Hydrocarbons, C4, 1,3-butadiene-free, polymd., tetraisobutylene fraction

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

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other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reference substance 003

Cas Number:

25167-70-8

Molecular formula:

C8H16

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 (Sprague-Dawley origin)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent, England
- Age at study initiation: 11 - 12 weeks
- Weight at study initiation: 352-401 g (males); 229-262 g (females)
- Housing: Stainless steel or high density polypropylene cages. Five animals of the same sex / cage pre-mating; 1 male:1 female for pairing; females housed individually during gestation and lactation.
- Diet: pelleted rodent diet (LAD 1 SQC, from Special Diets Services Limited, Witham, Essex, England ad libitum
- Water: Tap water ad libitum
- Acclimation period: 12 days
- Bedding: Lignocel 3/4 wood flakes (RS Biotech, Finedon, Northamptonshire, UK) during the littering phase

ENVIRONMENTAL CONDITIONS
- Temperature: 19.5-21.5°C
- Humidity: 49-74%
- Air changes: Approximately 15/hr
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 24 June 1996 To: 9 August 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 (Group 4) was prepared by mixing the test material with maize oil. The formulations for the other treated groups were prepared by serial dilution of the Group 4 formulation.
All efforts were taken to minimise vaporisation of the test material during the formulation procedure.

VEHICLE
- Concentration in vehicle: Prepared at the appropriate concentration in maize oil to permit administration at a constant volume-dosage of 5 mL/kg.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity, stability and achieved concentrations of the formulations were measured throughout the study.

Duration of treatment / exposure:

Dosing was for 15 days before pairing. Treatment was continued throughout mating, gestation and lactation to Day 3 of lactation for females and to termination after approximately six weeks of treatment for males.

Frequency of treatment:

Daily

Doses / concentrations

Remarks:

Doses / Concentrations:
100, 300 or 1000 mg/kg bw/day
Basis:
other: analytical conc.

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on the results of a preliminary seven-day toxicity study performed at these laboratories in which no evidence of toxicity was apparent at dosages up to and including 1000 mg/kg bw/day.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily during weeks 1-2, weekly during weeks 3-6

BODY WEIGHT: Yes
- Time schedule for examinations: Males were weighed on the day that treatment commenced, at weekly intervals thereafter, and before termination. Females were weighed on the day that treatment commenced, at weekly intervals until mating was detected, on Days 0, 7, 14 and 20 of gestation and on Days I and 4 of lactation.

FOOD CONSUMPTION : Yes
- Food consumption (by cage) determined until pairing and mean weekly diet consumption calculated as g food/kg body weight/day: Yes
- Food consumption for females was also recorded for the periods Days 0-3, 4-6, 7-10, 11-13, 14-16 and 17-19 of gestation and for the period Days 1-3 of lactation.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- Including a detailed examination of the cranial, thoracic, abdominal and pelvic cavities and their viscera
- The external and cut surfaces of the organs and tissues were examined before or after weighing, as appropriate
- Abnormalities, interactions and changes were noted and representative tissue samples preserved in fixative

HISTOPATHOLOGY: Yes
- The following tissue samples were examined histopathologically: abnormalities, epididymides, kidneys, liver, ovaries and testes
- The following tissue samples were preserved but not examined histopathologically: prostate gland, seminal vesicles, uterus and cervix and vagina

ORGAN WEIGHTS:
- The following organs were weighed: epididymides, kidneys, liver, ovaries, prostate, seminal vesicles, testes, uterus with cervix

Statistics:

Absolute bodyweights at the start of each phase, bodyweight gains and food consumption values were assessed by one-way analysis of variance. Whenever this was found to be significant, a Student's 't'-test was used.
For organ weights, homogeneity of variance was automatically tested using Bartlett's test. Whenever this was found to be statistically significant a Behrens-Fisher test was used to perform pairwise comparisons, otherwise a Dunnett's test was used.
Inter-group differences in macroscopic pathology and histopathology were assessed, on indication, using 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:

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

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

Details on results:

CLINICAL SIGNS AND MORTALITY:
One female receiving 2,4,4-Trimethyl pentene at 1000 mg/kg bw/day was killed in extremis on Day 2 of lactation due to signs including under-active behaviour, hunched posture, piloerection, slow respiration, brown-coloured urine and brown perigenital staining. In the absence of any similar signs or findings in other females on this study, it was considered that the death of this animal was incidental.
At 1000 mg/kg bw/day, animals showed transient salivation after dose administration. Single occurrences of transient salivation after dosing were observed for three males receiving 300 mg/kg bw/day and for one male receiving 100 mg/kg bw/day. At 1000 mg/kg bw/day, a number of animals showed brown staining on the dorsal and ventral body surfaces. In males this sign was first evident in Week 2 whilst in females it was generally first observed in Week 4.

ORGAN WEIGHTS:
The absolute and bodyweight-relative weights of the liver and kidneys were high, when compared with the Controls, for males that received 300 or 1000 mg/kg bw/day and for females that received 1000 mg/kg bw/day.

GROSS PATHOLOGY:
After approximately six weeks of treatment, all males and four females that received 1000 mg/kg/day had swollen livers or liver lobes. Two males that received 1000 mg/kg bw/day also had large kidneys.
No remarkable findings were apparent at dosages up to 300 mg/kg bw/day.

HISTOPATHOLOGY:
Treatment-related findings were confined to the kidneys of males. Basophilic cortical tubules were seen in all treated groups whilst proteinaceous casts and interstitial inflammatory cells were seen only at 300 or 1000 mg/kg bw/day. The severity of the basophilic cortical tubular changes was greater in males given 300 or 1000 mg/kg bw/day than in those given 100 mg/kg bw/day. Centriacinar fatty vacuolation was recorded in the livers of two females given 1000 mg/kg bw/day (one of these animals was an early decedent). This distribution of fat in the liver is more commonly associated with hepatotoxicity than periacinar vacuolation, but is considered equivocal in this instance.

Effect levels

Dose descriptor:

LOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: kidney enlargement (histopath: basophilic cortical tubular changes). Liver enlargement (histopath equivocal)

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

The absolute and bodyweight-relative weights of the liver and kidneys were high, when compared with the controls, for males that received 300 or 1000 mg/kg bw/day and for females that received 1000 mg/kg bw/day.

In the absence of any associated histopathological change, the increase in liver weight was considered to represent increased metabolism, as an adaptive response to the administration of a xenobiotic. Due to the effects on the kidneys at dosages of 100 mg/kg bw/day and higher, a no-observed-effect level (NOEL) was not demonstrated as part of this study.

Intergroup comparison of mean liver and kidney weight (g) relative to bodyweight

Dose level of 2,4,4-Trimethylpentene
Males					Females
	0	100	300	1000	0	100	300	1000 #
Liver	4.10	4.17	4.72*	6.65**	5.44	5.34	5.53	6.83**
Kidney	0.758	0.794	0.918**	0.981**	0.723	0.794*	0.745	0.845

# 9 animals (10 in other groups)

*p<0.05 **p<0.01

Applicant's summary and conclusion

Conclusions:

Due to the effects on the kidneys at dosages of 100 mg/kg bw/day and higher, a no-observed-effect level (NOEL) was not demonstrated.

Executive summary:

Groups of 10 males and 10 female CD rats were dosed orally with solutions of 2,4,4 -trimethylpentene in maize oil at dose levels of 0, 100, 300 or 1000 mg/kg/day in a combined reproductive toxicity / developmental toxicity screening test. . Oral administration of 2,4,4-Trimethylpentene at dosages of up to 1000 mg/kg bw/day was without adverse effect on the general condition of male and female rats. Treatment-related findings were confined to the kidneys of males; basophilic cortical tubules were seen in all treated groups whilst proteinaceous casts and interstitial inflammatory cells were seen only at 300 or 1000 mg/kg bw/day. The severity of the basophilic cortical tubular changes was greater in males given 300 or 1000 mg/kg bw/day than in those given 100 mg/kg bw/day. Due to the effects on the kidneys at dosages of 100 mg/kg bw/day and higher, a no-observed-effect level (NOEL) was not demonstrated as part of this study. Subsequent immunohistochemical investigations of alpha-2u-Globulin in kidneys has been reported.