Registration Dossier

Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2 December 2009 (animal arrival) -10 March 2010 (pathology completion)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
increased animal number
Principles of method if other than guideline:
The toxicity subgroup for this study comprised 5 male and 5 female rats per treatment group and the reproductive subgroup comprise 5 male and 10 female rats per treatment group. The five male rats in each treatment group of the toxicity subgroup were used for mating with the female reproductive subgroup animals of the corresponding treatment group. The ten female rats in each treatment group of the reproductive subgroup were used exclusively for the reproductive/developmental toxicity phase of the study. This deviation was undertaken to enhance the robustness of the study.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Tetrabutane
- Substance type: Industrial
- Physical state: clear liquid
- Impurities (identity and concentrations): no data
- Composition of test material, percentage of components: hydrocarbons C16 = 63.6 %, C20= 26.6 %, C24= 8.37% :low and high boiling components 0.38 % and 1.05 %, respectively
- Isomers composition: no data
- Purity test date: yes
- Lot/batch No.: 0649/82535
- Expiration date of the lot/batch: 8 June 2010
- Stability under test conditions: stable
- Storage condition of test material: room temperature

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: 9-10 weeks
- Weight at study initiation: 303-355 g
- Fasting period before study: none
- Housing: 5 animals/P2000 polycarbonate cage: males were housed singly with one female in RB3 modified polypropylene cages during mating.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23°C
- Humidity (%): 40-70 %
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hrs dark/ 12 hrs light

IN-LIFE DATES: From: 2 December 2009 (animal arrival) To: 20 January 2010 (necropsy completion)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was used as supplied. All formulations were prepared freshly weekly and were stored refrigerated (2-8°C) in the dark.

VEHICLE
- Justification for use and choice of vehicle (if other than water): no data
- Concentration in vehicle: 20, 60 or 200 mg/ml
- Amount of vehicle (if gavage): 5 ml/kg
- Lot/batch no. (if required): no data
- Purity: no data

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Before treatment commenced, the suitability of the proposed mixing procedure was determined and specimen formulations were analysed to assess the homogeneity and stability of the test material in the liquid matrix. The stability was assessed following storage at ambient temperature (nominally 21°C) for 0 hours, 4 hours (continual stirring) and 2 days, and refrigeration (nominally 2-8°C) for 2, 8 and 15 days. Prior to initial sampling on each day, the formulation was mixed by 20-fold inversion and magnetic stirring for a minimum of 5 minutes. At each time point, single samples (nominally 1 mL) were taken for assay from the top, middle and bottom of the magnetically stirred formulation. Stability was determined from the mean concentration of the analyte in the vehicle at each sampling point. Specimen formulations (typically 400 mL) were prepared at concentrations of 2 and 200 mg/mL and equally split between four amber glass screw-capped bottles and were confirmed for 15 days when refrigerated and for 48 hours at room temperature.
Samples of each formulation prepared for administration in the first week of the dosing procedure were analysed for achieved concentration of the test substance. Four samples were taken (nominally 1 mL accurately weighed); 2 assays from each group and 1 assay. The remainder was frozen (nominally -20°C) and retained as contingency for analysis if any result required confirmation.

The GC analytical procedure was validated with respect to linearity of detector response, precision of injection, specificity of chromatographic analysis, limit of detection, accuracy and precision.

The homogeneity and stability was confirmed for Tetrabutane in corn oil formulations at nominal concentrations of 2 mg/mL and 200 mg/mL during distribution between the bottles, during magnetic stirring for 4 hours, ambient temperature storage for 2 days and refrigerated storage for up to 15 days. The storage times represented the maximum time from preparation to completion of administration.

The mean concentrations of Tetrabutane in test formulations analysed for the study were within +10%/-15% of nominal concentrations, confirming accurate formulation
Duration of treatment / exposure:
5 weeks (daily)
Frequency of treatment:
All animals were dosed once each day, at approximately the same time each day, seven days per week for five weeks.
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300 or 1000 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
5 males and 5 females per dosage group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels of 100, 300 and 1000 mg/kg/day were selected in conjunction with the Sponsor with reference to previous work with this compound performed in these laboratories (Huntingdon Life Sciences Report Number: BBB0024). In that study, the CD rats received Tetrabutane at doses of 100, 300 or 1000 mg/kg/day for seven days, there were no findings which precluded the use of these dose levels on the subsequent 4 week general toxicity and reproductive developmental toxicity screening study.

- Rationale for animal assignment (if not random):
On arrival, the animals were removed from the transit boxes and allocated to study cages. Using the sequence of cages in the battery, one animal at a time was placed in each cage with the procedure being repeated until each cage held the appropriate number of animals. Each sex was allocated separately
- Rationale for selecting satellite groups: no satellite groups used
- Post-exposure recovery period in satellite groups: no post-exposure recovery period

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes

- Time schedule:
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of ill-health amongst the occupants. Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.
Daily during the first week of treatment, twice weekly during Weeks 2 to 4 (middle and end of each week) and weekly thereafter, detailed observations were recorded at the following times in relation to dose administration:
Immediately before dosing
Immediately after dosing on return of the animal to its cage
On completion of dosing of each group
Between one and two hours after completion of dosing of all groups
As late as possible in the working day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
Before treatment commenced and during each week of treatment, detailed physical examination and arena observations were performed on each animal. On each occasion, the examinations were performed at approximately the same time of day (before dosing during the treatment period), by an observer unaware (as far as practically possible) of the experimental group to which the animal belonged.
After removal from the home cage, animals were assessed for physical condition and behaviour during handling and after being placed in a standard arena. Any deviation from normal was recorded with respect to the nature and, where appropriate, degree of severity. Particular attention was paid to possible signs of neurotoxicity, such as convulsions, tremor and abnormalities of gait or behaviour.

Findings were either reported as "present" or assigned a severity grade - slight, moderate or marked.


BODY WEIGHT: Yes

- Time schedule for examinations:
All toxicity and reproductive subgroup males (10 animals per treatment group) were weighed weekly throughout the study. All toxicity and reproductive subgroup females (15 animals per treatment group) were weighed weekly for the first two weeks and the toxicity subgroup females (five animals per treatment group) were weighed during Weeks 3, 4 and 5.

FOOD CONSUMPTION: Yes

The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded during weeks 1 and 2 for all male (two cages of 5 animals per treatment group) and all female (three cages of 5 animals per treatment group) toxicity and reproductive subgroups. From these records the mean weekly consumption per animal (g/rat/week) was calculated for each cage.

FOOD EFFICIENCY: No

WATER CONSUMPTION : Yes

Fluid intake was assessed by daily visual observation. No effect was observed and, consequently, quantitative measurements were not performed

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: during week 5 of treatment
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: 5 males and 5 females per treatment group
- Parameters examined.
Blood samples (nominally 0.5 mL) were collected into tubes containing EDTA as anticoagulant and examined for the following characteristics:

The following were measured using a Bayer Advia 120 haematology analyser
Haematocrit (Hct)
Haemoglobin concentration (Hb)
Erythrocyte count (RBC)
Mean cell haemoglobin (MCH)
Mean cell haemoglobin concentration (MCHC)
Mean cell volume (MCV)
Total leucocyte count (WBC)
Differential leucocyte count
Neutrophils (N)
Lymphocytes (L)
Eosinophils (E)
Basophils (B)
Monocytes (M)
Large unstained cells (LUC)
Platelet count (Plt)

Morphology flags were generated by the Advia 120 analyser. The most common morphological changes, anisocytosis, micro/macrocytosis and hypo/hyperchromasia were recorded as follows:
- = no abnormalities detected
+ = slight
++ = moderate
+++ = marked

Blood film (prepared for all samples) - Romanowsky stain, examined for abnormalities by light microscopy, in the case of flags from the Advia 120 analyser. Confirmation or a written description from the blood film was made where appropriate.

Additional blood samples were taken into tubes containing citrate anticoagulant and examined in respect of:
Prothrombin time (PT) - using an ACL 3000 Plus analyser and IL PT-Fibrinogen reagent
Activated partial thromboplastin time (APTT) - using an ACL 3000 Plus Analyser and IL APTT reagent




CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: during week 5 of treatment (at the same time and using the same animals as for haematology)
- Animals fasted: Yes
- How many animals: 5 males and 5 females per treatment group
- Parameters examined.
Using lithium heparin as anticoagulant blood samples were mechanically agitated for at least two minutes and subsequently centrifuged at 2000 g for 10 minutes in order to separate the plasma. After separation, the plasma was examined using a Roche P Modular Analyser: in respect of:
Alkaline phosphatase (ALP)
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Gamma-glutamyl transpeptidase (gGT)
Total bilirubin (Bili)
Bile acids (BIAC)
Urea
Creatinine (Creat)
Glucose (Gluc)
Total cholesterol (Chol)
Sodium (Na)
Potassium (K)
Chloride (Cl)
Calcium (Ca)
Inorganic phosphorus (Phos)
Total protein (Total Prot)
Albumin (Alb) - by chemical assay

Albumin/globulin ratio (A/G Ratio) was calculated from total protein concentration and analysed albumin concentration.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
Sensory reactivity, grip strength and motor activity assessments were performed (before dosing) on five males and five females in each group during Week 5 of treatment. For sensory reactivity and grip strength, animals were tested by an observer who was unaware of the treatment group to which each animal belonged.

- Dose groups that were examined: all dose groups examined

- Battery of functions tested: sensory activity / grip strength / motor activity :

Approach response

A blunt probe was brought towards the animal’s head until it was close to the animal’s nose (but not touching the vibrissae). The animal’s reaction was recorded as:
1 No reaction or ignores probe
2 Normal awareness and reaction e.g. approaches and/or sniffs probe
3 Abnormally fearful or aggressive reaction

Touch response

The animal was stroked gently on the nape of the neck with a blunt probe and the reaction recorded as:
1 No reaction or ignores probe
2 Normal awareness and reaction e.g. turns towards or moves away
3 Abnormally fearful or aggressive reaction

Auditory startle reflex

The animal’s response to a sudden loud noise was assessed and scored as:
1 No response
2 Weak response e.g. ear twitch only
3 Normal response e.g. obvious flinch or startle
4 Exaggerated response e.g. all feet off floor

Tail pinch response

The animal’s tail was pinched sharply with forceps approximately one third from the tip and the response graded as:
1 No response
2 Weak response e.g. turns around slowly or weak vocalisation without moving away
3 Normal response e.g. jumps forward or turns around sharply, usually with vocalisation
4 Exaggerated response e.g. excessive vocalisation, body movement or aggression

Grip strength

Forelimb and hindlimb grip strength was measured using Mecmesin Force Indicators. Three trials were performed.
At any point during the observations, additional comments were made as free text where considered appropriate.

Motor activity

Motor activity was measured using a Rodent Activity Monitoring System, with hardware supplied by Pearson Technical Services and software developed and maintained by Huntingdon Life Sciences. Animals were tested individually in clear polycarbonate cages and motor activity was measured by counting infra-red beam breaks over ten 6-minute intervals (one hour total). Ten beams were set at two height levels (five low and five high) to detect cage floor and rearing activity respectively. All animals were not necessarily tested on the same day, but the numbers of animals and the times of testing were balanced across the groups on each day of testing.

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Five males and five females per group were killed after 5 weeks of treatment. All animals were killed by carbon dioxide asphyxiation.The sequence in which the animals were killed after completion of the study was selected to allow satisfactory inter-group comparison
.
All animals were subject to a detailed necropsy, which involved the following:

After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. After ventral mid-line incision, the neck and associated tissues and the thoracic, abdominal and pelvic cavities and their viscera were exposed and examined in situ. Any abnormal position, morphology or interaction was recorded. External and cut surfaces of the organs and tissues were examined as appropriate. Any abnormality in the appearance or size of any organ and tissue was recorded and the required tissue samples preserved in appropriate fixative.

The following organs, taken from five males and five females per treatnent group were dissected free of adjacent fat and other contiguous tissue and the weights recorded:

Adrenal glands Spleen
Brain Thymus
Heart Thyroid with parathyroids*
Kidneys Uterus with cervix
Liver
Ovaries with oviducts (L&R)
Pituitary


* Weighed after partial fixation

Organ weights were also adjusted for terminal bodyweight using the weight recorded before necropsy.


HISTOPATHOLOGY: Yes

Tissues were examined for 5 males and 5 females of the Control and 1000 mg/kg/day treatment groups, sacrificed on completion of the scheduled treatment period.

Adrenals Pituitary
Brain Peyer’s patches
Caecum Rectum
Colon Sciatic nerves+
Duodenum Skin
Heart Spinal cord
Ileum Spleen
Jejunum Sternum (with marrow bone)
Kidneys Stomach
Liver Thymus
Lungs Thyroid with parathyroids
Lymph nodes - mandibular Trachea
- mesenteric Urinary bladder
Mammary area - caudal Uterus with cervix
Marrow smear Vagina
Oesophagus
Ovaries with oviducts (L&R)








Statistics:
See free text field below

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
There were no deaths, clinical signs, arena observations or signs related to dosing considered to be related to treatment.
Mortality:
no mortality observed
Description (incidence):
There were no deaths, clinical signs, arena observations or signs related to dosing considered to be related to treatment.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The bodyweight and bodyweight change of males receiving 300 or 1000 mg/kg/day was slightly increased during Weeks 0-1, 1-2 and 3-4 when compared with that of the Control, resulting in a slightly increased overall bodyweight gain for males receiving 300 or 1000 mg/kg/day. The bodyweight change of males receiving 100 mg/kg/day was slightly increased in Week 3 of treatment when compared with that of the Control, however, this appeared to be an isolated incidence and is considered likely to be incidental.
The bodyweight and bodyweight change of females receiving 100, 300 or 1000 mg/kg/day was slightly increased during Weeks 0-1 and 1-2, and for toxicity subgroup females receiving 100 mg/kg/day during Week 4 when compared with that of the Control, resulting in a slightly increased overall bodyweight gain for females receiving 100, 300 or 1000 mg/kg/day.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The food consumption of males and females receiving 100, 300 or 1000 mg/kg/day was slightly increased during Weeks 1 and 2 of treatment when compared with that of the Control.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No visual effect observed, and, consequently, qualitative measurements were not performed.
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
There was no conclusive effect of treatment on haematology parameters.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There was no conclusive effect of treatment on blood chemistry parameters.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no effects of treatment on absolute or bodyweight relative organ weight.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no macropathology findings that were considered to be related to treatment with Tetrabutane
Neuropathological findings:
no effects observed
Description (incidence and severity):
There was no effect of treatment on the sensory reactivity, grip strength or motor activity.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no findings that were considered to be related to treatment with Tetrabutane.
Histopathological findings: neoplastic:
not examined

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effect for general toxicity seen at dosages up to 1000 mg/kg/day.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

TABLE 1

Bodyweight change - group mean values (g) for males

 

Group

:

 1

 2

 3

 4

Compound

:

Control

Tetrabutane

Tetrabutane

Tetrabutane

Dose (mg/kg/day)

:

0

100

300

1000

 

Group

 

Weeks

Weeks

Weeks

Weeks

Weeks

Weeks

/Sex

 

0-1

1-2

2-3

3-4

4-5

0-5

Statistical test:

 

 Wi

 Wi

 Wi

 Wi

 Wi

 Wi

1M

Mean

25

23

23

14

9

94

 

SD

6.5

8.3

6.6

5.3

10.4

16.6

 

N

10

10

10

10

10

10

 

 

 

 

 

 

 

 

2M

Mean

23

23

23

22*

7

98

 

SD

4.9

8.8

4.2

9.4

6.7

19.0

 

N

10

10

10

10

10

10

 

 

 

 

 

 

 

 

3M

Mean

28

31*

20

28**

9

116*

 

SD

8.0

6.1

8.3

7.1

9.4

23.5

 

N

10

10

10

10

10

10

 

 

 

 

 

 

 

 

4M

Mean

28

32*

24

24**

7

114*

 

SD

10.8

8.0

6.5

8.3

9.6

25.8

 

N

10

10

10

10

10

10

 

 

 

 

 

 

 

 

  Wi    Treated groups compared to Control using Williams’ test.

 

TABLE 1 - continued

 

Bodyweight change - group mean values (g) for females

 

 

Group

Weeks 

Weeks

Weeks

Weeks

Weeks

Weeks

/Sex

0 -1 

1 -2

2 -3

3 -4

4 -5

0 -5

Statistical test:

 Wi

Wi

 Wi

Du

 Wi

Wi

1F

Mean

10

12

20

6

-4

35

 

SD

7.8

6.4

6.7

2.9

4.2

9.1

 

N

15

15

5

5

5

5

 

 

 

 

 

 

 

 

2F

Mean

13

19

15

15*

-1

53

 

SD

8.8

7.8

6.0

8.2

1.8

3.3

 

N

15

15

5

5

5

5

 

 

 

 

 

 

 

 

3F

Mean

14

16

11

7

-4

43

 

SD

7.7

9.2

5.4

3.4

5.1

9.7

 

N

15

15

5

5

5

5

 

 

 

 

 

 

 

 

4F

Mean

13

18*

18

4

3*

59**

 

SD

6.3

7.6

3.0

2.9

5.5

19.4

 

N

15

15

5

5

5

5

 

 

 

 

 

 

 

 

  Du       Treated groups compared to Control using Dunnett’s test 

Wi     Treated groups compared to Control using Williams’ test.

 

TABLE 2

 

Food consumption - group mean values (g/animal/week) for males

 

Group

:

 1

 2

 3

 4

Compound

:

Control

Tetrabutane

Tetrabutane

Tetrabutane

Dose (mg/kg/day)

:

0

100

300

1000

 

Group

 

Week

Week

/Sex

 

1

2

Statistical test:

 

 Wi

 Wi

1M

Mean

159

146

 

SD

13.7

13.9

 

N

2

2

 

 

 

 

2M

Mean

163

153

 

SD

6.0

2.8

 

N

2

2

 

 

 

 

3M

Mean

177

164

 

SD

11.0

7.4

 

N

2

2

 

 

 

 

4M

Mean

173

170

 

SD

3.7

14.6

 

N

2

2

 

 

 

 

 

Wi    Treated groups compared to Control using Williams’ test.

 

 

 

 

TABLE 2 - continued

 

Food consumption - group mean values (g/animal/week) for females

 

 

Group

 

Week

Week

/Sex

 

1

2

Statistical test:

 

 Wi

 Wi

1F

Mean

114

113

 

SD

7.6

10.4

 

N

3

3

 

 

 

 

2F

Mean

117

122

 

SD

5.3

6.0

 

N

3

3

 

 

 

 

3F

Mean

120

119

 

SD

6.2

2.7

 

N

3

3

 

 

 

 

4F

Mean

122

125

 

SD

2.6

0.6

 

N

3

3

 

 

 

 

 

Wi    Treated groups compared to Control using Williams’ test.

 

Applicant's summary and conclusion

Conclusions:
Based on the results of this study, it was concluded that the No-Observed-Adverse-Effect-Level (NOAEL) for general toxicity in male and female rats was 1000 mg/kg/day Tetrabutane.
Executive summary:

A study was performed at the Laboratories of Huntingdon Life Sciences, Eye, on behalf of Evonik Oxeno GmbH., to investigate the sub-acute toxicity of the test substance Tetrabutane when administered to rats by oral gavage. Three groups, each comprising five male and five female rats, received Tetrabutane once daily at dosages of 100, 300 or 1000 mg/kg/day for a period of five complete weeks before termination. A similarly constituted Control group received the vehicle, corn oil, at the same volume-dose for the same duration.

Throughout the study, data was recorded on clinical condition, detailed physical and arena observations and bodyweight. Food consumption was recorded during Weeks 1 and 2 and sensory reactivity, grip strength, motor activity, haematology and blood chemistry during Week 4. Organ weight, macroscopic and microscopic pathology investigations were undertaken at termination. The study was conducted to GLP.  

There were no effects for general toxicity at dosages up to 1000 mg/kg/day. Slightly higher overall bodyweight and food consumption was recorded amongst males and females receiving 100, 300 or 1000 mg/kg/day, when compared with the controls, although the biological significance of these findings is uncertain.

Based on the results of this study, it was concluded that the No-Observed-Adverse-Effect-Level (NOAEL) for general toxicity in adult male and female rats was 1000 mg/kg/day Tetrabutane.

The study is considered acceptable for classification and satisfies the guideline requirements for a rat repeated dose oral toxicity screening test.