Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
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other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment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:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
not specified
GLP compliance:
yes (incl. certificate)
Species:
rat
Strain:
other: CD (Sprague-Dawley origin)
Sex:
male/female
Details on test animals 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 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.
Details on mating procedure:
- Impregnation procedure: co-housed
- M/F ratio per cage: 1:1
- Length of cohabitation: 6 days
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear; referred to as day 0 of pregnancy
- Any other deviations from standard protocol: Males and females paired on the fifteenth day of treatment.
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
Details on study schedule:
The animals were dosed for 15 days before pairing and throughout the study until termination. Males had received between 44 and 46 dose administrations, and females between 40 and 45 dose administrations. Females were not dosed if parturition was in progress.
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
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.
Parental animals: Observations and 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

Oestrous cyclicity (parental animals):
- Vaginal smears were taken for ten days before pairing from all females and examined to establish the regularity and duration of the oestrous cycle.
Litter observations:
PARAMETERS EXAMINED
- The following parameters were examined in offspring at approximately 24 hours after birth (day 1): number and sex of pups (live and dead), individual bodyweight (live offspring only), sex ratio, individual observations.

GROSS EXAMINATION OF DEAD PUPS:
- yes, for external and internal abnormalities.
Postmortem examinations (parental animals):
SACRIFICE
- 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 PATHOLOGY
- Gross necropsy consisted of external and internal examinations 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
- The number of corpora lutea and uterine implantation sites were recorded in all females
- Abnormalities, interactions and changes were noted and representative tissue samples preserved in fixative

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
Postmortem examinations (offspring):
SACRIFICE
- Killed on Day 4 of age

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations

HISTOPATHOLOGY
- Specimens of abnormal tissues indicated were retained
Statistics:
Absolute bodyweights at the start of each phase, bodyweight gains, food consumption values and litter sizes 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 pair-wise comparisons, otherwise a Dunnett's test was used. Inter-group differences in offspring survival, sex ratio and macroscopic pathology and histopathology were assessed, on indication, using Fisher's Exact test.
Reproductive indices:
- Mating performance and fertility
- Pre-coital interval
- Gestation length
- Group mean litter size
Offspring viability indices:
- Survival indices: pre-implantation survival index; post-implantation survival index; live birth index; viability index.
Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
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
Other effects:
not examined
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
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 (PARENTAL ANIMALS)
The absolute and bodyweight-relative weights of reproductive organs were similar in all groups and not affected by treatment. 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 (PARENTAL ANIMALS)
After approximately six weeks of treatment (Day 4 of lactation for females), 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 (PARENTAL ANIMALS)
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)
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
VIABILITY (OFFSPRING)
At 1000 mg/kg bw/day, one litter showed low offspring survival and the offspring were under-active, cold, unfed and all showed bodyweight loss. This was considered to have resulted from the flooding of the cage overnight on Days 2-3 of lactation, and therefore was not associated with parental treatment with 2,4,4-trimethyl pentene.

GROSS PATHOLOGY (OFFSPRING)
For the majority of offspring dying prematurely the necropsy findings observed were due to a lack of milk in the stomach.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: no treatment-related effects reported
Reproductive 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 parental 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

 

At dosages up to 1000 mg/kg/day, there were no effects on mating performance and fertility, or the birth and subsequent growth and survival of offspring to Day 4 of age. A parental dosage of 1000 mg/kg/day was therefore considered to be the no-observed-effect level (NOEL) for the offspring.

Conclusions:
The reproductive no-observed-effect level (NOEL) was 1000 mg/kg bw/day. Due to the effects on the kidneys at dosages of 100 mg/kg bw/day and higher, a parental 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-Trimethyl pentene at dosages of up to 1000 mg/kg bw/day was without adverse effect on the general condition or reproductive performance of male and female rats, or on the growth and viability of their offspring up to Day 4 of age. The dosage of 1000 mg/kg bw/day was therefore considered to represent the no-observed-effect level (NOEL) for these reproductive parameters. Due to the effects on the kidneys at dosages of 100 mg/kg bw/day and higher, a parental 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.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Adequate information is available to characterise the effects of this substance on reproduction
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Results of a combined reproductive toxicity / developmental toxicity screening test (OECD 421) are available for 2,4,4-trimethylpentene (HLS, 1997c). Male and female rats were administered 2,4,4–trimethylpentene (>95% pure) by gavage at 0, 100, 300 or 1000 mg/kg/day. All dose levels were without adverse effect on the general condition or reproduction/developmental performance in female rats, or on the growth and viability of their offspring up to day 4 of age. 1000 mg/kg bw/day was therefore considered to represent the NOEL for reproductive and developmental parameters.


Short description of key information:
A combined reproductive / developmental toxicity screen (OECD 421) did not highlight any reproductive toxicity potential associated with exposure of rats to 2,4,4-trimethylpentene

Justification for selection of Effect on fertility via oral route:
Available information indicates that this substance does not adversely affect reproduction (NOAEL exceeds 1000 mg/kg bw/day)

Effects on developmental toxicity

Description of key information
A combined reproductive / developmental toxicity screen (OECD 421) did not highlight any developmental toxicity potential associated with exposure of rats to 2,4,4-trimethylpentene
Link to relevant study records
Reference
Endpoint:
developmental toxicity
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:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
not specified
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
other: CD (Sprague-Dawley origin)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent, England
- Age at study initiation: 11-12 weeks
- Weight at study initiation: 229-262 g
- Housing: Individually in stainless steel or high density polypropylene cages.
- 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

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 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.
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1:1
- Length of cohabitation: 6 days
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear; referred to as day 0 of pregnancy
- Any other deviations from standard protocol: males and females paired on the fifteenth day of treatment.
Duration of treatment / exposure:
Dosing was for 15 days before pairing. Treatment was continued throughout mating, gestation and lactation to Day 3 of lactation.
Frequency of treatment:
Daily
Duration of test:
Until offspring were 4 days old.
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.
Maternal 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: 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 1 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
Ovaries and uterine content:
- The number of corpora lutea and uterine implantation sites were recorded in all females. Uterus and cervix were weighed.
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No
Statistics:
Absolute bodyweights at the start of each phase, bodyweight gains, food consumption values and litter sizes 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 pair-wise comparisons, otherwise a Dunnett's test was used. Inter-group differences in offspring survival, sex ratio and macroscopic pathology and histopathology were assessed, on indication, using Fisher's Exact test.
Indices:
Offspring viability indices: pre-implantation survival index; post-implantation survival index; live birth index; viability index.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
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, it was considered that the death of this animal was incidental.
At 1000 mg/kg bw/day, animals showed transient salivation after dose administration. At 1000 mg/kg bw/day, a number of animals showed brown staining on the dorsal and ventral body surfaces, generally first observed in Week 4.

ORGAN WEIGHTS:
The absolute and bodyweight-relative weights of reproductive organs were similar in all groups and not affected by treatment. The absolute and bodyweight-relative weights of the liver and kidneys were high, when compared with the Controls, for females that received 1000 mg/kg bw/day.

GROSS PATHOLOGY:
After approximately six weeks of treatment (Day 4 of lactation for females), four females that received 1000 mg/kg/day had swollen livers or liver lobes. No remarkable findings were apparent at dosages up to 300 mg/kg bw/day.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Basis for effect level:
other: Developmental toxicity
Abnormalities:
no effects observed
Developmental effects observed:
no
Conclusions:
The reproductive/developmental no-observed-effect level (NOEL) was 1000 mg/kg bw/day.
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 -trimethyl pentene at dosages of up to 1000 mg/kg bw/day was without adverse effect on the general condition or reproduction/developmental performance in female rats in this screening study, or on the growth and viability of their offspring up to Day 4 of age. The dosage of 1000 mg/kg bw/day was therefore considered to represent the no-observed-effect level (NOEL) for these reproductive parameters.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Adequate information is available to characterise the effects of this substance on the foetus
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information
Justification for selection of Effect on developmental toxicity: via oral route:
Available information indicates that this substance does not adversely affect foetal development (NOAEL exceeds 1000 mg/kg bw/day)

Justification for classification or non-classification

Based on the results of a guideline reproductive and developmental toxicity screen 2,4,4-trimethylpentene does not warrant classification under DSD or CLP.