Cineole

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

No data

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Eucalyptol

IUPAC Name:

Eucalyptol

Constituent 2

Reference substance name:

Cineole

EC Number:

207-431-5

EC Name:

Cineole

Cas Number:

470-82-6

Molecular formula:

C10H18O

IUPAC Name:

1,3,3-trimethyl-2-oxabicyclo[2.2.2]octane

Details on test material:

- Name of test material: Eucalyptol
- Analytical purity: 99.8 %
- Lot/batch No.: AZ00012750
- Expiration date of the lot/batch: 10 October 2012
- Storage condition of test material: Room temperature in teh dark (neat test item used under safety lightling).

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories UK Ltd, Blackthorn, Bicester, Oxon, UK
- Age at study initiation: (P) approximately 12 wks
- Weight at study initiation: (P) Males: 322 - 353 g; Females: 182 - 224 g
- Housing: In groups of 5 in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding. During the pairing phase, the animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis. Following evidence of successful mating, the males were returned to their original cages and the females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
- Use of restrainers for preventing ingestion: Not applicable
- Diet (e.g. ad libitum): Pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan Laboratories UK, Ltd.) was available ad libitum. The diet was not considered to contain any contaminant at a level that might have affected the purpose or integrity of the study.
- Water (e.g. ad libitum): Mains drinking water was available ad libitum. The water was not considered to contain any contaminant at a level that might have affected the purpose or integrity of the study.
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): The target ranges for the study were 50 ± 20 % relative humidity. Relative humidity exceeded the target range on occassions (acheived range (47 - 78 % RH), but these deviations were considered to have had no impact on the scientific integrity of the study.
- Air changes (per hr): At least 15
- Photoperiod (hrs dark / hrs light): 12 hours light/ 12 hours dark

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was prepared at the appropriate concentrations as a solution in Arachis oil BP. Formulations were therefore prepared fortnightly and stored at approximately 4 °C in the dark.

Details on mating procedure:

- M/F ratio per cage: 1 male:1 female
- Length of cohabitation: A maximum of 14 days
- Proof of pregnancy: Vaginal plug / sperm in vaginal smear referred to as Day 0 post coitum
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: No
- After successful mating each pregnant female was caged (how): The females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes. pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Litter size, offspring weight and sex, surface righting and clinical signs were recorded during this period.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability and homogeneity of the test item formulations were determined. Results show that the formulations were stable for at least twenty one days when stored at approximately 4 °C in the dark. The results indicate that the prepared formulations were within ± 4 % of the nominal concentration.

Duration of treatment / exposure:

Groups of ten males and ten females were treated daily, except for the females during parturition where applicable.

Frequency of treatment:

Daily throughout the study except for females during parturition. The first day of dosing was designated as Day 1 of the study.

Details on study schedule:

On Day 5 post partum, all females and offspring were killed and examined macroscopically. With the exception of high dosage females (allocated to a second mating phase), any females considered to be non-pregnant were killed on or after Day 25 post coitum. On day 43 of the study, any high dosage females failing to litter were re-paired. Males were killed and examined macroscopically after the second pairing phase was considered to have been completed.

Doses / concentrations

Remarks:

Doses / Concentrations:
30, 300 and 600 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

10/sex/dose

Control animals:

yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Immediately before dosing, soon after dosing, and one and five hours after dosing during the working week. Animals were observed soon after dosing, and one hour after dosing at weekends and public holidays, except for females during parturition, where applicable.
- Cage side observations included: Overt signs of toxicity, ill health and behavioural changes. All observations were recored. For each pregnant female, the date and time of observed start of parturition and date and time of observed completion of parturition were recorded along with the date of pairing and the date of mating.

BODY WEIGHT: Yes
- Time schedule for examinations: Day prior to dosing then weekly for males until termination and weekly for females until mating was evident. Body weights were then recorde for females on Days 0, 7, 14 and 20 post coitum and on Days 1 and 4 post partum.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes. Weekly food consumption was recorded for each cage of adults until pairing. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded during the lactation period (Days 1-4).
Weekly food efficiency (body weight gain/food intake) was calculated retrospectively for males and for females during the pre-pairing phase. Due to offspring growth and milk production for lactation, food efficiency for females could not be accurately calculated for females during gestation and lactation.


WATER CONSUMPTION AND COMPOUND INTAKE : Yes
- Time schedule for examinations: Water intake was observed daily by visual inspection of water bottles for any overt changes. Intergroup differences did not indicate any need for more formal gravimetric measurements.

Litter observations:

PARAMETERS EXAMINED
On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded. Offspring were individually identified within each litter by tattoo on Day 1 post partum. For each litter, the following was recorded:
Number of offspring born
Number of offspring alive recorded daily and reported on Days 1 and 4 post partum
Sex of offspring on Days 1 and 4 post partum
Clinical condition of offspring from birth to Day 5 post partum
Individual offspring weights on Days 1 and 4 post partum (litter weights were calculated retrospectively from this data)
All live offspring were assessed for surface righting reflex on Day 1 post partum.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: Adult males were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 52.
- Maternal animals: Adult females were killed by intraveous overdose of sodium pentobarbitone followed by exsanguination on Day 5 post partum.
Surviving offspring were terminated via intracardiac overdose of sodium pentobarbitone. Any females which failed to acheive pregnancy were killed on or after Day 25 post coitum unless allocated to a further mating phase.

GROSS NECROPSY
- Gross necropsy consisted of:
For all females, the uterus was examined for signs of implantation and the number of uterine implanations in each horn was recorded. This procedure was enhanced, as necessary, by staining the uteri with a 0.5 % ammonium polysulphide soution. The females dosed at 600 mg/kg bw/day that were not observed to have given birth to a litter were regarded as being non-pregnant from the first mating and all implantations were regarded as resulting from the second mating occasion. It is accepetd that where the number of uterine implantations exceeded the number of offspring born, some of these implantations would possibly have been from the earlier mating occasion. However, in view of the small differences observed, this was considered unlikely to have occurred and the presentation of this data as being completely from the second mating was considered valid.
All adult animals and offspring, including those dying during the study were subjected to a full external and internal examination and any macroscopic abnormalities.

HISTOPATHOLOGY / ORGAN WEIGHTS
Organ weights:
The pituitary, prostate, seminal vesicles, epididymides and testes were removed from adult males dissected free from fat and weighed before fixation. The additional requirements for the male pituitary, prostate and seminal vesicles were added to more fully assess the effect on male fertility.

Histopathology:
Samples of the following tissues were preserved from all aniamls from each dose group in buffered 10 % formalin, unless otherwise stated:
Coagulating gland
Epididymides*
Ovaries
Mammary gland (females only)
Pituitary
Prostate
Seminal Vesicles
Testes*
Uterus/cervix
Vagina
* preserved in Bouins fluid then transferred to 70 % Industrial Methylated Spirits approximately 48 h later.

The tissues from control and 1000 mg/kg bw/day dose group animals, any animals dying during the study and any animals which failed to mate or did not acheive a pregnancy were prepared as paraffin blocks, sectioned at a nominal thickness of 5 µm and stained with haematoxylin and eosin for subsequent microscopic examination. In addition, sections of testes and epididymides from all control and 600 mg/kg be/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined. In view of the lower pregnancy rate observed during the first scheduled mating phase at 600 mg/kg bw/day, detailed qualitative and quantitative examination of the testes was performed taking into account the tubular stages of the spermatogenic cycle.

Statistics:

Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was acheived at a level of p<0.05. Statistical analysis was performed on the following parameters:
Body weight, body weight change, food consumption during gestation and lactation, pre-coital interval, gestation length, litter size, litter weight, sex ratio, copora lutea, implantation sites, implantation losses, viability indices, offspring body weight, offspring bodyweight change, offspring surface righting, absolute organ weights, body weight-relative organ weights.
Data were analysed using the decision tree from the Povantis™ tables and statistics module as detailed below:
Where appropriate, data transformations were performed using the most suitable method. The homogeneity of variance from mean values was analysed using Bartlett's test. Intergroup variance were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covariates. Any transformed data were analysed fo find the lowest treatment level that showed a significant effect, using Williams Test for parametric data or the Shirley test for non-parametric data. If no dose response was found, but the data showed non-homogeneity of means, the data were analysed by a stepwise Dunnett's (parametric) or Steel (nonparametric) test to determine significant differences from the control group. Where the data were unsuitable for these analyses, pair-wise tests were performed using the Student t-test (parametric) or Mann-Whitney U test (non-parametric).

Reproductive indices:

Mating performance and fertility
The following parameters were calculated from individual data during the mating period of the parental generation:
Pre-coital interval - Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.
Fertility indices - Calculated for each group.

Gestation and parturition data
The following parameters were calculated from individual data during the gestation and parturition period of the parental generation:
Gestation length - Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.
Parturition index - Calculated for each group.

Offspring viability indices:

Litter responses:
The standard unit of assessment was considered to be the litter, therefore values were first calculated for each litter and the group mean was calculated using their individual litter values. Group mean values included all litters reared to termination (Day 5 of age).

Implantation losses (%) - Group mean percentile pre-implantation and post-implantation losse were calculated for each female/litter.
live birth and viability indices - Calculated for each litter.
Sex ratio (% males) - Calculated for each litter value for Days 1 and 4 post partum.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

See results

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

See results

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
There were no unscheduled deaths on the study.
Minor incidences observed on the study were considered not to represent a systemic toxic effect of the test item. Isolated findings were considered incidental and unrelated to treatment.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
At 600 mg/kg bw, weight gain of males was lower than control during the first week of treatment, with differences attaining statistical significance. Thereafter differences in body weight gain from control, although occassionally attaining statistical significance were considered to reflect normal biological variation. Although overeall gain at termination from the start of treatment was statistically significantly lower than control, overall gain from the second week of treatment was not statistically significantly different from control.
Differences in food consumption were considered incidental and unrelated to treatment.
There were no observed effects on food efficiency.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
There was no adverse effect of treatent on mating performance, as assessed by pre-coital interval and mating evidence at the times of conception at 30, 300 and 600 mg/kg bw/day.
At 600 mg/kg bw/day, only seven females delivered a litter following the initial pairing and these females were subsequently re-paired with proven males from the first pairing at this dosage. The apparent effect on fertility was suspected as being fortuitous and unrelated to treatment. Following the repairining, only one female at this dosage failed to acheive pregnancy, and this female was noted to have ovary findings at necropsy which may be congenital. It was therefore considered that the fertility of the females had been unaffected by treatment at 600 mg/kg bw/day. There was no effect of treatment on fertility at 30 and 300 mg/kg bw/day.
Gestation length was unaffected by treatment at 30, 300 and 600 mg/kg bw/day, with all littering females showing a gestation length between 22 and 23.5 days.

ORGAN WEIGHTS (PARENTAL ANIMALS)
There were no statistically significant differences from control for male reproductive organ weights at 30, 300 and 600 mg/kg bw/day.

GROSS PATHOLOGY (PARENTAL ANIMALS)
Increased pevic space in the kidneys was noted for one male at 30 mg/kg bw/day and one female at 600 mg/kg bw/day. This is a relatively common finding in this strain of rat and at the incidence observed in the study did not indicate any effect of treatment. A female at 600 mg/kg bw/day was found to have both ovaries encased in a fluid filled sac. This female did not acheive pregnancy and this finding for the ovaries is considered to be congenital in nature and unrelated to treatment.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Histopathological examinations did not indicate any treatment related effects in the tissues examined and, in particular, examinations, including assessment of the completeness of stages, cell populations and numbers of each tubular stages of the spermatogenetic cycle for the testes did not indicate any effect on male fertility.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

600 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Results: F1 generation

General toxicity (F1)

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:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Details on results (F1)

VIABILITY (OFFSPRING)
There was no effect of maternal treatment on the mean number of corpora lutea and implantations, pre- and post-implantation loss, number of offspring born or subsequent offspring survival to Day 4 of age, mean litter size or sex ratio on Day 1 and Day 4 of treatment at 30, 300 and 600 mg/kg bw/day.

CLINICAL SIGNS (OFFSPRING)
Offspring clinical signs were unremarkable and did not indicate any effect of maternal treatment on offspring development.

BODY WEIGHT (OFFSPRING)
At 600 mg/kg bw/day there was no effect of maternal treatment on initial offspring bodyweight for either sex or initial litter weight, but subsequent body weight gain to Day 4 was slightly lower than control; differences attaining statistical significance from control in mean offspring weight or litter weight at Day 4 of age.
There was no effect of maternal treatment on mean offspring body weight or litter weight on Day 1 or Day 4 at 30 and 300 mg/kg bw/day.

OTHER FINDINGS (OFFSPRING)
Surface righting ability of the offspring on Day 1 was unaffected by maternal treatment at 30, 300 and 600 mg/kg bw/day.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The No Observed Adverse Effect Level (NOAEL) for adult toxicity, reproduction and offspring survival, growth and development was considered to be 600 mg/kg bw/day.

Executive summary:

Introduction:

The study was performed to screen for potential adverse effects of the test item on reproduction including offspring development and provides an initial hazard assessment for effect on reproduction. The study is compatible with the requierments of the recommendations of the OECD Guidelines for Testing of Chemicals No. 421 "Reproduction/Developmental Toxicity Screening Test" (adopted 27 July 1995).

This study was also designed to be compatible with Commission Regulation (EC) No. 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No. 1907/2006 of the European Parliament and of the council of the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Methods:

The test item was administered by gavage to three groups, each of ten male and ten female Wistar Han (™): RCCHan(™): WIST strain rats, for up to eleven weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females) at dose levels of 30, 300 and 600 mg/kg bw/day. A control group of ten males and ten females was dosed with vehicle alone (Arachis oil BP).

Clinical signs, bodyweight change, dietary intake and water consumption were monitored during the study.

Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 5 of the study, with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation. An additional pairing for high dose females that failed to achieve pregnancy was performed to fully assess mating performance and fertility.

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex.

Adult males were terminated on Day 52 of the study following the completion of the second pairing at 600 mg/kg bw/day. Females and offspring were terminated on day 5 post partum. Any female which did not produce a pregnancy (unless allocated to a further mating phase) was terminated on or after Day 25 post coitum. All animals were subjected to a gross necropsy examination and histopathological evaluation of reproductive tissues was performed. Additional male organ weight and detailed histopathological examination of the testes were performed to more fully assess male fertility.

Results:

Adult response:

Mortality: There were no unscheduled deaths of the study.

Clinical observations: Transient post-dosing salivation was observed from Day 7 at 600 mg/kg bw/day and Day 13 at 300 mg/kg bw/day. This sign was observed regularly throughout the treatment period with all animals being affected at both dosages, although the incidence of this finding was greatest at the high dosage. Transient post dosing salivation was also observed at 30 mg/kg bw/day for two females on Day 2 and one male on Day 50 of this study.

Bodyweight: At 600 mg/kg bw/day body weight gain of males was statistically significantly lower than control during the first week of treatment. Subsequent body weight gains were considered to reflect normal biological varaition, but overall gain at termination remained lower than control.

Bodyweight gain of males at 30 and 300 mg/kg bw/day and for females at all dosages, throughout the pre-pairing, gestation and lactation phases of the study, were considered to have been unaffected by treatment.

Food consumption: Food consumption for both sexes was considered to have been unaffected by treatment throughout the study, and including gestation and lactation phases for females, at 30, 300 and 600 mg/kg bw/day.

Food efficiency: At 600 mg/kg bw/day food conversation efficiency for males was lower than control during week 1; subsequent food utilisation was similar to control.

Food conversation efficiency of males at 30 and 300 mg/kg bw/day and for females at all dosages, throughout the pre-pairing, gestation and lactation phases of the study, were considered to have been unaffected by the treatment.

Reproductive performance:

Mating: Pre-coital interval and mating evidence at the times of conception did not indicate any adverse effect of treatment on mating performance at 30, 300 and 600 mg/kg bw/day.

Fertility: At 600 mg/kg bw/day, only seven females delivered a litter following the initial pairing but subsequent re-mating and additional assessment of male organ weight and detailed testicular histopathology did not indicate any treatment related effect on fertility for either sex. There was also no effect of treatment on fertility at 30 and 300 mg/kg bw/day.

Gestation lengths: Gestation length was considered to be unaffected by treatment at 30, 300 and 600 mg/kg bw/day.

Litter responses:

Offspring litter size, sex ratio and viability: There was no effect of maternal treatment on corpora lutea and implantations counts, pre- and post-implantation loss, number of offspring born, sex ratio or subsequent survival to Day 4 of age at 30, 300 and 600 mg/kg bw/day.

Offspring growth and development: At 600 mg/kg bw/day initial offspring body weight was similar to control but weight gain to Day 4 was statistically significantly lower than control.

Mean offspring body weights, litter weight and weight gains to Day 4 of age were unaffected by maternal treatment at 30 and 300 mg/kg bw/day.

Offspring observations: Assessment of surface righting ability on Day 1; offspring clinical signs and necropsy findings and did not indicate any effect of maternal treatment.

Pathology:

Necropsy: Macroscpoic necropsy findings did not indicate any effect of treatment at 30, 300 and 600 mg/kg bw/day.

Organ weights: Male reproductive organ weights were unaffected by treatment at 30, 300 and 600 mg/kg bw/day and did not indicate any effect on fertility.

Histopathology: Histopathological examinations did not indicate any effect of treatment at 600 mg/kg bw/day and these examinations, including detailed assessment of the spermatogenetic cycle for the testes did not indicate any effect on male fertility.

Conclusion: Within the context of this study, the No Observed Adverse Effect Level (NOAEL) for adult toxicity, reproduction and offspring survival, growth and development was considered to be 600 mg/kg bw/day.