2,4,4-trimethylpentene

Endpoint summary

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other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment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

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

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 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

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. Further OECD 414 prenatal developmental toxicity studies in both rats and rabbits did not demonstrate adverse developmental effects.

Link to relevant study records

Reference

Reference 1

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 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)

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: 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.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01/02/2021 - 28/10/2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

This study has been conducted in line with Annex X of REACH, which states that an OECD 414 prenatal developmental toxicity study needs to be conducted in a non-rodent species.

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

See "Principles of method if other than the guideline"

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Deviations:

not specified

Principles of method if other than guideline:

The following deviations were recorded:

- On 3 days, the end of dosing went past the time determined for the stability of the test item, for no more than 60 minutes.
- The foetal weight for L11 No. 5 wasn't measured.
- The internal sex for foetus no. 40-R3 was not determined and was subsequently entered as unsexed.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

N/A

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

88 females were supplied by Charles River in Chatillon sur Chalaronne, France. Animals were mated at 17-19 weeks of age. The time allocated for acclimatisation was 5 days.

Route of administration:

oral: gavage

Vehicle:

arachis oil

Remarks on MMAD:

N/A

Details on exposure:

Dose formulations were prepared weekly for both the test material and the control. Test animals were dosed once daily from Day 7 to day 28 post-coitum.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical verification was conducted using ultra-performance liquid chromatography, with the following instrument parameters:

Instrument: Acquity UPLC system (Waters, Milford, MA, USA)
Detector: Acquity UPLC TUV detector (Waters)
Column: Acquity UPLC BEH C18, 50 50 mm x 2.1 mm i.d., dp =1.7 µm (Waters)
Column Temperature: 40°C +/- 1°C
Injection Volume: 5 µL
Mobile Phase: 70/30 (v/v) acetontrile/water
Flow: 0.6 mL/min
UV Detection: 210 nm

The solvent used was arachis oil, as this was determined to be the most relevant solvent for use in rabbits. This analytical method was verified in a separate study prior to the initiation of the primary study, and this concluded that the method met the following validation criteria:

- Specificity
- Calibration curve
- Accuracy and repeatability
- Limit of quantification
- Stability of analytical system and end solutions
- Stability of stock solutions

Details on mating procedure:

Animals were time-mated by the supplier between 0-2 days prior to animal delivery at the testing facility.

Duration of treatment / exposure:

Test animals were dosed once daily for 22 days, from Day 7 post-coitum.

Frequency of treatment:

Dosing was once daily.

Duration of test:

Maternal animals were euthanised on Day 29 post-coitum. Foetuses were euthanised on Day 29.

Dose / conc.:

0 mg/kg bw/day

Remarks:

(control)

Dose / conc.:

50 mg/kg bw/day

Dose / conc.:

150 mg/kg bw/day

Dose / conc.:

400 mg/kg bw/day

No. of animals per sex per dose:

22

Control animals:

yes, concurrent vehicle

Details on study design:

The vehicle was determined in a process prior to the in-life phase.

The New Zealand White Rabbit was chosen as the experimental animal due to the large historical control data set held by the CRO (Charles River Laboratories), including developmental toxicity specifically. Furthermore, this strain of rabbit is known to be sensitive to developmental toxins.

The study was designed to use the minimum number of animals required to produce statistically significant results.

Maternal examinations:

During the in-life phase, all maternal animals were examined twice daily for mortality, which was done via observation and further handling to confirm if necessary. Clinical observations were taken once daily, and during this animals were removed from their cages (which were also examined). Body weights for Day 0 were recorded by the supplier on Day 0, and by Charles River on Days: 7, 9, 12, 15, 18, 21, 24, 27 and 29. Food consumption was quantitatively measured daily after Day 3.

Ovaries and uterine content:

The following parameters were recorded following evaluation of the ovaries and uterine horn:

- The number of corpora lutea
- Uterus weight
- Number of implantation sites
- Number and distribution of live and dead foetuses
- Number and distribution of resorptions (early and late)
- Foetus sex

Anogenital distance was not recorded.

Blood sampling:

N/A

Fetal examinations:

Gross external examination was performed on late resorptions and foetuses of females found dead, or that were sacrified before planned necropsy. Additionally, this applied to all premature deliveries. All foetuses and late resorptions were fixed in 10% buffered formalin.
Scheduled foetal necropsy involved external, visceral and skeletal examinations, and findings from these were recorded as:

- Developmental variations - minor alterations in anatomy, structure or other deviations that are considered to have minimal biological effect.

- Malformations - anomalies that alter body conformity and that disrupt with bodily function in a way that will severely impact life, or be otherwise inviable.

Statistics:

Statistical analyses were conducted during the experimental phase. The following variables were calculated:

MATERNAL

- Body weight gains for 3 day intervals between days 7 and 29
- Corrected body weight gain for Day 29
- Overall food consumption for 2 or 3 day intervals between days 7 and 29
- Pregnancy rate
- Organ weight relative to body weight

OFFSPRING

- Number of male foetuses
- Number of female foetuses
- Pre-implantation loss
- Post-implantation loss
- Percentage of foetuses with abnormalities

Statistical tests were conducted at 5% significance level, and pairwise comparisons were reported at 1% or 5% levels.

For parametric analyses, Levene's test was used, as were ANOVA F-tests if Levene's was not significant, and Kruskal-Wallis if it was significant. For non-parametric analyses were performed using Kruskal-Wallis tests.

Indices:

N/A

Historical control data:

The historical control data used in this study is owned by Charles River Den Bosch.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Erect fur was observed at the top two dose groups. Reduced faeces production was observed across all treatment groups and was therefore not considered to be treatment-related. A single female (No. 45) was observed with splayed forelimbs from day 8 post-coitum. The animal seemed otherwise unaffected by this, and due to the lack of findings anywhere else, this was considered unrelated. No other specific effects have been recorded.

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

N/A

Mortality:

mortality observed, treatment-related

Description (incidence):

Four females had to be sacrificed during the study. Three of these were for reasons considered to be related to the test-item as follows:

- No. 72 (400 mg/kg bw/day) lost 10% of its body weight from Day 7 to 16 post-coitum, with concurrent reduction in food consumption, and displayed clinical signs including erected fur, skin pallor and gaunt appearance. This animal was sacrificed on Day 16 post-coitum.
- No. 73 (400 mg/kg bw/day) lost 10% of its body weight from Day 15 to 21 post-coitum, with concurrent reduction in food consumption, and displayed clinical signs including hunched posture, erected fur, thin appearance, decreased activity and reduced faeces production. This animal was sacrificed on Day 21 post-coitum.
- No. 84 (400 mg/kg bw/day) lost 11% of its body weight from Day 7 to 18 post-coitum, with concurrent reduction in food consumption, and displayed clinical signs including gaunt appearance and reduced faeces production. This animal was sacrificed on Day 19 post-coitum.

As these were at the top dose, they were considered to be treatment related.

Additionally, No. 48 (150 mg/kg bw/day) was found dead on Day 27 post-coitum, and its litter were all deceased. Red fluid was discovered on the gavage tube, and subsequent pathological examination revealed perforation of the oesophagus and the right lung, reduced heart size and ectopic spleen tissue. The death was attributed to a gavage error.

No. 50 (150 mg/kg bw/day) was sacrificed following a gavage error. The prevalence of gavage errors is attributed to the use of arachis oil instead of corn oil for the test material.

See Table 5.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No pertubations in body weights and weight gain were recorded across all doses. See Table 1 to Table 3.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Animals in the top dose experienced a lower mean level of food consumption initially, but this had recovered by Day 18 post-coitum (Table 4).

Food efficiency:

not examined

Description (incidence and severity):

N/A

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

not examined

Description (incidence and severity):

N/A

Ophthalmological findings:

not examined

Description (incidence and severity):

N/A

Haematological findings:

not examined

Description (incidence and severity):

N/A

Clinical biochemistry findings:

not examined

Description (incidence and severity):

N/A

Endocrine findings:

not examined

Description (incidence and severity):

N/A

Urinalysis findings:

not examined

Description (incidence and severity):

N/A

Behaviour (functional findings):

not examined

Description (incidence and severity):

N/A

Immunological findings:

not examined

Description (incidence and severity):

N/A

Organ weight findings including organ / body weight ratios:

not examined

Description (incidence and severity):

N/A

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Multiple minor effects were seen but were not associated with any one treatment group, nor were they observed with dose-response. Any effects observed were therefore considered unrelated to treatment with the test item.

Neuropathological findings:

not examined

Description (incidence and severity):

N/A

Histopathological findings: non-neoplastic:

not examined

Description (incidence and severity):

N/A

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

N/A

Other effects:

not examined

Description (incidence and severity):

N/A

Details on results:

N/A

Number of abortions:

no effects observed

Description (incidence and severity):

No abortions or stillbirths were observed.

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

No effects were observed generally. Post-implantation loss was higher in the control group when compared with historical control data. Minor increase in pre-implantation loss was noted in the 150 mg/kg bw/day group, however was not considered to be treatment-related due to the lack of dose-response. See Table 6.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

N/A

Early or late resorptions:

no effects observed

Description (incidence and severity):

No early or late resoprtions were observed.

Dead fetuses:

no effects observed

Description (incidence and severity):

No dead fetuses were observed.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

No changes in the duration of pregnancy were observed.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

No changes in the number pregnant were observed.

Other effects:

no effects observed

Description (incidence and severity):

N/A

Details on maternal toxic effects:

The primary adverse effects observed were the mortalities at 400 mg/kg bw/day, and these will drive the NOAEL for maternal animals.

Key result

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

mortality

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Fetal body weights were not affected by the test material (Table 6).

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

Litter sizes were not affected by the test material (Table 6).

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Sex ratio was not affected by the test material

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

Litter sizes were not affected by the test material.

Anogenital distance of all rodent fetuses:

not examined

Description (incidence and severity):

Anogenital distance was not measured.

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

N/A

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

One animal in the 150 mg/kg bw/day dose group had various malformations including cleft lip, gastroschisis, ectrodactyly and malrotated hindlimbs (Table 7 to Table 8).

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

Foetuses with misalignment of ilium were increased in the top-dose group compared with control and so this finding was considered to be treatment related. However, as this concerns a variation, this finding was considered non-adverse.
Other minor malformations across all dose groups were not considered to be treatment-related.
See Table 7 and Table 8.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Minor malformations were seen across all dose groups, including small eyes with red ocular orbits and nondescript malformations in the cardiovascular system, lungs and kidneys. All findings were not considered to be treatment related. See Table 7 and Table 8.

Other effects:

no effects observed

Description (incidence and severity):

N/A

Details on embryotoxic / teratogenic effects:

N/A

Key result

Dose descriptor:

NOAEL

Effect level:

> 400 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

skeletal: pelvic girdle

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

400 mg/kg bw/day

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

no

Relevant for humans:

no

Table 1              Summary of bodyweight (g) – gestation

Sex: Female		Day(s) Relative to Mating (Litter: A)
		0	7	9	12	15	18	21	24	27	29
0  mg/kg/day Group 1	Mean  SD  N	3456.4 427.6 21	3427.1 366.1 21	3446.5 351.0 21	3514.8 348.3 21	3592.6 326.7 21	3611.2 319.5 21	3648.4 313.5 21	3699.7 319.7 21	3750.2 314.3 21	3801.2 316.0 21
50  mg/kg/day Group 2	Mean  SD  N %Diff	3685.6 411.9 22    6.6	3666.1 363.8 22    7.0	3682.0 369.7 22    6.8	3745.2 360.1 22    6.6	3830.6 344.8 22    6.6	3865.1 349.7 22    7.0	3896.0  335.3  22    6.8	3927.7 325.4 22    6.2	3981.5 330.6 22    6.2	4032.7 325.5 22    6.1
150  mg/kg/day Group 3	Mean  SD  N  %Diff	3453.1 312.2 18    -0.1	3426.9 267.0 18    0.0	3452.1 270.2 17    0.2	3516.9 274.2 17    0.1	3625.6 296.8 17    0.9	3665.4 287.7 17    1.5	3666.4  273.9 17    0.5	3725.5 286.7 17    0.7	3779.2 266.8 17    0.8	3868.9 273.8 16    1.8
400  mg/kg/day Group 4	Mean  SD  N %Diff	3555.7 424.1 19    2.9	3531.5 384.9 19    3.0	3532.6 346.9 19    2.5	3565.3 338.6 19    1.4	3590.2 324.9 19    -0.1	3639.1 354.5 18    0.8	3669.9  384.8 17    0.6	3740.8 344.5 16    1.1	3783.8 332.3 16    0.9	3858.3 348.1 16    1.5

Anova & Dunnett

 

Table 2              Summary of bodyweight gains (g) – gestation

Sex: Female		Day(s) Relative to Mating (Litter: A)
		7 ¿ 9 [G]	9 ¿ 12 [G]	12 ¿ 15 [G]	15 ¿ 18 [G]	18 ¿ 21 [G]	21 ¿ 24 [G1]	24 ¿ 27 [G]	27 ¿ 29	7 ¿ 29
0  mg/kg/day  Group 1	Mean  SD N	19.4 56.4 21	68.2 42.1 21	77.8 79.1 21	18.7 55.5 21	37.1 37.1 21	51.3 50.9 21	50.5 63.9 21	51.0 46.4 21	374.1 235.2 21
50  mg/kg/day  Group 2	Mean  SD  N	15.9 49.5 22	63.1 44.5 22	85.5 74.6 22	34.5 68.3 22	30.9 84.7 22	31.7 67.4 22	53.8 39.3 22	51.3 27.1 22	366.6 142.6 22
150  mg/kg/day  Group 3	Mean  SD  N	27.4 58.6 17	64.9 34.8 17	108.6 60.9 17	39.8 50.2 17	1.0 71.1 17	59.1 40.9 17	53.7 77.4 17	70.5 36.7 16	420.4 201.7 16
400  mg/kg/day  Group 4	Mean  SD  N	1.2 56.9 19	32.7 58.5 19	24.9 100.5 19	33.0 91.8 18	26.8 61.9 17	23.6 40.3 16	43.0 49.0 16	74.6 52.1 16	339.3 156.3 16

[G] - Anova & Dunnett

[G1] - Kruskal-Wallis & Dunn

 

Table 3              Summary of Gravid Uterine Weights and Corrected Body Weights: Gestation

Sex: Female		0 mg/kg/day Group 1	50 mg/kg/day Group 2	150 mg/kg/day Group 3	400 mg/kg/day Group 4
Bodyweight on Day 7 (g) [G]	Mean SD N %Diff	3427.1   366.1   21       -	3666.1   363.8   22      7.0	3448.6   265.3   16      0.6	3519.0   387.1   16      2.7
Terminal Body Weight (g) [G]	Mean SD N %Diff	3801.2   316.0   21      -	4032.7   325.5   22      6.1	3868.9   273.8   16      1.8	3858.3   348.1   16      1.5
Gravid Uterus Weight (g) [G]	Mean SD N %Diff	494.78 116.28 21      -	502.75 104.84 22      1.61	535.66 106.04 16      8.26	478.82 87.31 16      -3.23
Adjusted BWG (7-abw) (g) [G]	Mean SD N %Diff	-120.7   234.6   21           -	-136.2   177.3     22      12.8	-115.3   194.5   16      -4.5	-139.5   141.7   16      15.6

[G] - Anova & Dunnett

 

Table 4              Summary of Food Consumption: Gestation

Food Mean Daily Consumption (g/animal/day)

Sex: Female		Day(s) Relative to Mating (Litter: A)
		7 ¿ 9	9 ¿ 12	12 ¿ 15	15 ¿ 18	18 ¿ 21	21 ¿ 24	24 ¿ 27	27 ¿ 29	7 ¿ 29
0  mg/kg/day  Group 1	Mean  SD  N	136.57 21.91 21	128.75 24.07 21	99.86 40.24 21	114.75 34.54 21	119.63 25.71 21	104.71 28.72 21	104.14 35.01 21	117.81 33.60 21	114.74 20.84 21
50  mg/kg/day Group 2	Mean  SD  N  %Diff	135.09 24.27 22      -1.08	131.59 22.19 22      2.21	107.52 38.17 22      7.67	116.64 34.16 22      1.65	123.08 29.38 22      2.88	111.15 29.58 22      6.15	115.11 22.76 22      10.53	118.57 23.28 22      0.64	119.21 19.08 22      3.89
150  mg/kg/day Group 3	Mean  SD  N  %Diff	122.09 23.45 17      -10.60	126.88 27.47 17      -1.45	96.94 38.68 17      -2.92	108.71 35.23 17      -5.26	120.37 28.35 17      0.62	125.76 22.60 17      20.10	114.75 31.01 17      10.18	130.22 21.10 16      10.53	117.81 21.75 16      2.68
400  mg/kg/day Group 4	Mean  SD  N  %Diff	103.89** 35.72 19      -23.93	97.25** 34.24 19      -24.47	68.47* 37.76 19      -31.43	87.46* 35.91 18      -23.78	110.06 33.68 17      -8.00	104.94 24.06 16      0.21	106.75 15.43 16      2.50	120.56 25.08 16      2.34	104.05 14.18 16      -9.32

Anova & Dunnett: * = p = 0.05; ** = p = 0.01

 

Table 5              Summary of Maternal Performance and Mortality

Sex: Female		0 mg/kg/day Group 1	50 mg/kg/day Group 2	150 mg/kg/day Group 3	400 mg/kg/day Group 4
Group size		22	22	22	22
Number of females found dead [f]	N %	0 0.0	0 0.0	1 4.5	0 0.0
Unscheduled euthanasia [f]	N %	0 0.0	0 0.0	1 4.5	3 13.6
Terminal euthanasia [f]	N %	22 100.0	22 100.0	20 90.9	19 86.4
Number of females pregnant [f]	N %	21 95.5	22 100.0	18 81.8	19 86.2
Females with live fetuses [f]	N %	21 100.0	22 100.0	17 94.4	19 100.0
Total resorptions [f]	N %	0 0.0	0 0.0	0 0.0	0 0.0
Females with all non-viable [f]	N %	0 0.0	0 0.0	1 5.6	0 0.0

[f] - Fisher's Exact

 

Table 6              Summary of ovarian and uterine examinations and litter observations

Sex: Female		0 mg/kg/day Group 1	50 mg/kg/day Group 2	150 mg/kg/day Group 3	400 mg/kg/day Group 4
Pre-implantation loss (%) [k]	Mean SD N %Diff	11.06 19.54 21 -	6.53 9.48 22 -40.98	12.89 15.56 16   16.54	6.75 10.57 16 -38.91
Post-implantation loss (%) [k]	Mean SD N %Diff	14.47 16.88 21 -	12.35 12.92 22 -14.65	11.30 15.24 16 -21.89	4.94 11.09 16 -65.87
Number of dead fetuses [k]	Mean SD N %Diff	0.3 0.7 21 -	0.2 0.5 22 -45.5	0.1 0.5 16 -62.5	0.0 0.0 16 -100.0
Number live fetuses, male [k]	Mean SD N %Diff	4.2 1.7 21 -	3.9 1.8 22 -8.8	3.6 1.5 16 -14.5	4.6 2.2 16 9.1
% live fetuses, male [k]	Mean SD N %Diff	50.99 18.72 21 -	44.30 16.97 22 -13.12	42.83 20.02 16 -15.99	49.52 16.26 16 -2.87
Number live fetuses, female [k]	Mean SD N %Diff	4.1 2.3 21 -	4.5 1.8 22 8.6	5.3 2.6 16 26.7	4.4 1.6 16 7.1
% live fetuses/litter, female [k]	Mean SD N %Diff	45.49 17.10 21 -	53.04 17.42 22 16.59	55.92 19.10 16 22.92	50.48 16.26 16 10.95
Fetal weight, male [G]	Mean SD N %Diff	37.74 5.79 21 -	39.93 4.77 22 5.80	38.85 5.36 16 2.95	38.31 6.34 16 1.50
Fetal weight, female [G]	Mean SD N %Diff	38.25 6.77 21 -	38.79 3.79 22 1.41	38.75 5.62 16 1.29	38.11 5.71 16 -0.37
Fetal weight, all [G]	Mean SD N %Diff	37.82 5.66 21 -	39.29 3.97 22 3.91	38.72 5.31 16 2.40	38.09 5.75 16 0.73

[k] - Kruskal-Wallis & Dunn

[G] - Anova & Dunnett

 

Table 7              Number of fetuses submitted for morphological examinations

Group No.	1	2	3	4
External examination   Visceral examination   Skeletal examination	183 (21)	189 (22)	144 (16)	145 (16)
	183 (21)	189 (22)	144 (16)	145 (16)
	183 (21)	189 (22)	144 (16)	145 (16)

 

Table 8              Summary of malformations – individual descriptions

Dose Level (mg/kg/day)	Female No.	Foetus No.	Malformation(s)#
0	10	L1	Lumbar vertebra, 1 or more, Supernumerary (S)
		L3	Lumbar vertebra, 1 or more, Supernumerary (S)
	12	L2	Lumbar vertebra, 1 or more, Supernumerary (S)
	14	R4	Subclavian artery, Right, Retroesophageal (V)
	16	R2	Lumbar vertebra, 1 or more, Supernumerary (S)
		R3	Lumbar vertebra, 1 or more, Supernumerary (S)
		R6	Lumbar vertebra, 1 or more, Supernumerary (S)
		R7	Lumbar vertebra, 1 or more, Supernumerary (S)
	19	L2	Kidney, Both, Malpositioned (V) Kidney, Both, Small (V) Sternebra, 1 or more, Supernumerary (S)
		L3	Lumbar vertebra, 1 or more, Supernumerary (S)
50	25	L1	Lung Lobe, Right cranial, fused (V)
		L6	Lumbar vertebra, 1 or more, Supernumerary (S)
		R10	Lumbar vertebra, 1 or more, Supernumerary (S)
		R11	Lumbar vertebra, 1 or more, Supernumerary (S)
	27	L2	Rib, 1 or more, Branched (S)
		L3	Cervical arch, 1 or more, Fused (S)
	29	L5	Lumbar vertebra, 1 or more, Supernumerary (S)
		L6	Lumbar vertebra, 1 or more, Supernumerary (S)
		R9	Nasal, Both, Fused (S)
	31	R5	Rib, 1 or more, Supernumerary (S) Thoracic arch, 1 or more, Supernumerary (S)
	36	L4	Lung Lobe, Left cranial, fused (V)
		R10	Sternebra, 1 or more, Supernumerary (S)
	43	L4	Aortic arch, Dilatation (V) Heart, Membranous Ventricular Septal Defect (V)
		R7	Aortic arch, Dilatation (V)
	44	L5	Nasal, Both, Fused (S)
		R9	Lumbar vertebra, 1 or more, Supernumerary (S)
150	46	L1	Lumbar vertebra, 1 or more, Supernumerary (S)
		L3	Lumbar vertebra, 1 or more, Supernumerary (S)
		L7	Lumbar vertebra, 1 or more, Supernumerary (S)
	51	L1	Lip, Cleft Lip (E) Hindlimb, Both, Malrotated (E) Hindpaw, Right, Ectrodactyly (E) Trunk, Gastroschisis (E) Eye, Both, Small (V) Nasal, Both, Fused (S) Premaxilla, Both, Fused (S) Premaxilla, Both, Split (S) Fibula, Right, Absent (S) Hindpaw phalanges, 1 or more, Absent (S) Metatarsal, Right, Absent (S) Talus, Right, Absent (S) Rib, 1 or more, Fused (S) Rib, 1 or more, Supernumerary (S) Thoracic arch, 1 or more, Absent (S) Thoracic arch, 1 or more, Fused (S) Thoracic arch, 1 or more, Supernumerary (S) Thoracic centrum, 1 or more, Absent (S) Lumbar centrum, 1 or more, Absent (S) Lumbar centrum, 1 or more, Fused (S) Lumbar centrum, 1 or more, Supernumerary (S)
		R12	Thoracic centrum, 1 or more, Fused (S) Thoracic vertebra, 1 or more, Hemivertebra (S)
400	75	L3	Lumbar vertebra, 1 or more, Supernumerary (S)
		R7	Lumbar vertebra, 1 or more, Supernumerary (S)
	81	R7	Cervical centrum, 1 or more, Absent (S)
	82	L3	Lumbar vertebra, 1 or more, Supernumerary (S)
	85	L2	Lumbar vertebra, 1 or more, Supernumerary (S)

#: Including external (E), visceral (V) and skeletal (S) examinations.

 

Conclusions:

Following administration of the test material, the substance was determined to cause generalised maternal toxicity (based upon mortality), and the maternal NOAEL is set as 150 mg/kg bw/day. The testing material was not determined to cause any adverse effects on development and the developmental NOAEL was determined to be greater than 400 mg/kg bw/day.

Executive summary:

In an OECD 414 guideline study, the prenatal developmental toxicity of 2,4,4 -Trimethylpentene was evaluated in time-mated New Zealand White Rabbits. Test animals were dosed with either 50, 150 or 400 mg/kg bw/day of the test item, or with just the arachis oil vehicle as a negative control, between Days 7 and 28.

 

In the top dose-group, three animals died after demonstrating clinical signs such as erected fur and skin pallor. Food consumption was reduced in all three animals, and concurrent body weight loss was also observed. These effects were considered to be related to administration of test item. Two further animals died due to gavage errors, and these were attributed to the use of arachis oil as a vehicle. Food consumption was reduced generally in the top dose group, but this was recovered by Day 18. No additional maternal toxicity was observed and the maternal NOAEL was set at 150 mg/kg bw/day.

Minor effects in the foetuses were observed, including multiple animals in the 400 mg/kg bw/day dose group that had misalignment of the ilium. This effect was considered to be treatment-related, due to its prevalence at the top-dose. One animal in the 150 mg/kg bw/day group had multiple external malformations, and further minor skeletal and visceral malformations were also noted across all dose-groups, but were not considered to be treatment related. No other developmental toxicity was observed, and the NOAEL was set to >400 mg/kg bw/day.

Due to the absence of any adverse effects attributable to the test item, 2,4,4 -trimethylpentene is not considered to be a prenatal developmental toxin in rabbits, and no classification is required.

Reference 3

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04/11/2020 - 15/10/2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

See "Principles of method if other than the guideline"

Principles of method if other than guideline:

The following deviations were recorded:

- Uterus weights from two females in the 100 mg/kg bw/day group were not determined following necropsy.

The following deviations were recorded in the dose-range finder:

- Terminal body weights were not determined for all animals. Data from previous days were used in their place.
- Formulations on post-coitum day 19 may not have acclimatised for 30 minutes prior to dosing.
- No clinical observations were recorded on post-coitum day 2.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

N/A

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

88 females were supplied by Charles River Deutschland in Sulzfeld, Germany. Animals were mated at 11-15 weeks of age. The time allocated for acclimatisation was 5 days.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Dose formulations were prepared weekly for both the test material and the control. Test animals were dosed once daily from day 6 to day 20 post-coitum. Dosing was oral, as is this is considered to be the most relevant route of exposure for the test material, which is a liquid hydrocarbon at room temperature.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical verification was conducted using ultra-performance liquid chromatography, with the following instrument parameters:

Instrument: Acquity UPLC system (Waters, Milford, MA, USA)
Detector: Acquity UPLC TUV detector (Waters)
Column: Acquity UPLC BEH C18, 50 50 mm x 2.1 mm i.d., dp =1.7 µm (Waters)
Column Temperature: 40°C +/- 1°C
Injection Volume: 5 µL
Mobile Phase: 70/30 (v/v) acetontrile/water
Flow: 0.6 mL/min
UV Detection: 210 nm

The solvent used was arachis oil, as this was determined to be the most relevant solvent for use in rabbits. This analytical method was verified in a separate study prior to the initiation of the primary study, and this concluded that the method met the following validation criteria:

- Specificity
- Calibration curve
- Accuracy and repeatability
- Limit of quantification
- Stability of analytical system and end solutions
- Stability of stock solutions

Details on mating procedure:

Animals had been time-mated prior to receipt at the testing site.

Duration of treatment / exposure:

Test animals were dosed once daily for 15 days, from Day 6 post-coitum.

Frequency of treatment:

Dosing was once daily.

Duration of test:

Maternal animals were euthanised on Day 21 post-coitum. Foetuses were euthanised on Day 21.

Dose / conc.:

0 mg/kg bw/day

Remarks:

(control)

Dose / conc.:

100 mg/kg bw/day

Dose / conc.:

300 mg/kg bw/day

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

22

Control animals:

yes, concurrent vehicle

Details on study design:

The vehicle was determined in a process prior to the in-life phase.

The Wistar Rat was chosen as the experimental animal due to the large historical control data set held by the CRO (Charles River Laboratories), including developmental toxicity specifically. Furthermore, this strain of rat is known to be sensitive to developmental toxins.

The study was designed to use the minimum number of animals required to produce statistically significant results.

Maternal examinations:

During the in-life phase, all maternal animals were examined twice daily for mortality, which was done via observation and further handling to confirm if necessary. Cage-side observations were taken once daily, and detailed examinations were made weekly during which animals were removed from their cages (which were also examined). Body weights for Day 0 were recorded by the supplier on Day 0, and by Charles River on Days: 2 ,6, 9, 12, 15, 18 and 21. Food consumption was quantitatively measured over days 2-6, 6-9, 9-12, 12-15, 15-18 and 18-21. Water consumption was measured by visual inspection across the duration of the study.

Additional endocrine and thyroid analyses were performed to determine T3, T4 and TSH levels, and both macroscopic and microscopic analyses of the thyroid gland were undertaken.

Ovaries and uterine content:

The following parameters were recorded following evaluation of the ovaries and uterine horn:

- The number of corpora lutea
- Uterus weight
- Number of implantation sites
- Number and distribution of live and dead foetuses
- Number and distribution of resorptions (early and late)
- Foetus sex, based on anogenital distance (where possible)

Blood sampling:

Blood samples were collected on the day of euthanasia, and animals were not fasted overnight. Animal selection was randomised for blood sample collection.

Fetal examinations:

Gross external examination was performed on late resorptions and foetuses of from a female that delivered prematurely, or that were sacrified before planned necropsy. Additionally, this applied to all premature deliveries. All foetuses and late resorptions were fixed in 10% buffered formalin.

Scheduled foetal necropsy will involve external, visceral and skeletal examinations, and findings from these will be recorded as:

- Developmental variations - minor alterations in anatomy, structure or other deviations that are considered to have minimal biological effect.

- Malformations - anomalies that alter body conformity and that disrupt bodily function in a way that will severely impact life, or be otherwise inviable.

Statistics:

Statistical analyses were conducted during the experimental phase. The following variables were calculated:

MATERNAL

- Body weight gains for 3 day intervals between days 6 and 21
- Corrected body weight gain for 3 day intervals between days 6 and 21
- Overall food consumption for 3 day intervals between days 6 and 21
- Pregnancy rate
- Organ weight relative to body weight

OFFSPRING

- Number of male foetuses
- Number of female foetuses
- Pre-implantation loss
- Post-implantation loss
- Percentage of foetuses with abnormalities

Statistical tests were conducted at 5% significance level, and pairwise comparisons were reported at 1% or 5% levels.

For parametric analyses, Levene's test was used, as were ANOVA F-tests if Levene's was not significant, and Kruskal-Wallis if it was significant. For non-parametric analyses were performed using Kruskal-Wallis tests. Dunnett's test was also employed for both parametric and non-parametric analyses.

Historical control data:

The historical control data used in this study is owned by Charles River Den Bosch.

Clinical signs:

no effects observed

Description (incidence and severity):

Any clinical signs observed were within the boundaries of the historical control data, and were not statistically significant.

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

N/A

Mortality:

no mortality observed

Description (incidence):

No mortality was observed in the study.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Minor changes in body weight were observed at the top dose (1000 mg/kg bw/day) at around 4-6%. Lower body weights were observed from the start of treatment. Due to this, these effects are considered unrelated to treatment with the test item. Body weight data can be found in Table1 - Table 3.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Animals in the top dose experienced a lower mean level of food consumption between days 9-12, and treatment-related effect could not be excluded. These effects were concurrent with weight loss. Food consumption data can be found in Table 4.

Food efficiency:

not examined

Description (incidence and severity):

N/A

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

not examined

Description (incidence and severity):

N/A

Ophthalmological findings:

not examined

Description (incidence and severity):

N/A

Haematological findings:

not examined

Description (incidence and severity):

N/A

Clinical biochemistry findings:

not examined

Description (incidence and severity):

N/A

Endocrine findings:

no effects observed

Description (incidence and severity):

No effect on thyroid hormones was observed during the study (Table 5).

Urinalysis findings:

not examined

Description (incidence and severity):

N/A

Behaviour (functional findings):

not examined

Description (incidence and severity):

N/A

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):

At the highest dose (1000 mg/kg bw/day), a treatment-related increase in relative, but not absolute, liver weight was observed (7% increase above control - Tables 6 & 7). No other statistically significant changes in absolute or relative organ weights were found.

Gross pathological findings:

no effects observed

Description (incidence and severity):

N/A

Neuropathological findings:

not examined

Description (incidence and severity):

N/A

Histopathological findings: non-neoplastic:

not examined

Description (incidence and severity):

N/A

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

N/A

Other effects:

not examined

Description (incidence and severity):

N/A

Details on results:

N/A

Number of abortions:

no effects observed

Description (incidence and severity):

No effects were observed.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

No effects were observed.

Total litter losses by resorption:

effects observed, non-treatment-related

Description (incidence and severity):

One animal in the control group and one in the top dose were observed to have no resorptions or foetuses and had only implantation sites. This was not attributed to the test item.

Early or late resorptions:

no effects observed

Description (incidence and severity):

No effects were observed.

Dead fetuses:

no effects observed

Description (incidence and severity):

No effects were observed.

Changes in pregnancy duration:

effects observed, non-treatment-related

Description (incidence and severity):

One animal in the 300 mg/kg bw/day dose group delivered its litter on the day of necropsy, but this early delivery was not attributed to the test item.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

No effects were observed.

Other effects:

no effects observed

Description (incidence and severity):

No effects were observed.

Details on maternal toxic effects:

N/A

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

For all dose groups, mean male fetal weights were significantly lower than in the concurrent control group (Table 8). However, the decreases was by less than 7% and no dose-dependent trend was evident.

For all dose groups, mean female fetal weights were lower than the concurrent control group but the changes were not statistically significant. Moreover, no dose-dependent trend was evident.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

Litter sizes were not affected by the test material.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Sex ratio was not affected by the test material

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

Litter sizes were not affected by the test material.

Anogenital distance of all rodent fetuses:

no effects observed

Description (incidence and severity):

Anogenital distance was unaffected by the test material (Table 9).

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

N/A

External malformations:

no effects observed

Description (incidence and severity):

No external malformations were observed.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

An instance of skeletal malformation was observed in each dose group except the high-dose group, but all of these instances were different to one another (Table 10). A lack of dose-response indicated that these were not attributable to the test item.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Minor variations such as supernumerary liver lobes and convoluted ureters were observed, but these were not considered to be related to the test item.

Other effects:

no effects observed

Description (incidence and severity):

N/A

Details on embryotoxic / teratogenic effects:

N/A

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

skeletal: sternum

skeletal: vertebra

Key result

Developmental effects observed:

no

Table 1              Summary of bodyweight (g) - gestation

Sex: Female		Day(s) Relative to Mating (Litter: A)
		2 [G]	6 [G]	9 [G1]	12 [G1]	15 [G]	18 [G]	21 [G]
0  mg/kg/day Group 1	Mean  SD  N	212.5 16.6 22	225.0 16.1 22	231.3 16.2 22	244.2 21.0 22	258.7 21.6 22	288.0 27.9 22	322.8 34.3 22
100  mg/kg/day Group 2	Mean  SD  N  %Diff	208.1 21.3 22    -2.1	219.0 21.4 22    -2.7	225.7 22.5 22    -2.4	240.6 23.7 22    -1.5	255.1 25.6 22    -1.4	287.0 30.2 22    -0.4	324.8 33.6 22    0.6
300  mg/kg/day Group 3	Mean  SD  N  %Diff	210.0 14.8 22    -1.2	220.9 17.1 22    -1.9	228.7 15.6 22    -1.1	241.0 16.8 22    -1.3	257.5 17.0 22    -0.5	289.0 21.0 22    0.3	329.1 25.0 21    2.0
1000  mg/kg/day Group 4	Mean  SD  N  %Diff	203.7 13.8 22    -4.2	213.9 12.2 22    -5.0	218.9 13.2 22    -5.4	230.6 12.2 22    -5.6	245.6 15.3 22    -5.0	273.9 17.7 22    -4.9	309.0 24.6 22    -4.3

 [G] - Anova & Dunnett

[G1] - Kruskal-Wallis & Dunn

 

Table 2              Summary of bodyweight gains (g) - gestation

Sex: Female		Day(s) Relative to Mating (Litter: A)
		6 ¿ 9	9 ¿ 12	12 ¿ 15	15 ¿ 18	18 ¿ 21	6 ¿ 21
0  mg/kg/day Group 1	Mean  SD  N	6.2 5.4 22	13.0 7.5 22	14.5 5.2 22	29.3 8.7 22	34.8 10.3 22	97.7 23.4 22
100 mg/kg/day Group 2	Mean  SD  N	6.7 5.2 22	14.9 4.3 22	14.5 5.9 22	31.8 6.7 22	37.9 8.2 22	105.8 17.6 22
300  mg/kg/day Group 3	Mean  SD N	7.8 4.8 22	12.3 3.9 22	16.5 5.0 22	31.5 6.4 22	38.6 7.6 21	107.1 15.6 21
1000  mg/kg/day Group 4	Mean  SD N	5.0 5.8 22	11.7 4.5 22	15.0 5.6 22	28.3 7.7 22	35.1 8.8 22	95.2 24.5 22

Anova & Dunnett

 

Table 3              Summary of Gravid Uterine Weights and Corrected Body Weights: Gestation

Sex: Female		0 mg/kg/day Group 1	100 mg/kg/day Group 2	300 mg/kg/day Group 3	1000 mg/kg/day Group 4
Bodyweight on Day 6 (g) [G]	Mean SD N %Diff	225.0   16.1   22       -	219.0   21.4   22      -2.7	222.0   16.7   21      -1.4	213.9   12.2   22      -5.0
Terminal Body Weight (g) [G]	Mean SD N %Diff	322.8   34.3   22           -	324.8   33.6   22      0.6	329.1   25.0   21      2.0	309.0   24.6   22      -4.3
Gravid Uterus Weight (g) [G]	Mean SD N %Diff	68.58 20.65 21           -	75.31 14.46 21      9.82	78.09 12.32 21      13.87	73.00 17.85 22      6.46
Adjusted BWG (6-abw) (g) [G]	Mean SD N %Diff	29.14 9.79 21             -	30.40 8.73 21      4.35	29.06 6.78 21      -0.28	22.18* 8.27 22      -23.89

[G] - Anova & Dunnett: * = p = 0.05

 

Table 4              Summary of Food Consumption: Gestation

Food Mean Daily Consumption (g/animal/day)

Sex: Female				Day(s) Relative to Mating (Litter: A)
		6 ¿ 9	9 ¿ 12	12 ¿ 15	15 ¿ 18	18 ¿ 21	6 ¿ 21
0  mg/kg/day Group 1	Mean  SD  N	17.88 2.22 22	18.97 2.18 22	18.97 2.00 22	20.71 2.57 22	20.09 2.84 22	19.32 2.00 22
100  mg/kg/day Group 2	Mean  SD  N  %Diff	18.30 2.64 22      2.37	19.06 2.23 22      0.48	19.06 2.54 22      0.48	21.79 3.05 22      5.19	20.30 2.31 22      1.06	19.70 2.10 22      1.96
300  mg/kg/day Group 3	Mean  SD  N  %Diff	18.15 1.75 22      1.53	18.17 1.85 22      -4.23	18.53 1.64 22      -2.32	20.77 2.62 22      0.29	19.76 2.42 22      -1.66	19.08 1.60 22      -1.29
1000  mg/kg/day Group 4	Mean  SD  N  %Diff	16.83 2.59 22      -5.85	16.94** 2.03 22      -10.70	17.65 2.12 22      -6.95	19.82 1.82 22      -4.32	19.30 2.49 22      -3.92	18.11 1.53 22      -6.29

Anova & Dunnett: ** = p = 0.01

 

Table 5              Summary of Thyroid Hormone Values

Sex: Female		Reporting Special Chemistry
		T3 (ng/mL) [G]	TSH (mU/L) [G]	T4 (ng/mL) [G]
0  mg/kg/day Group 1	Mean SD N	0.389   0.069   22	0.3078   0.1813   22	21.50   4.71 22
100  mg/kg/day Group 2	Mean SD N tCtrl	0.388   0.066   22          1.00	0.3681   0.2023   22            1.20	21.15   3.50   22        0.98
300  mg/kg/day Group 3	Mean SD N tCtrl	0.364   0.061   21          0.94	0.3254   0.1741   21            1.06	20.05   3.14   21        0.93
1000  mg/kg/day Group 4	Mean SD N tCtrl	0.354   0.102   22          0.91	0.3344   0.1513   22            1.09	21.29   9.27   22        0.99

[G] - Anova & Dunnett

 

Table 6              Summary of Absolute Organ Weights

Sex: Female		0 mg/kg/day Group 1	100 mg/kg/day Group 2	300 mg/kg/day Group 3	1000 mg/kg/day Group 4
Terminal Body Weight (g) [G]	Mean SD N %Diff	322.8         34.3         22                -	324.8         33.6         22                0.6	329.1         25.0         21   2.0	309.0         24.6         22            -4.3
Gland, Thyroid Weight (g) [G1]	Mean SD N %Diff	0.01371 0.00335 22                -	0.01289 0.00346 22            -5.96817	0.01310 0.00320 21  -4.47771	0.01265 0.00237 22            -7.75862
Liver Weight (g) [G1]	Mean SD N %Diff	10.6884   1.3811   22              -	10.7349   1.4148   22   0.4346	11.0303   1.0633   21   3.1986	10.9547   1.0405   22   2.4917

 [G] - Anova & Dunnett

[G1] - Anova & Dunnett

 

Table 7              Summary of Organ Weights Relative to Body Weight

Sex: Female		0 mg/kg/day Group 1	100 mg/kg/day Group 2	300 mg/kg/day Group 3	1000 mg/kg/day Group 4
Gland, Thyroid (%) [G]	Mean SD N %Diff	0.00429 0.00108 22                -	0.00400 0.00106 22            -6.77142	0.00401 0.00106 21            -6.47838	0.00413 0.00093 22            -3.66172
Liver (%) [G]	Mean SD N %Diff	3.31282 0.26220 22              -	3.30675 0.28360 22            -0.18330	3.35090 0.17554 21              1.14955	3.55355** 0.31318 22              7.26662

 [G] - Anova & Dunnett: ** = p = 0.01

 

 

Table 8              Summary of mean fetal weights

Sex: Female		0 mg/kg/day Group 1	100 mg/kg/day Group 2	300 mg/kg/day Group 3	1000 mg/kg/day Group 4
Mean Fetal Weight males (g) [G]	Mean SD N %Diff	5.524 0.314 21 -	5.187** 0.175 22 -6.095	5.207** 0.269 21    -5.743	5.149** 0.204 21 -6.792
Mean Fetal Weight females (g) [G1]	Mean SD N %Diff	5.097 0.276 21 -	4.948 0.240 22 -2.925	5.004 0.242 21    -1.827	4.887 0.251 21 -4.120
Mean Fetal Weight all (g) [G1]	Mean SD N %Diff	5.318 0.261 21 -	5.084** 0.179 22 -4.403	5.089** 0.242 21 -4.311	5.025** 0.187 21 -5.520

 [G] - Kruskal-Wallis & Dunn: ** = p = 0.01

[G1] - Anova & Dunnett: ** = p = 0.01

 

Table 9              Summary of mean normalized anogenital distance

Sex: Female		0 mg/kg/day Group 1	100 mg/kg/day Group 2	300 mg/kg/day Group 3	1000 mg/kg/day Group 4
Mean Normalized Fetal AGD m [G]	Mean SD N %Diff	1.638 0.141 21 -	1.610 0.165 22 -1.745	1.667 0.109 21 1.778	1.689 0.124 21 3.085
Mean Normalized Fetal AGD f [G]	Mean SD N %Diff	0.802 0.112 21 -	0.814 0.146 22 1.455	0.802 0.090 21 -0.067	0.837 0.128 21 4.306

 [G] - Anova & Dunnett

 

Table 10            Summary of Malformations - Individual Descriptions

Dose Level (mg/kg/day)	Female No.	Fetus No.	Malformation(s)#
0	6	L5	Sternebra, 1 or more, Supernumerary (S)
100	26	R10	Lumbar vertebra, 1 or more, Supernumerary (S)
300	49	L1	Sternebra, 1 or more, Sternoschisis (S)

#: Including skeletal (S) examinations.

Conclusions:

Following administration of the test material, the substance was determined not to cause any maternal or developmental toxicity, and the NOAELs are set as >1000 mg/kg bw/day.

Executive summary:

In an OECD 414 guideline study, the prenatal developmental toxicity of 2,4,4 -Trimethylpentene was evaluated in time-mated Wistar Han rats. Test animals were dosed with either 100, 300 or 1000 mg/kg bw/day of the test item, or with just the corn oil vehicle as a negative control, between Days 6 and 20.

Multiple effects were observed in maternal animals at the top dose (1000 mg/kg bw/day), including an increase in relative liver weight, but as there were no corresponding macroscopic and microscopic changes recorded, this effect was considered non-adverse. A decrease in food consumption was observed and attributed to the test item, but was not considered adverse due to the absence of concurrent toxicity. No additional maternal toxicity was observed and the maternal NOAEL was set at >1000 mg/kg bw/day.

Minor effects in the foetuses were observed, including a reduction in body weights, sporadic skeletal malformations and visceral variations. None of these effects were seen with a dose-response and were thus not considered to be adverse and relevant. No other developmental toxicity was observed, and the NOAEL was set to >1000 mg/kg bw/day.

Due to the absence of any adverse effects attributable to the test item, 2,4,4 -trimethylpentene is not considered to be a prenatal developmental toxin in rats, and no classification is required.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

400 mg/kg bw/day

Study duration:

subacute

Species:

rabbit

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

In an OECD 414 guideline study, the prenatal developmental toxicity of 2,4,4 -Trimethylpentene was evaluated in time-mated New Zealand White Rabbits. Test animals were dosed with either 50, 150 or 400 mg/kg bw/day of the test item, or with just the arachis oil vehicle as a negative control, between Days 7 and 28.

 

In the top dose-group, three animals died after demonstrating clinical signs such as erected fur and skin pallor. Food consumption was reduced in all three animals, and concurrent body weight loss was also observed. These effects were considered to be related to administration of test item. Two further animals died due to gavage errors, and these were attributed to the use of arachis oil as a vehicle. Food consumption was reduced generally in the top dose group, but this was recovered by Day 18. No additional maternal toxicity was observed and the maternal NOAEL was set at 150 mg/kg bw/day.

Minor effects in the foetuses were observed, including multiple animals in the 400 mg/kg bw/day dose group that had misalignment of the ilium. This effect was considered to be treatment-related, due to its prevalence at the top-dose. One animal in the 150 mg/kg bw/day group had multiple external malformations, and further minor skeletal and visceral malformations were also noted across all dose-groups, but were not considered to be treatment related. No other developmental toxicity was observed, and the NOAEL was set to >400 mg/kg bw/day.

Due to the absence of any adverse effects attributable to the test item, 2,4,4 -trimethylpentene is not considered to be a prenatal developmental toxin in rabbits, and no classification is required.

In an OECD 414 guideline study, the prenatal developmental toxicity of 2,4,4 -Trimethylpentene was evaluated in time-mated Wistar Han rats. Test animals were dosed with either 100, 300 or 1000 mg/kg bw/day of the test item, or with just the corn oil vehicle as a negative control, between Days 6 and 20.

Multiple effects were observed in maternal animals at the top dose (1000 mg/kg bw/day), including an increase in relative liver weight, but as there were no corresponding macroscopic and microscopic changes recorded, this effect was considered non-adverse. A decrease in food consumption was observed and attributed to the test item, but was not considered adverse due to the absence of concurrent toxicity. No additional maternal toxicity was observed and the maternal NOAEL was set at >1000 mg/kg bw/day.

Minor effects in the foetuses were observed, including a reduction in body weights, sporadic skeletal malformations and visceral variations. None of these effects were seen with a dose-response and were thus not considered to be adverse and relevant. No other developmental toxicity was observed, and the NOAEL was set to >1000 mg/kg bw/day.

Due to the absence of any adverse effects attributable to the test item, 2,4,4 -trimethylpentene is not considered to be a prenatal developmental toxin in rats, and no classification is required.

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 CLP.