related liver and kidney changes, examination was subsequently extended to include similarly prepared sections of the liver and kidneys from all animals in the other treatment groups. The Mallory Heidenhain stain was also performed on kidney sections fromall males.

Microscopic examination was conducted by the Study Pathologist. All findings were entered into the ROELEE Pathology computerisation system for tabulation and report production. Findings were reported in the summary table by exception ( where a finding was seen it was reported but if
no finding was seen, the organ was not listed at all).
 
 
 
 
 
 
 
 
 
 
 
Other examinations:
No
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). Probability 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.
Kruskal-Wallis one way non-parametric analysis of variance for the comparison of severity grades for the more frequently observed graded
conditions. Probability values (p) are presented as follows:
p<0.001 +++ --- ***
p<0.01 ++ -- **
p<0.05 + - *
p<0.1 (+) (-) (*)
p>0.1 N.S. (not significant)
Plus signs indicate positive differences from the control group and minus signs indicate negative differences. Asterisks refer to overall between
group variation which is non-directional.

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):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
There were no unscheduled deaths and no clinically observable signs of toxicity were detected in test or control animals throughout the study period.
Increased salivation was evident in 750 mg/kg/day animals throughout the treatment period. Isolated incidents of increased salivation were also
evident in 200 and 50 mg/kg/day animals. Observations of this nature are often reported following oral administration of an unpalatable or slightly
irritant test material formulation and considered not to be an indication of systemic toxicity. Generalised red/brown stained fur and wet fur was
evident in a number of treated animals throughout the dosing period. Such observations are commonly observed in laboratory maintained rats and in view of the sporadic nature of these findings were considered to be entirely incidental and considered of no toxicological significance. One male
treated with 50 mg/kg/day had an open wound on Day 28 followed by scab formation on Days 29 to 31. One control male showed abnormal gait on
Day 76. These were isolated incidental findings and unrelated to treatment.


BODY WEIGHT AND WEIGHT GAIN
No toxicologically significant effects on bodyweight development were detected.
Males treated with 750 mg/kg/day showed a statistically significant reduction in bodyweight gain during Weeks 8 and 11 of treatment. Recovery 750 mg/kg/day males showed a statistically significant increase in bodyweight gain during Week 16 (Week 3 of the treatment free period). In the absence
of any effect on overall bodyweight gain, differences in weekly bodyweight change were considered to reflect normal biological variation and to be of no toxicological significance.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study) No feeding study
FOOD CONSUMPTION: There was no adverse effect on food consumption during the study period.


FOOD EFFICIENCY: Food efficiency (the ratio of bodyweight gain to dietary intake) was similar to that of controls.


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study) No drinking water study

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


OPHTHALMOSCOPIC EXAMINATION
There were no treatment-related ocular effects.


HAEMATOLOGY
There were no toxicologically significant changes in the haematological parameters measured.
Non-recovery males from all treatment groups showed a statistically significant increase in erythrocyte count and a reduction in mean cell
haemoglobin. In the absence of any semblance of a dose related response the intergroup differences were considered to be of no toxicological
importance. Non-recovery females treated with 750 and 200 mg/kg/day showed a statistically significant reduction in activated partial
thromboplastin time. In the absence of an associated effect on clotting time the intergroup differences were considered to be of no toxicological
significance. Recovery 750 mg/kg/day males showed a statistically significant reduction in haemoglobin and haematocrit whilst recovery females
showed a statistically significant increase in platelet count following twenty eight days without treatment. In the absence of similar effects for
non-recovery animals at the end of the dosing period, the intergroup differences were considered to be incidental and ofno toxicological importance.


CLINICAL CHEMISTRY
Non-recovery males treated with 750 mg/kg/day showed statistically significant increases in plasma urea, calcium concentration, creatinine and
cholesterol with the majority of individual values for cholesterol and creatinine outside of the respective normal ranges for rats of the strain and age used. The effect on urea and creatinine extended to the 200 mg/kg/day non-recovery male dose group, with five male creatinine values being outside of the respective normal range.
No such effects were detected in non-recovery females treated with 750 or 200 mg/kg/day, non-recovery animals of either sex treated with
50 mg/kg/day or recovery animals following twenty eight days without treatment.
The remaining statistically significant intergroup differences were considered to be of no toxicological importance. Non-recovery females treated
with 750 and 200 mg/kg/day showed a statistically significant reduction in plasma glucose. The majority of individual values were within the
respective normal range and, in isolation, were regarded as incidental. Aspartate aminotransferase and alkaline phosphatase were reduced for
750 mg/kg/day non-recovery males. The majority of individual values were within the respective normal ranges and, in any case, only an increase in
these parameters are indicative of tissue destruction in the liver and kidneys and as such the reduction cannot be regarded as toxicologically
important. An increase in total protein was also detected in non-recovery females treated with 50 mg/kg/day. There was no dose related response
and as such the intergroup difference was considered to be fortuitous.


URINALYSIS
not examined


NEUROBEHAVIOUR
- Behavioural Assessments: there were no toxicologically significant changes in the behavioural parameters measured.
Hunched posture and tiptoe gait were evident in one male treated with 200 mg/kg/day during the open-field assessments performed in Week 9. In the absence of a dose related response or any similar effects being detected during the daily clinical observations this intergroup difference was
considered to be of no toxicological importance.
- Functional Performance Tests: there were no treatment-related changes in the functional performance parameters measured.
Females treated with 750 mg/kg/day showed a statistically significant increase in mean hind limb grip strength. This effect was confined to one out of the three tests for this parameter and in the absence of any supporting clinical observations to suggest an effect of neurotoxicity, this finding was
considered to be of no toxicological significance. Males treated with 200 mg/kg/day showed a statistically significant increase in overall activity. In
the absence of a dose-related response or any supporting clinical observations to suggest an effect of neurotoxicity, these findings were considered to be of no toxicological significance.
- 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 were of no toxicological importance.


ORGAN WEIGHTS
Males treated with 750 mg/kg/day showed a substantial increase in liver and kidney weight, both absolute and relative to terminal bodyweight,
compared with controls. Statistical significance was achieved and the majority of individual values were outside the respective normal ranges. The
effect on kidney weight extended to the 200 and 50 mg/kg/day male dose groups, again with many values outside the normally expected ranges.
Liver weights were also increased for 750 mg/kg/day females and in 200 mg/kg/day males.
The increased kidney weights persisted in recovery 750 mg/kg/day males following twenty eight days without treatment, however the intergroup
difference was not as substantial as in the non-recovery animals.
No toxicologically significant effects were detected in females treated with 200 or 50 mg/kg/day or in recovery 750 mg/kg/day females following a
twenty eight days treatment free period.
Non-recovery and recovery females treated with 750 mg/kg/day showed a statistically significant increase in kidney weight both absolute and relative to terminal bodyweight. In the absence of any histological correlates the intergroup differences were considered not to be of any toxicological
significance. Recovery 750 mg/kg/day females also showed a reduction in absolute and relative thymus weight. In the absence of similar effects for
non-recovery animals at the end of the dosing period, the intergroup differences were considered to be incidental and of no toxicological importance.

GROSS PATHOLOGY
Four non recovery males treated with 750 mg/kg/day showed enlarged kidneys at necropsy. One of the affected males also showed a speckled
appearance on the kidney surface and increased renal pelvic cavitation. Enlarged kidneys were also evident in one 200 mg/kg/day male.
The effect on the kidneys continued into recovery 750 mg/kg/day males following twenty eight days without treatment, with four males showing a
mottled appearance on the kidney surface.
No such effects were detected in females treated with 750 or 200 mg/kg/day, in animals of either sex treated with 50 mg/kg/day or in recovery 750 mg/kg/day females following the treatment free period.


HISTOPATHOLOGY: NON-NEOPLASTIC
LIVER: Centrilobular hepatocyte enlargement was observed in relation to treatment for animals of either sex, but more especially for males, treated
with 750 mg/kg/day (P <0.001 for males and NS for females), and for males only treated with 200 mg/kg/day (P <0.001).
Three males treated with 50 mg/kg/day were also affected and the possibility that hepatocyte enlargement was related to treatment at this dose level cannot be excluded although such changes are occasionally seen among untreated rats as a spontaneous change.
Hepatocyte enlargement is commonly observed in the rodent liver following the administration of xenobiotics and, in the absence of associated
inflammatory or degenerative changes, is generally considered to be adaptive in nature. This was evidenced by the regression of hepatocyte
enlargement among recovery 750 mg/kg/day animals following an additional twenty eight days without treatment.

KIDNEY: Tubular basophilia, tubular degeneration/necrosis and accumulations of globular eosinophilic material in the tubular epithelium were all
seen as a consequence of treatment for males only treated with 750 mg/kg/day (P <0.001 for all observations), or at 200 mg/kg/day (P <0.001 for
all observations). The majority of males treated with 50 mg/kg/day were affected only with accumulations of globular eosinophilic material in the
tubular epithelium (P <0.01).
Globular accumulations of eosinophilic material in the tubular epithelium are consistent with the presence of hydrocarbon nephropathy, which
results from the excessive accumulation of a2-microglobulin in renal proximal tubular epithelial cells. In more severe cases tubular basophilia and
tubular degeneration/necrosis are associated with a2-microglobulin accumulation. a2-Microglobulin is found only in the proximal tubular
epithelium of adult male rats.
Although globular accumulations of eosinophilic material had regressed to background control levels among recovery 750 mg/kg/day males, there was no evidence of regression of tubular basophilia or tubular degeneration/necrosis (P <0.001 for both conditions) following an additional
twenty eight days without treatment.
Further sections of kidney stained with Mallory Heidenhain were also examined. This staining technique demonstrates protein droplets in the renal
tubules and epithelium and correlated reasonably well with the eosinophilic accumulations reported from haematoxylin and eosin stained sections.
No adverse effects in male and female reproductive organs were detected.
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.

HISTOPATHOLOGY: NEOPLASTIC: No effects

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: adaptive changes to the liver
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: adaptive changes to the liver and minor, reversible blood chemistry alterations

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Data which only significant deviate or necessary for justification of the validity of this study's results are summarized in Tables 1 and 2:

Table1: Group mean blood chemical values for non-recovery animals

A. Males

Group (Sex)

 

Urea mg/dL

Glucose mg/dL

Tot. Prot. g/dL

Ca++ mmol/L

ASAT IU/L

AP IU/L

Creat mg/dL

Chol mg/dL

1(M)

Mean

22.6

149.1

7.568

2.532

72.9

279.3

0.790

76.2

S.D.

4.9

17.4

0.530

0.047

8.0

77.2

0.067

7.5

2(M)

Mean

23.7

159.9

7.437

2.588

73.7

275.5

0.830

76.2

S.D.

3.3

16.3

0.504

0.047

21.5

68.7

0.053

13.0

3(M)

Mean

26.6*

148.9

7.492

2.571

67.5

239.8

0.842*

79.6

S.D.

4.6

13.7

0.440

0.079

4.7

41.5

0.063

15.5

4(M)

Mean

29.0**

152.0

7.473

2.607*

64.9*

189.2**

0.869**

89.6*

S.D.

6.4

15.8

0.283

0.080

5.9

49.4

0.068

12.2

B. Females

Group (Sex)

 

Urea mg/dL

Glucose mg/dL

Tot. Prot. g/dL

Ca++ mmol/L

ASAT IU/L

AP IU/L

Creat mg/dL

Chol mg/dL

1(F)

Mean

29.7

146.1

7.015

2.611

73.9

183.7

0.846

77.2

S.D.

6.3

20.3

0.365

0.093

11.5

67.0

0.071

14.1

2(F)

Mean

31.9

136.4

7.543*

2.685

81.1

188.8

0.903

82.2

S.D.

6.2

10.2

0.257

0.053

15.6

73.2

0.106

21.6

3(F)

Mean

30.1

133.1*

6.972

2.619

78.3

184.6

0.828

82.3

S.D.

5.8

7.4

0.357

0.102

11.3

77.4

0.070

16.7

4(F)

Mean

27.8

129.9**

7.130

2.583

78.0

159.7

0.813

87.4

S.D.

4.1

8.8

0.569

0.104

13.5

52.3

0.046

19.7

Dose Levels: Group 1 - 0(Control) Group 2 - 50 mg/kg/day Group 3 - 200 mg/kg/day Group 4 - 750 mg/kg/day

M-males; F-females; 10 animals of each sex were used for every parameter tested. Asterixes indicate significance level.

Table 2: Group Mean with Corresponding Relative (% of Bodyweight) Organ Weights

A. Non-recovery animals

 

MALES

FEMALES

 

 

0 Control

50 mg/kg/day

200 mg/kg/day

750 mg/kg/day

0 Control

50 mg/kg/day

200 mg/kg/day

750 mg/kg/day

Kidneys

Mean (g)

3.15912

3.48128*

4.06481**

4.37218**

1.78743

1.68603

1.85559

1.81901*

S.D.

0.39093

0.24750

0.65549

0.71916

0.17446

0.19862

0.19720

0.21460

Mean (%)

0.543

0.594*

0.721**

0.785**

0.590

0.597

0.612

0.644*

S.D.

0.046

0.050

0.095

0.114

0.056

0.039

0.053

0.039

Liver

Mean (g)

18.0617

19.3558

19.8321**

22.3936**

10.3658

9.95510

10.5018

10.9495*

S.D.

1.87998

2.16781

2.51897

3.29017

1.18067

1.18859

0.92810

1.67206

Mean (%)

3.107

3.289

3.508**

4.013**

3.420

3.531

3.463

3.884*

S.D.

0.164

0.240

0.167

0.390

0.388

0.356

0.216

0.514

Thymus

Mean (g)

0.46297

0.46903

0.48859

0.46179

0.38588

0.36019

0.38674

0.35193

S.D.

0.12032

0.07361

0.14691

0.08700

0.08699

0.07082

0.08189

0.10885

Mean (%)

0.080

0.080

0.086

0.083

0.127

0.128

0.127

0.124

S.D.

0.021

0.012

0.021

0.017

0.028

0.024

0.020

0.032

 

B. Recovery animals

 

 

MALES

FEMALES

 

 

0 Control

750 mg/kg/day

0 Control

750 mg/kg/day

Kidneys

Mean (g)

3.50237

3.97069**

1.71979

1.94648*

S.D.

0.38627

0.43261

0.14790

0.12362

Mean (%)

0.558

0.672**

0.585

0.608*

S.D.

0.042

0.073

0.056

0.035

Liver

Mean (g)

19.3049

18.2855

9.99168

10.4391

S.D.

1.89168

1.33113

1.04258

1.04399

Mean (%)

3.077

3.093

3.397

3.261

S.D.

0.139

0.208

0.370

0.335

Thymus

Mean (g)

0.45715

0.41172

0.34710

0.30426*

S.D.

0.06759

0.07793

0.06566

0.06431

Mean (%)

0.073

0.070

0.117

0.095*

S.D.

0.009

0.013

0.018

0.019

M-males; F-females; 10 animals of each sex were used for every parameter tested. Asterixes indicate significance level

Applicant's summary and conclusion

Conclusions:
It was concluded that the NOAEL for males, excluding male rat hydrocarbon nephropathy, was 50 mg/kg/day, based upon adaptive changes to the liver and minor, reversible blood chemistry alterations. The NOAEL was 200 mg/kg/day in females, based only upon adaptive liver changes. For human health hazard assessment, liver changes that are adaptive in nature due to metabolism of large amounts of a xenobiotic are generally not considered to be adverse. For the purpose of hazard evaluation, the NOAEL is therefore regarded as 750 mg/kg/day.
Executive summary:

A 90-day oral (gavage) toxicity study has been carried out using 'Naphtha (Fischer-Tropsch), light, C4 -10 - branched and linear', following OECD Test Guideline 408 and conducted according to GLP. 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 750 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. There were no unscheduled deaths and no clinically observable signs of toxicity were detected in test or control animals throughout the study period. There were no toxicologically significant or treatment-related findings in functional performance tests, sensory reactivity assessments, bodyweight measurements, food and water consumption, opthalmoscopy or haematology.

Treatment-related liver and kidney effects were observed in males at all dose levels and in females at 750 mg/kg/day only. Statistically significant blood chemistry effects (increased plasma urea, calcium concentration, creatinine and cholesterol) were observed in males only at 200 and 750 mg/kg. These effects were not observed at the end of the recovery period, indicating reversibility. In the liver, centrilobular hepatocyte enlargement was observed in animals of both sexes at 750 mg/kg, but in the absence of associated inflammatory or degenerative changes was considered to be adaptive in nature. This view is supported by the regression of hepatocyte enlargement during a 28-day recovery period.

In the kidneys, tubular basophilia, tubular degeneration/necrosis and accumulations of globular eosinophilic material in the tubular epithelium were all seen as a consequence of treatment for males only treated with 750 mg/kg/day and 200 mg/kg/day. The majority of males at 50 mg/kg/day were affected only with accumulations of globular eosinophilic material in the tubular epithelium.

Although globular accumulations of eosinophilic material had regressed to background control levels among recovery group animals, there was no evidence of regression of tubular basophilia or tubular degeneration/necrosis.

The kidney changes identified in males at all dose levels were consistent with well documented light hydrocarbon-induced male rat nephropathy, changes that are peculiar to the male rat in response to treatment with certain hydrocarbons. This effect was therefore considered irrelevant for the assessment of hazard to human health. In males, no NOEL could be established, based on liver effects. The report concluded that the NOAEL for males, excluding male rat hydrocarbon nephropathy, was 50 mg/kg/day, based upon adaptive changes to the liver and minor, reversible blood chemistry alterations. The NOAEL was 200 mg/kg/day in females, based only upon adaptive liver changes. For human health hazard assessment, liver changes that are adaptive in nature due to metabolism of large amounts of a xenobiotic are generally not considered to be adverse. For the purpose of hazard evaluation, the NOAEL is therefore regarded as 750 mg/kg/day.