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Administrative data

Description of key information

In the general toxicity element of an OECD 422 screening test in rats, Tetrabutane elicited no systemic toxic effect at dosages of 100, 300 or 1000 mg/kg/day. The NOAEL for repeat dose oral toxicity is 1000 mg/kg/day.
90-day repeated dose toxicity studies are available for structurally related source substances.

Repeated dose studies have not been undertaken by the dermal or inhalation routes. The physical properties of Tetrabutane and its low toxicity by oral administration, indicates that the substance represents a low toxic risk by both the dermal and inhalation routes.

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
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
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
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
Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals or test system 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)
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.
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
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
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
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.
Critical effects observed:
not specified

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.

 

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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Subacute studies

Studies conducted with the target substance

A Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test according to OECD TG 422 was performed to investigate the subacute 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. 

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.

 

Supporting studies conducted with the source substances

A Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test according to OECD TG 422 was also performed with the source substance Oxooil LS9 at dose levels of 0 (control), 100, 300 or 1000 mg/kg/day for a period of five complete weeks before termination (5 animals/sex/dose). An additional five male and ten female rats per treatment group were used to investigate the reproductive toxicity potential of Oxooil LS9. Pertinent observations and findings in these animals are included in the assessment of the general systemic toxicity of Oxooil LS9.

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. Three of ten males receiving 1000 mg/kg/day were killed for reasons of animal welfare during weeks 3 or 4 of treatment. Due to the sudden deterioration of individuals within this treatment group, treatment of all males receiving 1000 mg/kg/day was terminated in week 4 and all males (including reproductive group animals) were subsequently killed. These males were observed to be of thin build and had brown staining on their body surface.

Bodyweight gain and food consumption was low during the first week of treatment, and bodyweight loss was apparent during weeks 2 and 3 when compared with Controls.

Macroscopic examination revealed a high number of males with abnormal pink colouration of the peripheral nerve(s), sciatic nerve, seminal vesicles, pale jejunum and pale and thickened duodenum, they were also of thin build and their fur was stained. A few males had abnormal colouration of the skin, mesenteric lymph node, adipose tissue, prostate and urinary bladder which was also distended for two males. Dark and enlarged mandibular lymph nodes, small thymus, non glandular thickened stomach, thickened and abnormal contents of the jejunum was also observed at a lower incidence. Minimal hepatocellular centrilobular hypertrophy was observed in the liver of 4/6 males and cortical tubular basophilia, cortical tubular dilatation and hyaline droplets were respectively observed in 4/6, 3/6 and 6/6 males given 1000 mg/kg/day.

There were no clinical signs for females receiving 1000 mg/kg/day or animals receiving 300 or 100 mg/kg/day and sensory reactivity observation, grip strength values and motor activity scores were unaffected by treatment with Oxooil LS9.

Overall mean bodyweight gain was slightly low for males (84%) and females (69%) treated at 300 mg/kg/day and females (56%) treated at 1000 mg/kg/day, when compared with Controls.

Water intake assessed during Week 4 of treatment was increased for males receiving 100 mg/kg/day or above, and females receiving 1000 mg/kg/day when compared with Controls.

Haematological investigations performed during Week 5 of treatment revealed, when compared with Controls, prolonged activated partial prothrombin time (APTT) in females receiving 1000 mg/kg/day. Biochemical examination of the blood plasma indicated when compared with Controls that alkaline phosphatase, alanine aminotransferase and aspartate aminotransferase activities were lower for females receiving 1000 mg/kg/day. Alanine aminotransferase activity was low in females receiving 300 mg/kg/day and aspartate aminotransferase activity was low in males receiving 300 mg/kg/day. Total plasma cholesterol levels were higher in animals receiving 1000 mg/kg/day. In males receiving 300 mg/kg/day and animals receiving 1000 mg/kg/day, there was an increase of plasma albumin concentration and albumin to globulin ratio was increased in males receiving 1000 mg/kg/day.

The analysis of organ weights after 5 weeks of treatment indicated, when compared with Controls, high mean absolute and adjusted liver and kidney weights among males that received 300 mg/kg/day and females that received 1000 mg/kg/day. Seminal vesicles absolute and adjusted weights were slightly low in males that received 300 mg/kg/day.

Females that received 1000 mg/kg/day and males that received 300 mg/kg/day were observed to have abnormal pink colouration of the sciatic nerve(s) and peripheral nerve(s), when compared with Controls. Males that received 300 mg/kg/day were observed to have abnormal pink colouration of the seminal vesicles. The abnormal colouration of these tissues did not correlate with any microscopic changes, and the toxicological importance of this finding is unknown.

Microscopic examination revealed the presence of hepatocellular centrilobular hypertrophy at a dose-related incidence in males that received 100 or 300 mg/kg/day and females that received 1000 mg/kg/day. Renal findings consisted of a dose related increase in the incidence and severity of hyaline droplets in the proximal convoluted tubules in the males given 100 or 300 mg/kg/day and cortical tubular basophilia and tubular dilatation were seen in the males given 100 or 300 mg/kg/day. Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No cell or stage specific abnormalities were noted.

Based on the results of this study, it was concluded that the No-Observed-Adverse-Effect-Level (NOAEL) for systemic toxicity in adult male and female rats treated with Oxooil LS9 for up to 5 weeks is 100 mg/kg/day.

 

For the source substance Oxooil LS13, a Reproduction / Developmental Toxicity Screening Test in accordance with OECD Guideline 421 with limited assessment of general toxicity is also available:

All doses (0, 150, 500 and 1000 mg/kg/day) were administered orally, by gavage. The control group received corn oil (vehicle).

Males were treated for 2 weeks prior to pairing and during pairing with females until the day before necropsy, for a total of 31/32 days.

Females were treated for 2 weeks prior to pairing, and thereafter during pairing, post coitum and post partum periods until Day 13 post partum (for 41-44 days).

The following investigations were performed: mortality check, clinical signs, body weight, food consumption, oestrous cycle, mating performance, litter data, sex ratios, macroscopic observations and organ weights.

Histopathological examination was performed on control and high dose groups, on all abnormalities detected during post mortem observation at the end of treatment period and on animals of the intermediate groups on target organs selected on the basis of the first protocol based examination.

Salivation was the most relevant clinical sign recorded for males and females, observed in all treated groups with a dose-related incidence. This sign was seen starting from Day 10 of treatment up to the end of the study.

Body weight growth of treated males, at all dose levels, was similar to controls. During the pre-mating, mating, gestation and post partum periods, body weight of females was similar in treated and control animals, with the exception of reductions observed in females treated at 1000 mg/kg/day on Days 20 post coitum and 7 post partum.

Food consumption of treated males, at all dose levels, was similar to controls. No relevant differences were seen in food consumption of females during pre-mating, mating, gestation and post partum periods.

No changes were observed on terminal body of study animals of both sexes, when compared to the control group.

A statistically significant increase was observed in mean liver weight of animals of both sexes receiving 500 and 1000 mg/kg/day, in mean kidneys weight of males receiving 500 and 1000 mg/kg/day and in mean adrenals weight of females receiving 500 and 1000 mg/kg/day, when compared to controls.

The only relevant change observed following gross pathology examination was enlarged kidneys of two males treated at 500 mg/kg/day, when compared to the controls.

Treatment-related changes, associated with the oral administration of the test item, were present in the non-glandular region of the stomach of animals of both sexes receiving 500 and 1000 mg/kg/day, in liver (minimal up to moderate centrilobular hepatocellular hypertrophy in all males and two females treated at 1000 mg/kg/day, and from minimal to mild degree in males treated at 500 mg/kg/day) and kidneys (increased severity of hyaline droplets accumulation in kidneys from mild to marked degree in males receiving 1000 mg/kg/day and from minimal to mild degree in males receiving 500 mg/kg/day). As consequence of the above mentioned treatment-related findings, the cortical hypertrophy of the adrenals of females receiving 500 and 1000 mg/kg/day could be considered stress-related. No other treatment-related changes were noted in animals sacrificed at the end of the treatment period.

On the basis of the results obtained in this study, the NOAEL (No Observed Adverse Effect Level) for general toxicity was considered to be 150 mg/kg/day for males and females, as at this level no histopathological changes were observed.

 

Subchronic studies

No subchronic repeated dose toxicity study is available for the target substance tetrabutane. However, subchronic studies were     conducted with the source substances Oxooil LS9 and Oxooil LS13. A justification for read-across is attached to Iuclid section 13.

 

The oral toxicity of Oxooil LS9 in Sprague Dawley rats, following daily oral administration at dose levels of 50, 100 and 200 mg/kg/day for 13 consecutive weeks, and recovery from any treatment-related effect during a period of 4 weeks, were investigated in this study.

No clinical signs related to treatment were recorded at the daily and weekly observations performed throughout the study.

Body weight and food consumption were not affected by treatment.

Statistically significant decreases were observed at functional tests performed at the end of treatment: landing foot splay in mid- and high dose males and grip strength in high dose males. Landing foot splay was still statistically significantly decreased at the end of the recovery period, as well as a significant decrease was noted in motor activity measurements after 4 weeks of recovery.

No lesions were recorded at ophthalmological examination.

No changes of toxicological relevance were observed in haematology, clinical chemistry and urinalysis.

At the end of treatment period, liver weight was statistically significantly increased in males treated at 100 and 200 mg/kg/day and kidney weight was statistically significantly increased in males treated at 200 mg/kg/day.

No treatment-related findings were reported at post mortem macroscopic observations performed at final and recovery sacrifices.

At histopathology, changes considered treatment-related were present in the liver of males dosed with 200 mg/kg/day and kidneys of males treated at all dosages (50, 100 and 200

mg/kg/day).

In the liver, hepatocellular hypertrophy was seen in all high dose males. This change correlated with an increased liver mean weight observed in males and was considered an adaptive change and not adverse. In addition, the further evaluation of the intermediate and low dose treated animals revealed no treatment-related pathomorphological changes. After 4 weeks of recovery, the treatment-related changes in the liver were completely reversible.

In the kidneys, there was an increased severity, dose-related, of hyaline droplets accumulation in the proximal tubular epithelial cell in males from all treated groups in comparison to controls. This hyaline droplets accumulation was accompanied by moderate exacerbation of spontaneous nephropathy in the high and mid-dose animals with a dose response relationship. On the other hand, the incidence and the severity of this change in the low dose males was comparable to the controls. After a 4-week recovery period, although the hyaline droplets were not observed, there was still an increase for incidence and severity of nephropathy.

On the basis of the above mentioned results, it can be concluded that the high dose of 200 mg/kg/day may be considered as the No Observed Adverse Effect Level (NOAEL) for females and the low dose of 50 mg/kg/day the NOAEL for male.

In conclusion, Oxooil LS9 induced lesions observed in the proximal tubules of the male rat kidney were considered most likely associated with lysozymal accumulation of alpha 2µ-globulin and constitute a distinctive pathological entity. In fact, alpha 2µ-globulin response, following chemical administration, appears to be unique to the male rat. This observation is not relevant for humans.

Thus, the high dose of 200 mg/kg bw/d is considered the overall NOAEL.

 

In a subchronic toxicity study Oxooil LS13 was given to rats (10 animals/sex/dose) by daily oral administration via gavage at dose levels of 100, 300 or 1000 mg/kg b.w./day for 90 days. The study was conducted according to OECD TG 408.

No deaths were noted, all animals treated orally with 100,300, or 1000 mg Oxooil LS13/kg b.w./day survived until their scheduled sacrifice at study termination.

No test item-related changes were noted in behaviour, external appearance, body posture, or movement and coordination capabilities at any dose level.

The consistency of all animals' faeces was normal throughout the course of the study. No test item-related effects were noted during the observational screening and the functional tests.

No test item-related influence on Body weight and body weight gain, Food and drinking water consumption, Haematology and coagulation, Clinical chemistry, Urinalysis, Ophthalmological and

auditory examination, Macroscopic post mortem findings was observed.

Starting at the intermediate dose of 300 mg Oxooil LS13/kg b.w./day, statistically significantly increased organ weights were noted for the kidneys of the male animals, and for the liver of the male and female animals.

The increased kidney weights can be correlated with the histopathological findings of tubular basophilia, cystic tubular degeneration, lymphocytic infiltration, and hyaline droplets in the proximal tubuli in the kidneys of nearly all male animals of the high dose group and a few animals in the intermediate dose group. The increased liver weights for the male high dose animals correspond to a hepatocyte hypertrophy noted for a few animals upon histopathological examination.

Morphological lesions considered to be related to the test item Oxooil LS13 were noted in form of tubular basophilic, cystic tubular degeneration, lymphocytic infiltration, and hyaline droplets in the proximal tubuli in the kidneys of the male animals and in form of hyperplasia (epithelium), hyperkeratosis (epithelium) and granulocytic infiltration in the stomach of the male and female animals at the high dose.

The test item related changes in the kidneys of the male animals are classified as Alpha 2µ-globulin nephropathy.

Further, 4 of 10 male high dose animals revealed a test item-related hepatocyte hypertrophy, correlating with the slightly increased liver weights.

In conclusion, the experimental no-observed-adverse-effect level (NOAEL) was 300 mg Oxooil LS13/kg b.w. by daily oral administration, in particular as no test item-related changes were noted at histopathological examination at this dose level.

 

Conclusion

The same type of effects typical forlow molecular weight hydrocarbons – i.e. centrilobular hypertrophy, which can be considered an adaptive change and not adverse, andhydrocarbon nephropathy in male rats – was observed in studies conducted with the source substances Oxooil LS9 and Oxooil LS13 with, however, lower effect levels in the studies with Oxooil LS9. These effects were already observed in the subacute studies conducted with both source substances. Whereas no such effects were noted in the OECD TG 422 study conducted with Tetrabutane up to the limit dose of 1000 mg/kg bw/d.

These results demonstrate a clear trend towards higher effect levels with increasing molecular weight.This is in line with the hypothesis, that based on lower molecular weight, lower log Kow and higher water solubility, a higher bioavailability of the source substance Oxooil LS9 can be expected and the source substance Oxooil LS9 represents a worst case.

The subchronic studies conducted with the source substances Oxooil LS9 and Oxooil LS13 further show, that qualitatively no additional effects occur after prolonged exposure to the substances compared with the subacute studies. From a quantitative point of view, the NOAELs obtained in the subchronic studies were lower by a factor of 2 compared to the NOAELs from the subacute studies.

Therefore, the NOAEL of 1000 mg/kg bw/d obtained in the OECD TG 422 study conducted with the target substance Tetrabutane can be used as starting point for DNEL derivation when applying the default assessment factors for time extrapolation (factor 3 for extrapolation from subacute to subchronic).

Justification for classification or non-classification

No classification for repeated dose toxicity is indicated according to the classification, labelling and packaging (CLP) regulation (EC) No 1272/2008.

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