Registration Dossier

Administrative data

Endpoint:
specific investigations: mechanistic studies
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Non-GLP, non-guideline, expert interpretation of finding from guideline study
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004

Materials and methods

Principles of method if other than guideline:
The kidney sections from the original reproductive /developmental toxicity screening study were stained histochemically, to determine if 2,4,4-trimethyl pentene-induced kidney effects were caused by alpha-2u-globulin accumulation in male rats.
GLP compliance:
no
Type of method:
other: histochemical staining of kidney sections

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 (Sprague-Dawley origin)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS - as original study (HLS (1997): 2,4,4-Trimethyl Pentene: Reproductive Developmental Toxicity Screening Test by Oral Gavage Administration to CD Rats. Huntingdon Life Sciences Ltd., UK; study report 96/SOC009/1097)

- 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 exposure:
As original study (HLS (1997): 2,4,4-Trimethyl Pentene: Reproductive Developmental Toxicity Screening Test by Oral Gavage Administration to CD Rats. Huntingdon Life Sciences Ltd., UK; study report 96/SOC009/1097)

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
Basis:
analytical conc.
mg/kg bw/day
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
other: positive control kidney sections from male Fisher 344 rats gavage dosed with d-limonene (150 mg/kg bw/day, 4 days) were included with each set of study slides to be processed.
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/day.

Examinations

Examinations:
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.

POST-MORTEM EXAMINATIONS: Yes
- Male animals: All surviving animals killed after successful littering of the parental females (after approximately 6 weeks of treatment)
- Maternal animals: All surviving animals killed on Day 4 of lactation.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
- Organs examined histopathologically: all abnormalities, epididymides, kidneys, liver, ovaries, testes.
- Organs stored: prostate, seminal vesicles, uterus, cervix and vagina.
- Organs weighed: epididymides, kidneys, liver, ovaries, prostate, seminal vesicles, testes, uterus and cervix.

Results and discussion

Details on results:
Renal lesions associated with 2,4,4-trimethyl pentene-induced alpha-2u-globulin nephropathy are not considered relevant for human risk assessment.

Any other information on results incl. tables

The original oral developmental / reproductive toxicity study on 2,4,4-trimethyl pentene demonstrated an increased incidence of hyaline droplets in the proximal tubules of the kidney in male, but not female rats receiving the test compound. This study utilized the kidney sections from the original study to determine if the hyaline droplets observed in the original study were composed of alpha-2u-globulin using mouse anti-alpha-2u-globulin monoclonal antibodies. All slides were evaluated without knowledge of treatment and graded for the extent of immunostaining. All positive and negative controls gave the expected response.

None of the kidney sections from female control or high dose rats exhibited any positive staining for alpha-2u-globulin. In contrast, all slides from male animals exhibited some positive staining for alpha-2u-globulin. When the results were decoded, the average score for control males was 1.2, with all but two animals having a score of 1.0, and two animals having a score of 2.0. All of the high dose males exhibited moderate to severe staining for alpha-2u-globulin. All but two of the male high dose rats exhibited positive staining granular casts, with numerous specific positive staining droplets. When the results were decoded, the average score for the low dose males was 2.2, with a range from 2.0 to 3.0. The average score for the middle dose males was 3.6, with a range from 2.0 to 4.0, while the high dose males had an average score of 3.8, with a range from 3.0 to 4.0.

Immunohistochemical staining of alpha-2u-globulin in male and female kidney sections

Group

negative

Grade 1

Grade 2

Grade 3

Grade 4

Average grade

Gp 1 females (vehicle control)

10/10

0/10

0/10

0/10

0/10

0

Gp 4 females (1000 mg/kg/day)

10/10

0/10

0/10

0/10

0/10

0

Gp 1 males (vehicle control)

0/10

8/10

2/10

0/10

0/10

1.2

Gp 2 males (100 mg/kg/day)

0/10

0/10

8/10

2/10

0/10

2.2

Gp 3 males (300 mg/kg/day)

0/10

0/10

1/10

2/10

7/10

3.6

Gp 4 males (1000 mg/kg/day)

0/10

0/10

0/10

2/10

8/10

3.8

All positive and negative controls gave the expected response.

 

Applicant's summary and conclusion

Conclusions:
Based on the results of this study and the prior research on alpha-2u-globulin nephropathy, the renal lesions associated with 2,4,4-trimethyl pentene -induced alpha-2u-globulin nephropathy in rats are considered not to be relevant for human risk assessment.
Executive summary:

Oral administration of 2,4,4-trimethyl pentene to rats at a daily dose of 100, 300 or 1000 mg/kg/day induced alpha-2u-globulin nephropathy. The severity of the disease induced was moderate to severe which was identical to other chemicals such as d-limonene or the close structural analog, 2,2,4-trimethyl pentane, which have also been shown to induce moderate to severe alpha-2u-globulin nephropathy.