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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report Date:
1997

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 2,4,4-trimethylpentene (also known as diisobutylene and diisobutene)
- Physical state: clear, colourless liquid
- Analytical purity: 95.19%
- Lot/batch No.: Batch No. 2 (a 50:50 mixture of two original batches of 2,4,4-trimethylpentene - the details of which are as follows: Batch No. R11 supplied by Shell and Batch No. 155833 supplied by Erdolchemie).
- Expiration date of the lot/batch: 29 April 1997
- Storage condition of test material: Under nitrogen, protected from light, in a cool store.

Test animals

Species:
rat
Strain:
other: CD
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent, England
- Age at study initiation: 35-42 days
- Weight at study initiation (9 days after arrival): 116-119 g
- Fasting period before study: no
- Housing: Stainless steel Type TR18 cages (54 x 38 x 20 cm). Five animals of the same sex / cage.
- Diet: Complete pelleted rodent diet (RM1(E) SQC, from Special Diets Services Limited, Witham, Essex, England ad libitum
- Water: Tap water ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-25°C
- Humidity: 40-70%
- Air changes: At least 15/hr
- Photoperiod (12 hrs dark / 12 hrs light):

IN-LIFE DATES: From: 26 June 1996 To: 24 July 1996

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
maize oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The formulation for the high dosage group was prepared by mixing the test material with maize oil. The formulations for the other treated groups were prepared by serial dilution of the high dose formulation.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Acceptable homogeneity of the formulation was demonstrated at 200 mg/mL and 2,4,4-trimethyl pentene was shown to be stable at this level in maize oil for at least six hours during the first trial mix. The low concentration (1 mg/mL) mix was not homogeneous and at a significantly lower concentration than intended (59.3%). A second trial mix was performed using a higher level of 6 mg/mL. Acceptable homogeneity was obtained and the formulation was shown to be stable for at least six hours, but the concentration was still significantly lower than intended (62.5%). A third trial mix was performed to assess the concentration at 20 mg/mL (the lowest dosage group on this study) and this was satisfactory. Analysis of samples of each formulation obtained in Weeks 1 and 4 of treatment indicated that, with the exception of the high dosage group formulation in Week 4, the achieved concentration were slightly lower than intended. The values achieved were between 80 and 87% of intended.
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg/day
Basis:
other: nominal in maize oil
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale: Based on the results of a preliminary study (Schedule No. SOC/008) where 3 groups of 3 rats/sex/group dosed by gavage at 100, 300 or 1000 mg/kg/day by gavage for 7 days.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice/day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Before and after dosing daily during week 1 and twice/week during weeks 2-4. (The detailed observations were made on the specified days at the following times: 1: Pre-dose observation, 2: As each animal was returned to its home cage, 3: At the end of each dosing group, 4: Between 1 and 2 hours after completion of dosing of all groups, 5: As late as possible in the working day.

BODY WEIGHT: Yes
- Time schedule for examinations: during acclimatisation, day 1, weekly throughout study and day of termination.

FOOD CONSUMPTION: weekly

FOOD EFFICIENCY: weekly

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: during week 4
- Anaesthetic used for blood collection: Yes (halothane/nitrous oxide)
- Animals fasted: Yes
- How many animals: All
- Parameters examined (peripheral blood): Packed cell volume (PCV), Haemoglobin concentration (BB), Erythrocyte count (RBC), Mean cell haemoglobin concentration (MCHC), Mean cell haemoglobin (MCH), Mean cell volume (MCV), Total and differential leukocyte count (WBC), Platelet count (PLAT), Prothrombin time (PT), Blood film prepared and examined for abnormal morphology and unusual cell types. Bone marrow smears prepared from all animals. Smears from controls and high dose examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: during week 4
- Anaesthetic used for blood collection: Yes (halothane/nitrous oxide)
- Animals fasted: Yes
- How many animals: All
- Parameters examined: Alkaline phosphatase activity (ALP), Alanine amino-transferase activity (ALT), Aspartate amino-transferase activity (AST), Gamma-glutamyl transpeptidase activity (GGT), Urea concentration, Glucose concentration (GLUC), Total cholesterol concentration (CHOL), Creatinine concentration (CREA), Total protein concentration (TP), Albumin concentration (CHEM ALB), Albumin/globulin ratio (A/G), Sodium (Na), Potassium (K)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Before treatment and once/week
- Dose groups that were examined: All
- Battery of functions tested: Open field observations: comprising locomotor activity / alertness / behaviour / convulsion / defecation / exophthalmos / fur appearance / gait / grooming frequency / lachrymation / palebral closure / piloerection / posture / pupil size / removal from cage / respiratory rate / salivation / tremor / urination.
Sensory reactivity test: comprising auditory pinna reflex / auditory startle reflex / body temperature/ flexor (withdrawal) reflex / landing footsplay / pain (tail pinch) response / pupil closure response / reaction to handling / righting reflex.

GRIP STRENGTH AND MOTOR ACTIVITY: Yes
- Time schedule for examinations: During week 4
- Dose groups that were examined: All
- Functions tested: fore limb grip strength / hind limb grip strength / motor activity
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (all animals)

ORGAN WEIGHTS: Yes (all animals)
- Tissues: Adrenals / Liver / Brain / Spleen / Epididymides / Testes / Heart / Thymus / Kidneys

HISTOPATHOLOGY: Yes (macroscopically abnormal tissues, kidneys, livers and lungs were examined from all animals. All other tissues listed below were examined from controls and high dose animals only).
- Tissues: Adrenals / Brain / Caecum / Colon / Duodenum / Epididymides / Heart / Ileum / Jejunum / Kidneys / Liver / Lungs with mainstem bronchi / Lymph nodes - mandibular and mesenteric / Ovaries / Prostate / Sciatic nerve - one only / Spinal cord / Spleen / Stomach / Testes / Thymus / Thyroid with parathyroids / Trachea / Urinary bladder / Uterus with cervix
Statistics:
Standard deviations were calculated, as considered appropriate, using the sample statistic. The following statistical tests were used: Bartlett's test, Shapiro-Wilk test, Kruskal-Wallis test, Wilcoxon Rank Sum test, one-way analysis of variance (ANOVA), Dunnett's test, Student's t-test using a pooled error variance, Behrens-Fisher test and Fisher's Exact test.


Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
effects observed, treatment-related
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
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:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not specified
Details on results:
CLINICAL SIGNS AND MORTALITY: No mortalities. Brown staining of the coat in males and females at 1000 mg/kg/day. Females at 1000 mg/kg/day had an ungroomed appearance. Salivation after administration occurred on isolated occasions in animals receiving 1000 mg/kg/day. No other treatment-related signs.

BODY WEIGHT AND WEIGHT GAIN: Slightly increased weight gain in females at 1000 mg/kg/day. Slightly increased weight gain in females at 300 mg/kg/day, but considered not to be toxicologically significant. Bodyweight gains of males and females receiving 100 mg/kg/day and males receiving 300 mg/kg/day were unaffected.

FOOD CONSUMPTION / FOOD EFFICIENCY: Slightly increased food intake and food conversion efficiency for females receiving 1000 mg/kg/day. A slight increase in food intake was also recorded for males receiving 1000 mg/kg/day and a slightly higher food conversion efficiency recorded for females receiving 300 mg/kg/day. These trends were, however, were considered not to be toxicologically significant. Food consumption of males and females receiving 100 or 300 mg/kg/day was essentially similar to that of the controls.

HAEMATOLOGY: There were no toxicologically significant haematological findings in Week 4 and the composition of the bone marrow was unaffected by treatment.

CLINICAL CHEMISTRY: Biochemical changes in the plasma in Week 4 comprised: high urea, total protein and albumin concentrations in males receiving 1000 mg/kg/day; low glucose concentrations in females receiving 1000 mg/kg/day; low urea concentrations in females receiving 300 or 1000 mg/kg/day.

NEUROBEHAVIOUR: Open field observations indicated yellow/brown staining of the coat from Week 2 in males and females receiving 1000 mg/kg/day. No treatment-related changes were identified in the sensory reactivity tests or grip strength and motor activity measurements performed in Week 4.

ORGAN WEIGHTS: Absolute and bodyweight-relative kidney weights were significantly higher in males given 1000 mg/kg/day and absolute and bodyweight-relative liver weights were significantly higher in males and females given 1000 mg/kg/day compared to controls.

GROSS PATHOLOGY: No treatment-related effects.

HISTOPATHOLOGY: No treatment-related effects

Effect levels

Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: increased liver and kidney weights, no histopathological changes, at 1000 mg/kg/day

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

The administration of 2,4,4-trimethylpentene to CD rats at a dosage of 1000 mg/kg/day was generally well tolerated. The liver and kidneys were identified as target organs. The liver weights of animals given 1000 mg/kg/day were raised compared to controls but there was no associated histopathological change. The increase in liver weight is considered to represent an adaptive response to the administration of a xenobiotic. Variations in plasma protein, glucose and, possibly, urea concentrations may be due to an alteration in liver metabolism following treatment.

Males receiving 1000 mg/kg/day had increased kidney weights compared to controls but there was no associated histopathological change. The increased urea concentration may be related to a minor alteration in renal function.

No significant findings were observed among animals receiving 100 or 300 mg/kg/day and the NOAEL is considered to be 300 mg/kg/day in this study. In addition, as there were no adverse signs or responses in the battery of neurological tests on this study, it is considered that 2,4,4-trimethylpentene did not cause any change to normal nervous system function when administered by gavage at dosages up to 1000 mg/kg/day.

Applicant's summary and conclusion

Conclusions:
The NOAEL is considered to be 300 mg/kg/day. As there were no adverse signs or responses in the battery of neurological tests on this study, it is considered that 2,4,4-trimethylpentene did not cause any change to normal nervous system function when administered by gavage at dosages up to 1000 mg/kg/day.
Executive summary:

Groups of five male and five female CD rats received 2,4,4-trimethylpentene orally, by gavage, at dosages of 100, 300 or 1000 mg/kg/day for four weeks. A similarly constituted control group received the vehicle (maize oil) at the same volume-dosage. The administration of 2,4,4-trimethylpentene to CD rats at a dosage of 1000 mg/kg/day was generally well tolerated. The liver and kidneys were identified as target organs; organ weights were increased but there were no histopathological changes. The NOAEL is considered to be 300 mg/kg/day in this study. In addition, as there were no adverse signs or responses in the battery of neurological tests on this study, it is considered that 2,4,4-trimethylpentene did not cause any change to normal nervous system function when administered by gavage at dosages up to 1000 mg/kg/day.