2,4,4-trimethylpentene

Endpoint summary

Description of key information

Adequate information is available to characterise the hazards of this substance following repeated exposure, with a NOEL of 300mg/kg/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

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other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: CD

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: 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

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

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.

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.

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

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

Critical effects observed:

not specified

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.

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.

 

Reference 2

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

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

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

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

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

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.

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.

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.

Dose descriptor:

LOAEL

Effect level:

100 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

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

Critical effects observed:

not specified

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

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.

Reference 3

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18th Nov 2020 - 07th Jan 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

No deviations that affect the integrity of the study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

Adopted 25th June 2018.

Deviations:

no

Principles of method if other than guideline:

Additional immunohistochemical analyses have been performed to confirm the presence of a2µ-globulin nephropathy in male animals. This is discussed further in "Overall Remarks".

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

N/A

Species:

rat

Strain:

other: Wistar Han

Details on species / strain selection:

The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test animals:

Crl: WI(Han), Outbred, SPF-Quality, Charles River Deutschland, Sulzfeld, Germany or Charles River Laboratories France, L'Arbresle Cedex, France (actual source not specified in report).
-Age at initiation of dosing: 6-7 weeks old
-Body weight range at initiation of dosing:170 – 205g males, 112 – 151g females
-Number of acclimation days: 14 days

Housing:

Polycarbonate cages (Makrolon type IV, height 18 cm or Makrolon type 2000P, height 21.5 cm) containing sterilized wooden fibers as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles. Up to 5 animals of the same sex and same dosing grouped together. Cages were arranged on the racks according to a Latin square model.

Diet and water:

Food was provided ad libitum. Pelleted SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany. Results of analysis for nutritional components and environmental contaminants are provided by the supplier and are on file at the Test Facility. It is considered that there are no known contaminants in the feed that would interfere with the objectives of the study. Municipal tap water was made freely available to the animals via water bottles. Periodic analysis of the water is performed, and results of these analyses are on file at the Test Facility. It is considered that there are no known contaminants in the water that could interfere with the outcome of the study.

Environmental conditions:

Daily mean temperature during the study period: 21-22°C
Daily mean relative humidity: 34-55%. The value that was outside the targeted range occurred for 1 day with a minimum of 34% and was without a noticeable effect on the clinical condition of the animals or on the outcome of the study

Route of administration:

oral: gavage

Details on route of administration:

The oral route of exposure was selected because this is a possible route of human exposure during manufacture, handling, or use of the test item.

Vehicle:

corn oil

Remarks:

Supplier: Sigma-Aldrich, Steinheim, Germany; batch number: MKCM3364

Details on oral exposure:

Dose formulations were prepared at least weekly. They were stored under conditions set to maintain a temperature of 2 °C to 8 °C. Dose formulations were divided into aliquots where required to allow to be dispensed on each dosing occasion. An adjustment was made for specific gravity of the test item and vehicle. No correction was made for the purity/composition of the test item.

Analytical verification of doses or concentrations:

yes

Remarks:

N/A

Details on analytical verification of doses or concentrations:

Chemical analyses of formulations were conducted on Weeks 1, 6 and 13 to assess accuracy and homogeneity. Analysis was based on the analytical method validated for the test item in vehicle in Test Facility Reference No. 20251978 using Acquity UPLC System.

Acceptance criteria were as follows:
1) mean sample concentration results within or equal to ± 10% of nominal value.
2) For homogeneity, relative standard deviation (RSD) of concentrations was =<10% for each group.

For the formulation of Group 3 (intermediate dose group) prepared for use in Week 6, the mean accuracy was slightly above the target concentration (i.e. 112% of target). All other tested formulations met the acceptance criteria. No test item was detected in the group 1 (control) formulations.

Duration of treatment / exposure:

90 days

Frequency of treatment:

daily seven days every week

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

N/A

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Not corrected for purity.

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Not corrected for purity.

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Not corrected for purity.

No. of animals per sex per dose:

20 animals (10 males and 10 females)

Control animals:

yes, concurrent vehicle

Details on study design:

RATIONALE FOR DOSE SELECTION:

The dose levels were selected based on information provided by the Sponsor and results of a 28-day repeated dose toxicity study with oral exposure of 2,4,4-trimethylpentene in rats. In this 28-day study, the NOAEL was established at 300 mg/kg/day. 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, with increased organ weights but without histopathological changes. In addition, there were no adverse signs or responses in the battery of neurological tests in this study, therefore it is considered that 2,4,4-trimethylpentene did not cause any changes to normal nervous system function when administered by gavage at dosages up to 1000 mg/kg/day.

The dose levels in the current study were chosen in an attempt to produce graded responses to the test item. The high-dose level was anticipated to produce some toxic effects, but not excessive lethality that would prevent meaningful evaluation. The mid-dose level was expected to produce minimal to moderate toxic effects. The low-dose level should produce no observable indications of toxicity.

ANIMAL ASSIGNMENT:

Animals were to be assigned to groups at random at the discretion of the biotechnician. Body weight variation was not to exceed ± 20% of the sex mean.

Positive control:

None

Observations and examinations performed and frequency:

GENERAL IN-LIFE OBSERVATIONS:

See Table 1 for details of general in-life observations, including:

i. Mortality
ii. Cage side observations
iii. Detailed clinical observations
iv. Arena observations
v. Individual body weights
vi. Food consumption
vii. Water consumption

OPHTHALMOSCOPIC EXAMINATION:

i. Frequency:
Pretreatment Period - All Study animals once (including spare animals)
Dosing Period - All Group 1 and 4 Study animals during Week 13.
ii. Procedure: The eyes were examined using an ophthalmoscope after application of a mydriatic agent (tropicamide 0.5%).

FUNCTIONAL TESTS:

i. Animals: The first 5 animals per sex per group.
ii. Frequency: Once during Week 12-13. The tests were performed after clinical observations (including arena observation, if applicable).
iii. Tests:
1. hearing ability, pupillary reflex and static righting reflex (score 0 = normal/present, score 1 = abnormal/absent).
2. fore- and hind-limb grip strength will be recorded as the mean of three measurements.
3. locomotor activity (recording period: 1 hour under normal laboratory light conditions, using a computerized monitoring system).

ESTROUS STAGE DETERMINATION:

i. Frequency: On the day of necropsy, a vaginal smear was taken to determine the stage of estrus from all female study animals.
ii. Procedure: Estrous stage was evaluated by examining vaginal cytology.

CLINICAL PATHOLOGY: See Table 2 for sample collection.

i. HAEMATOLOGY parameters evaluated are listed in Table 3.
ii. COAGULATION parameters evaluated are listed in Table 4.
iii. CLINICAL CHEMISTRY parameters evaluated are listed in Table 5.

Sacrifice and pathology:

At termination, study animals were deeply anesthetized using isoflurane and subsequently exsanguinated and subjected to a full post-mortem examination. Animals were fasted (overnight with a maximum of 24 hours) before their scheduled necropsy.

NECROPSY:

Animals as detailed in Table 6 were subjected to a complete necropsy examination, which included evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues.

ORGAN WEIGHTS:

The organs detailed in Table 7 were weighed at necropsy.

TISSUE COLLECTION AND PRESERVATION:

Representative samples of tissues were collected and preserved in 10% neutral buffered formalin, except when stated otherwise, as detailed in Table 6 and associated Table 7.

HISTOLOGY:

Tissues as detailed in Table 6 and associated Table 7 were embedded in paraffin, sectioned, and stained with haematoxylin and eosin.

MICROSCOPIC EVALUATION:

Tissues as detailed in Table 6 and associated Table 7 were evaluated histopathologically by a board-certified toxicological pathologist.

Optional endpoint(s):

None.

Other examinations:

None.

Statistics:

DESCRIPTIVE STATISTICS:

Means, standard deviations (or % coefficient of variation or standard error, when deemed appropriate), percentages, numbers, and/or incidences were reported as appropriate.

INFERENTIAL STATISTICS:

Statistical tests were conducted at the 5% significance level. Pairwise comparisons against the control group used two-sided tests and were reported at the 1% and 5% levels, unless otherwise noted.

INFERENTIAL STATISTICS IN PROVANTIS
1. Analyses were performed according to Table 8 when possible but excluded any group with less than 3 observations.
2. Parametric/non-parametric: Levene’s test was used to assess homogeneity of variance. The groups were compared using an overall one-way ANOVA if Levene’s test was not significant or the Kruskal-Wallis test if it was significant. If the overall ANOVA or Kruskal-Wallis test was significant, then pairwise comparisons used Dunnett’s or Dunn’s test, respectively.
3. Non-parametric: The groups were compared using an overall Kruskal-Wallis test. If significant, then pairwise comparisons used Dunn’s test.
4. Incidence: Fisher’s exact test was used to conduct pairwise group comparisons of interest.

INFERENTIAL STATISTICS IN TOXDATA
1. Analyses were performed according to Table 8 when possible, but excluded semi-quantitative data, and any group with less than 2 observations.
2. Parametric: Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test. For the motor activity data set (at least 3 groups) ANOVA tests on group means were applied with Bonferroni correction for multiple testing. Mixed modelling techniques, comparing six different covariance structures, were used in order to select the best fitting model.
3. Non-parametric datasets with at least 3 groups were compared using a Steel-test.
4. Incidence: An overall Fisher’s exact test was used to compare all groups. Pairwise comparisons used Fisher’s exact test if the overall test was significant.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Salivation seen after dosing at 100 (1/10 males), 300 (2/10 males and 6/10 females) and 1000 mg/kg/day (6/10 males and all females) was considered not toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response rather than a sign of systemic toxicity. Salivation was also seen on two days in one control animal (male, No. 3).

Male No. 36, at 1000 mg/kg/day was observed on Day 76 with slight tremors, closed eyes and abnormal gait just after dosing, on Day 78 with slight tremors of the head just before dosing, and on Day 80 with slight tremors of the head and partly closed eyes just after dosing. These clinical signs lasted for a very short period of time only, and fully recovered afterwards. At the incidence observed (in a single animal), the timing and the temporary nature of the clinical signs and in absence of differences in body weight, clinical pathology and microscopic findings compared with other animals in this dosing group, these clinical signs were considered to be not related to the treatment with the test item, but rather a conditioned response to the dosing procedure.

Hunched posture was noted in a single male at 1000 mg/kg/day on Day 59. As this occurred only once in a single animal, this is considered to be unrelated to the treatment with the test item.
Other clinical signs (e.g. scabs and thin fur cover, and salivation in one control animal) noted during the Dosing Period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose related trend. At the incidence observed, these were considered to be unrelated to treatment with the test item.

Mortality:

no mortality observed

Description (incidence):

N/A

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Any changes in body weight gains were considered to be unrelated to treatment with the test item since no trend was apparent regarding dose and duration of treatment.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 100 (males and females) and 300 (females) mg/kg/day, food consumption was comparable to controls. For males at 300 mg/kg/day, there was a trend towards increased food consumption from Week 5 onwards. As overall food consumption was only 4.4% higher than the control group (not statistically significant) this was considered to be of no biological significance.

At 1000 mg/kg/day, overall food consumption was increased over the entire study period (14.6 and 13.6% higher than control in males and females, respectively). Food consumption up to 22.1 and 10.3% higher than control (not statistically significant) was noted in males and females, respectively from Week 3 and 4 onwards.

Food efficiency:

not examined

Description (incidence and severity):

N/A

Water consumption and compound intake (if drinking water study):

not specified

Description (incidence and severity):

N/A

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No ophthalmology findings were noted that were considered to be related to treatment with the test item.
The nature and incidence of ophthalmology findings noted during the Pretreatment Period was similar among the groups and occurred within the range considered normal for rats of this age and strain. Findings in Week 13 were only noted in control animals. These findings were therefore considered to be unrelated to treatment with the test item.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related haematology parameter changes were observed in males and females up to 300 mg/kg/day.
At 1000 mg/kg/day, changes in males comprised an increase in monocyte (1.81x of control) and large unstained cells count (1.87x of control), and platelet count (1.23x of control), and a decrease in mean corpuscular hemoglobin (0.95x of control) and mean corpuscular hemoglobin concentration (0.98x of control). In females, only an increase in large unstained cell count (1.78x of control) was noted. These differences were considered to have arisen by chance or in the absence of corroborative finding and are considered to be of no toxicological significance.
The remaining differences in hematology parameters, regardless of statistical significance, were considered not test item-related based on the absence of a dose response, general overlap of individual values with the range of control, and/or were of a magnitude of change commonly observed in rats under similar study conditions.
Coagulation parameters of treated rats were considered not to have been affected by dosing with the test item.

It should be noted that a very long activated partial thromboplastin time (APTT) was noted in Animal No. 72 at 1000 mg/kg/day (72.8 sec), which should be interpreted with caution. No reason could be found for this extreme value. However, as no changes were noted in any concurrent parameter and as it concerns a single animal, this was considered to be of no toxicological relevance.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The changes distinguished animals dosed with the test item and from control animals are presented Table 9.
Alkaline phosphatase (ALP) activity was increased in males at 1000 mg/kg/day. Inorganic phosphate and total protein concentrations were increased in males at 300 and 1000 mg/kg/day, and albumin in males at 1000 mg/kg/day only. Total bilirubin concentration was decreased in females at 1000 mg/kg/day. Furthermore, dose response-related changes in concentrations of urea (increase) and triglyceride (decrease) were noted in males of all groups, and of calcium (increase) in males and females of all groups. Cholesterol (including HDL and LDL) and potassium concentrations were also increased in males at 1000 mg/kg/day, and in females of all groups.

The decreased concentrations of chloride in males at all dose levels is the result of slightly high control values and is therefore considered not test item-related. Remaining differences (including decreased aspartate aminotransferase (AST) activity) in clinical chemistry parameters, regardless of statistical significance, were considered not test item-related based on the absence of a dose response, general overlap of individual values with the range of control values, and/or were of a magnitude of change commonly observed in rats under similar study conditions.

Endocrine findings:

effects observed, treatment-related

Description (incidence and severity):

T3 concentration was decreased in males and females at 1000 mg/kg/day, and T4 concentration was decreased in males at 1000 mg/kg/day only.

Urinalysis findings:

not examined

Description (incidence and severity):

N/A

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

No effect on motor activity was seen in males at 100 and 300 mg/kg/day and in females at all dose levels.
Motor activity in males at 1000 mg/kg/day is lower when compared to control animals for the number of total movement (0.56x, of control) as well as the number of ambulations (0.47x). It should be noted that the low activities were mainly observed for Male Nos. 31 and 32, which attributed to this effect. As these changes were not supported by clinical observations, other functional observation tests or corroborating findings, this was considered of no toxicological significance.
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength was similar between control and high dose animals.

Immunological findings:

not examined

Description (incidence and severity):

N/A

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Test item-related higher liver and kidney weights (absolute and relative to body weights) were noted in the 300 and 1000 mg/kg/day group males and in the 1000 mg/kg/day group females, higher heart weights (absolute and relative to body weights) were noted in the 1000 mg/kg/day group males and test item-related lower thymus weights (absolute and relative to body weights) were noted in the 1000 mg/kg/day group males (Table 10).

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Test item-related gross observations were present in the kidney consisting of pale discoloration in 1/10 males at 300 mg/kg/day and 4/10 males at 1000 mg/kg/day.
Test item-related gross observations were present in the liver consisting of enlargement in 9/10 males, dark brown discoloration in 8/10 males and dark red discoloration in 1/10 males and 2/10 females dosed at 1000 mg/kg/day.
The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

Neuropathological findings:

not examined

Description (incidence and severity):

N/A

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings after treatment with the test item were noted in the liver and spleen of males and females and the kidney of males and are summarised in Tables 11 and 12.
In the liver, centrilobular hepatocellular hypertrophy was present in males starting at 300 mg/kg/day and in females at 1000 mg/kg/day up to mild degree. In the spleen, an increased incidence and severity of hemosiderin pigment was present in males and females treated at 1000 mg/kg/day up to moderate degree. In the kidney, granular casts day up to moderate degree, an increased incidence and severity of tubular basophilia up to moderate degree and hyaline droplet accumulation up to moderate degree were present all in males starting at 100 mg/kg/day. Further immunohistochemical staining analyses (performed on the controls and highest-dose group) demonstrated that both the granular casts and hyaline droplets were positive for alpha 2u-globulin.
The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

not specified

Description (incidence and severity):

N/A

Other effects:

not examined

Description (incidence and severity):

N/A

Details on results:

N/A

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

N/A

Key result

Dose descriptor:

NOAEL

Effect level:

< 100 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

N/A

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

not determinable

Remarks:

Adverse effects were observed even at the low dose.

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day (nominal)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Table 9                Test Item-Related Clinical Chemistry Changes at End of Treatment

	Males			Females
Dose level (mg/kg/day):	100	300	1000	100	300	1000
Alkaline Phosphatase	–	–	1.56x	–	–	–
Total Protein	–	1.04x	1.08x	–	–	–
Albumin	–	–	1.07x	–	–	–
Total Bilirubin	–	–	–	–	–	0.83x
Urea	1.09x	1.25x	1.52x	–	–	–
Cholesterol	–	–	1.29x	–	–	1.47x
HDL Cholesterol	–	–	1.24x	1.10x	1.14x	1.48x
LDL Cholesterol	–	–	1.55x	1.17x	1.20x	1.72x
Triglycerides	0.89x	0.86x	0.64x	–	–	1.20x
Potassium	–	–	1.08x	1.04x	1.05x	1.12x
Calcium	1.02x	1.07x	1.08x	1.05x	1.04x	1.09x
Inorganic Phosphate	–	1.06x	1.15x	–	–	–
Triiodothyronine (T3)	–	–	0.76x	–	–	0.74x
Thyroxine (T4)	–		0.55x	–	–	–
M = Males F = Females A dash (—) indicates absence of change. Numerical values indicate fold change of the treated group mean value relative to the control group mean value. Bolded values indicate the mean value was statistically different from controls.

 

Table 10             Mean Percent Organ Weight Differences from Control Groups

	Males					Females
Dose level (mg/kg/day):	100		300		1000	100	300	1000
Kidney
Absolute	5		29**		50**	8	5	23**
Relative to body weight	8		26**		58**	7	8	22**
Liver
Absolute	3		21*		63**	1	1	35**
Relative to body weight	5		19**		71**	0	4	32**
Heart
Absolute	-2		5		10*	3	3	8
Relative to body weight	0		2		15**	2	6	6
Thymus
Absolute	-9		-6		-23**	7	2	22
Relative to body weight	-7		-9		-19**	6	4	21
*: P<0.05, **: P<0.01

 

Table 11             Summary of test item-related Microscopic Findings – Males and Females

		Males					Females
	Dose level (mg/kg/day):	0	100	300		1000	0	100	300	1000
	Liver ^a	10	10	10		10	10	10	10	10
	Centrilobular hepatocellular     hypertrophy
	Minimal	-	-	5		2	-	-	-	4
	Mild	-	-	1		8	-	-	-	3
	Spleen ^a	10	10	10		10	10	10	10	10
	Hemosiderin pigment
	Minimal	6	5	7		3	8	8	7	5
	Mild	-	-	-		3	1	1	2	4
	Moderate	-	-	-		1	-	-	-	1
^a  =  Number of tissues examined from each group.

 

Table 12             Summary of Test Item-related Microscopic Findings – Males

	Males
Dose level (mg/kg/day):	0	100	300	1000
Kidney ^a	10	10	10	10
Casts, granular
Minimal	-	1	3	3
Mild	-	-	2	2
Moderate	-	-	2	-
Tubular basophilia
Minimal	3	6	6	3
Mild	-	1	3	4
Moderate	-	-	1	2
Hyaline droplet accumulation
Minimal	4	4	4	-
Mild	2	6	5	5
Moderate	-	-	1	4
^a  =  Number of tissues examined from each group.

Conclusions:

In this study, NOAEL was considered to be at least 1000 mg/kg/day for females and no NOAEL could be determined for males.

Executive summary:

In a key Guideline (OECD 408) oral repeated dose toxicity study, the test material (2,4,4-trimethylpentene; CAS No. 25167-70-8) was administered to Han-Wistar rats (10/sex/dose) by gavage at dose levels of 100,300, and 1000 mg/Kg bw/day for 90 consecutive days. A control group of ten males and ten females was dosed with vehicle alone (Corn oil).

 

The following parameters and end points were evaluated: mortality, clinical signs, body weights, food consumption, ophthalmology, functional observation tests, estrous stage, clinical pathology parameters (hematology, coagulation, and clinical chemistry), gross necropsy findings, organ weights, and histopathologic examinations.

 

No mortality was observed through the study period and there were no treatment-related clinical signs. There were no treatment-related effects on body weight or body weight changes. Increased food consumption was observed in males and females at 1000 mg/kg/day. As no effects were observed on body weight and in absence of corroborating findings, this was considered to be not adverse.

 

There were no treatment-related ophthalmological or functional findings.

 

Regarding clinical pathology, there were no treatment-related haematological or coagulation findings. There were a number of treatment-related changes in clinical biochemistry parameters. However, in the absence of histopathological correlates, none of these findings was considered adverse.

 

Regarding thyroid function, T3 concentration was decreased in males and females at 1000 mg/kg/day, and T4 concentration was decreased in males at 1000 mg/kg/day only. However, in the absence of histopathological correlates, none of these findings was considered adverse.

 

At gross necropsy, test item-related gross observations were present in the kidney consisting of pale discoloration in 1/10 males at 300 mg/kg/day and 4/10 males at 1000 mg/kg/day.

 

Test item-related gross observations were present in the liver consisting of enlargement in 9/10 males, dark brown discoloration in 8/10 males and dark red discoloration in 1/10 males and 2/10 females dosed at 1000 mg/kg/day.

 

Test item-related higher liver and kidney weights (absolute and relative to body weights) were noted in the 300 and 1000 mg/kg/day group males and in the 1000 mg/kg/day group females, higher heart weights (absolute and relative to body weights) were noted in the 1000 mg/kg/day group males and test item-related lower thymus weights (absolute and relative to body weights) were noted in the 1000 mg/kg/day group males.

 

Histopathological findings were as follows:

 

Correlating with increased liver weights, centrilobular hepatocellular hypertrophy was present in males starting at 300 mg/kg/day and in females at 1000 mg/kg/day up to mild degree. In the absence of any degenerative finding and at the low severity of the hepatocellular hypertrophy this finding was considered to be non-adverse.

 

In the spleen, an increased incidence and severity of hemosiderin pigment was present in males and females treated at 1000 mg/kg/day up to moderate degree. This finding was considered non-adverse.

 

In the kidney, granular casts day up to moderate degree, an increased incidence and severity of tubular basophilia up to moderate degree and hyaline droplet accumulation up to moderate degree were present in all males starting at 100 mg/kg/day. The combination of higher kidney weight, increased incidence/severity of hyaline droplet accumulation, tubular basophilia and presence of granular casts consistent with ‘hyaline droplet nephropathy’ were considered to be adverse. Subsequent immunohistochemical staining confirmed that the presence of hyaline droplet accumulation and granular casts were due to alpha 2u-globulin nephropathy, a phenotype which is not considered to be relevant in humans.

 

The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

 

Based on the results for the kidney, the No Observed Adverse Effect Level (NOAEL) was considered to be at least 1000 mg/kg/day for females and no NOAEL could be determined for males (<100 mg/kg/day). However, the male rat-specific nature of the effects seen in the kidney are considered not relevant to human.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Adequate information is available to characterise the hazards of this substance following repeated exposure

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

There are 3 key studies investigating repeat-dose toxicity following oral administration of 2,4,4 -trimethylpentene (HLS, 1997b; HLS, 1997c, CRL 2021).

In the first study (HLS, 1997b) male and female CD rats received 2,4,4 -trimethylpentene, by gavage, at 0, 100, 300 or 1000 mg/kg/day for four weeks. The administration of 2,4,4 -trimethylpentene to CD rats was generally well tolerated. The liver and kidneys were identified as target organs with organ weight increases although this was in the absence of histopathological change. There were no adverse signs or responses in the battery of neurological tests on this study. The NOAEL was 300 mg/kg/day (based on organ weight changes at 1000mg/kg/day).

In a second guideline study (HLS, 1997c) male and female CD rats 2,4,4 -trimethylpentene was administered by gavage at dose levels of 0, 100, 300 or 1000 mg/kg/day in a combined reproductive toxicity / developmental toxicity screening test 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 and a no-observed-effect level (NOEL) was not demonstrated as part of this study. However in subsequent investigations (Swenberg and Schoonhoven, 2004) on kidney samples from this study, including immunohistochemical investigations of alpha-2u-Globulin and comparison with prior research on alpha-2u-globulin nephropathy, the renal lesions were attributed to alpha-2u-globulin nephropathy and thus considered not to be relevant for human risk assessment.

In a third guideline study (performed according to the OECD 408 guideline) oral repeated dose toxicity study, the test material (2,4,4-trimethylpentene; CAS No. 25167-70-8) was administered to Han-Wistar rats (10/sex/dose) by gavage at dose levels of 100,300, and 1000 mg/Kg bw/day for 90 consecutive days. A control group of ten males and ten females was dosed with vehicle alone (Corn oil).

 

No mortality was observed through the study period and there were no treatment-related clinical signs. There were no treatment-related effects on body weight or body weight changes. Increased food consumption was observed in males and females at 1000 mg/kg/day. As no effects were observed on body weight and in absence of corroborating findings, this was considered to be not adverse. Regarding thyroid function, T3 concentration was decreased in males and females at 1000 mg/kg/day, and T4 concentration was decreased in males at 1000 mg/kg/day only. However, in the absence of histopathological correlates, none of these findings was considered adverse.

 

At gross necropsy, test item-related gross observations were present in the kidney consisting of pale discoloration in 1/10 males at 300 mg/kg/day and 4/10 males at 1000 mg/kg/day. Test item-related gross observations were present in the liver consisting of enlargement in 9/10 males, dark brown discoloration in 8/10 males and dark red discoloration in 1/10 males and 2/10 females dosed at 1000 mg/kg/day. Test item-related higher liver and kidney weights (absolute and relative to body weights) were noted in the 300 and 1000 mg/kg/day group males and in the 1000 mg/kg/day group females, higher heart weights (absolute and relative to body weights) were noted in the 1000 mg/kg/day group males and test item-related lower thymus weights (absolute and relative to body weights) were noted in the 1000 mg/kg/day group males. In the kidney, granular casts day up to moderate degree, an increased incidence and severity of tubular basophilia up to moderate degree and hyaline droplet accumulation up to moderate degree were present in all males starting at 100 mg/kg/day. The combination of higher kidney weight, increased incidence/severity of hyaline droplet accumulation, tubular basophilia and presence of granular casts consistent with ‘hyaline droplet nephropathy’ were considered to be adverse. Subsequent immunohistochemical staining confirmed that the presence of hyaline droplet accumulation and granular casts were due to alpha 2u-globulin nephropathy, a phenotype which is not considered to be relevant in humans.

 

Based on the results for the kidney, the No Observed Adverse Effect Level (NOAEL) was considered to be at least 1000 mg/kg/day for females and no NOAEL could be determined for males (<100 mg/kg/day). However, the male rat-specific nature of the effects seen in the kidney are considered not relevant to human.

In conclusion 2,4,4 -trimethylpentene, like other structural analogues, produced alpha-2u-globulin nephropathy in male rats - an effect that is not relevant for human risk assessment. Neurological tests were negative and only liver and kidney weight increases, in the absence of histopathological change, were reported at the highest dose level (1000mg/kg/day). Therefore excluding the male rat-specific alpha-2u-globulin nephropathy, 2,4,4 -trimethylpentene is generally well tolerated in the studies available. The overall NOEL, appropriate for human risk assessment, was 300mg/kg/day.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Available information indicates that this substance is generally well tolerated following repeated exposure although in common with other structural analogues it causes alpha-2u-globulin nephropathy in male rats.

Justification for classification or non-classification

The results of repeat-dose oral studies on 2,4,4 -trimethylpentene in rats do not warrant classification under CLP.