3,9-dicyclohex-3-enyl-2,4,8,10-tetraoxaspiro[5.5]undecane

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

No fertility study is available.

Effect on fertility: via oral route

Endpoint conclusion:

no study available

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

No data

Effects on developmental toxicity

Description of key information

A GLP guideline study according OECD TG 414 is available.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Principles of method if other than guideline:

The test item was administered by gavage to three groups each of twenty-four time mated Sprague-Dawley Crl:CD® (SD) IGS BR strain rats, between Days 5 and 19 of gestation inclusive at dose levels of 250, 500, and 1000 mg/kg bw/day. A further group of twenty-four time mated females was exposed to the vehicle only (Arachis oil) to serve as a control.

Clinical signs, body weight change, food and water consumptions were monitored during the study.

All females were terminated on Day 20 of gestation and subjected to gross necropsy including examination of the uterine contents. The number of corpora lutea, number, position and type of implantation, placental weight, fetal weight, sex and external and internal macroscopic appearance were recorded. Half of each litter were examined for detailed skeletal development and the remaining half were subjected to detailed visceral examination.

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Identification : 3,9-Di-3-cyclohexen-1-yl-2,4,8,10-tetraoxaspiro[5.5]undecane (CAS No. 6600-31-3)
Physical State/Appearance : Solid, beige to brown
Purity : 95.5%
Storage Conditions : Room temperature in the dark; used/formulated in light

Species:

rat

Strain:

other: Sprague-Dawley Crl:CD® (SD) IGS BR

Details on test animals or test system and environmental conditions:

A total of ninety-six time-mated female Sprague-Dawley Crl:CD® (SD) IGS BR strain rats were obtained from Charles River (UK) Limited, Margate, Kent. Animals were delivered in two batches containing females prior to Day 3 of gestation. The day that positive evidence of mating was observed was designated Day 0 of gestation. On arrival the females weighed 176g to 277g.

The animals were housed individually in solid-floor polypropylene cages with stainless steel mesh lids furnished with softwood flakes (Datesand Ltd., Cheshire, UK). The animals were allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK) was used. Mains drinking water was supplied from polycarbonate bottles attached to the cage. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK). The diet, drinking water, bedding and environmental enrichment was considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

The animals were housed in a single air-conditioned room within the Envigo Research Limited, Shardlow, UK Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerized system, and print-outs of hourly mean temperatures and humidity are included in the study records. The Study Plan target ranges for temperature and relative humidity were 22 ± 3 ºC and 50 ± 20% respectively; there were no deviations from these targets.

The animals were randomly allocated to treatment groups using a randomization procedure based on stratified body weight to ensure similarity between the treatment groups. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.

Route of administration:

oral: gavage

Vehicle:

arachis oil

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For the purpose of the study, the test item was prepared at the appropriate concentrations as a solution in Arachis Oil. On each day of formulation preparation, for each concentration the required amount of Test Item was weighed out and added to a volume of vehicle. Additional vehicle was added to the resulting formulation to make it up to the required volume and shaken/mixed to give a homogeneous bulk formulation. These bulk formulations were subsequently divided into the required daily aliquots and stored at 4°C in the dark until the day of use. As the test item formulations appeared to have a tendency to solidify after storage in the refrigerator, these formulations were heated to approximately 50°C on the day of use and allowed to cool to room temperature prior to dosing.

The stability and homogeneity of the test item formulations were determined by Envigo Research Limited, Shardlow, UK Analytical Services as part of another study (Envigo Study Number 41500054). Formulations were shown to be stable for at least fifteen days when stored at 4°C in the dark. Formulations for this study were made and used within the known stability period.

Representative samples of dosing formulations were analyzed for concentration of 3,9-Di-3-cyclohexen-1-yl-2,4,8,10-tetraoxaspiro[5.5]undecane (CAS NO. 6600-31-3) at Envigo Research Limited Analytical Laboratory, Shardlow. The results indicate that the prepared formulations were within 82-113 % of nominal concentration.

Details on mating procedure:

Animals were delivered in two batches containing females prior to Day 3 of gestation. The day that positive evidence of mating was observed was designated Day 0 of gestation.

Duration of treatment / exposure:

The test item was administered daily, from Day 5 to Day 19 of gestation, by gavage. Control animals were treated in an identical manner with the vehicle alone.

Frequency of treatment:

daily

Duration of test:

All animals were killed on Day 20 of gestation.

Dose / conc.:

0 mg/kg bw/day

Dose / conc.:

250 mg/kg bw/day

Dose / conc.:

500 mg/kg bw/day

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

24 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

The study was performed and designed to investigate the effects of the test item on embryonic and fetal development, following repeated administration by gavage at dose levels of 250, 500 and 1000 mg/kg bw/day to the Sprague-Dawley Crl:CD® (SD) IGS BR strain rat during gestation, including the period of organogenesis.

The dose levels were selected based on available toxicity data including a preliminary pre-natal development study (Envigo Study Number 41500054), where dose levels up to 1000 mg/kg bw/day were well tolerated. The oral route was selected as the most appropriate route of exposure for this kind of studyas the most appropriate route of exposure, based on the physical properties of the test item, and the results of the study are believed to be of value in predicting the likely potential toxicity of the test item to man.

The study was performed between 02 December 2015 and 04 May 2016. The in-life phase of the study was conducted between 06 December 2015 (first day of treatment) and 23 December 2015 (final day of necropsy).

Maternal examinations:

Clinical Observations
Following arrival, all animals were examined for overt signs of toxicity, ill-health or behavioral changes once daily during the gestation period. Additionally, during the dosing period, observations were recorded immediately before and soon after dosing and one hour post dosing. All observations were recorded.

Body Weight
Individual body weights were recorded on Day 3 (before the start of treatment) and on Days 5, 6, 7, 8, 11, 14 and 17 of gestation. Body weights were also recorded for animals at terminal kill (Day 20).

Food Consumption
Food consumption was recorded for each individual animal at Day 3, 5, 8, 11, 14, 17 and 20 of gestation.

Water Consumption
Water intake was observed daily by visual inspection of the water bottles for any overt changes.

Ovaries and uterine content:

Necropsy
All animals were killed by carbon dioxide asphyxiation followed by cervical dislocation on Day 20 of gestation. All animals were subjected to a full external and internal
examination and any macroscopic abnormalities were recorded. The ovaries and uteri of pregnant females were removed, examined and the following data recorded:
i) Number of corpora lutea
ii) Number, position and type of intrauterine implantation
iii) Fetal sex
iv) External fetal appearance
v) Fetal weight
vi) Placental weight
vii) Gravid uterus weight
The uteri of any apparently non-pregnant females were immersed in 0.5% ammonium polysulphide solution to reveal evidence of implantation.
Implantation types were divided into:
Early Death: No visible distinction between placental/decidual tissue and embryonic tissue
Late Death: Separate embryonic/fetal and placental tissue visible
Dead Fetus: A fetus that had died shortly before necropsy. These were included as late deaths (one fetus from litter 42 (250 mg/kg bw/day) and one fetus from litter 74
(1000 mg/kg bw/day)) for reporting purposes
All implantations and viable fetuses were numbered according to their intrauterine position as follows (as an example):

Left Horn Cervix Right Horn

L1 L2 L3 L4 L5 L6 L7 L8 R1 R2 R3 R4 R5 R6 R7 R8
V1 V2 V3 V4 V5 V6 V7 V8 V9 V10 V11 V12 V13 V14 V15 V16

V = viable fetus

The fetuses were killed by subcutaneous injection of sodium pentobarbitone. Fetuses from each litter were divided into two groups and examined for either skeletal
alterations (skeletal examinations) or soft tissue alterations (visceral examinations).

Fetal examinations:

Visceral Examinations
At necropsy, alternate fetuses were identified using an indelible marker and placed in Bouin’s fixative. These fetuses were subsequently transferred to distilled water prior to examination
for visceral anomalies under a low power binocular microscope and following examination transferred to 10% Buffered Formalin for storage.
Visceral examinations commenced with an external assessment of general appearance, including the limbs, digits, genitals, anus, tail and umbilicus. Once completed the head, and
subsequently the tongue was removed, for examination of the tongue, palate and surrounding tissue including the teeth, genioglossus and nasopharynx. Serial sections were made of the
head and lower jaw to enable a detail examination of brain morphology and the lower jaw was also sectioned to allow examination of the incisors, multicuspid teeth and genioglossus
muscle.
The skin was opened up to allow further visceral examination and the internal sex of the fetuses was confirmed. The position of the umbilical artery and the liver, stomach spleen,
pancreas and intestines assessed. These tissues were then removed to enable examination of the under lying abdominal tissues including the kidneys which were transversely cut to
enable assessment of the cortex, medulla and papilla.
Following these examinations the thoracic tissues were examined, commencing first with the diaphragm lungs, azygo us vein and thymus. The lungs and thymus were subsequently
removed to allow assessment of the heart and cervicothoracic blood vessels. Examination of the heart included the external size and shape of the ventricles and atria, the entry and exit of
the blood vessels around the heart and assessment of the foramen ovale, atrio-ventricular valves, semi-lunar valves, ventricular septum and general proportions of ventricular walls and
cavities.

Skeletal Examinations
At necropsy, fetuses not allocated to visceral examinations were identified using cardboard tags marked with chinagraph pencil and placed in 70% IMS in distilled water. The fetuses
were subsequently eviscerated, processed and the skeletons stained with alizarin red S before being transferred to 50% glycerol for examination of skeletal development and alterations
and storage.
For assessment, fetuses were placed in a petri dish containing glycerol and examined using a microscope. Fetuses were examined whole but for evaluation and reporting purposes the
skeleton was divided into the following regions: skull, vertebral column, ribs, sternebrae, pectoral girdle, pelvic girdle, fore limbs and hind limbs. A peer review (approximately 10%
of the total number of litters examined) was performed as part of the overall skeletal examination procedures for the study.

Statistics:

The following parameters were analyzed statistically, where appropriate, using the test methods outlined below:
Body weight and body weight change (including adjustment for the contribution of the gravid uterus), food consumption, gravid uterus weight, litter data and fetal, litter and placental
weights and external, visceral and skeletal observations.
Data were first analysed using Shapiro Wilk normality test and Bartlett’s test for homogeneity of variance. Where there was no significance, parametric methodology was
applied using one way analysis of variance and, if significant, Dunnett’s multiple comparison
test. Where statistical significance was observed, non parametric methodology was applied using Kruskal-Wallis nonparametric analysis of variance; and, if significant, pairwise
analysis of control values against treated values using the Mann-Whitney ‘U’ test. Due to the non-normal distribution of the data, external, visceral and skeletal observations were
generally analyzed using non-parametric methodology.

Indices:

Pre and Post Implantation Loss
Percentage pre-implantation loss was calculated as:
[(number of corpora lutea - number of implantations) : (number of corpora lutea)] x 100

Percentage post-implantation loss was calculated as:
[(number of implantations - number of live fetuses) : (number of implantations)] x 100

Sex Ratio
Sex ratio was calculated as:
% male fetuses (sex ratio) = [(Total number of fetuses : Number of male fetuses)] x 100

Historical control data:

Historical control data are available for:
Normal Range Data for Pre-Natal Study Gestation Body Weights in the Sprague-Dawley Crl:CD® (SD) IGS BR Rat
Normal Range Data for Pre-Natal Study Gestation Food Consumption in the Sprague-Dawley Crl:CD® (SD) IGS BR Rat
Normal Range Data for Pre-Natal Litter Data in the Sprague-Dawley Crl:CD® (SD) IGS BR Rat
Normal Range Data for Pre-Natal Study External Fetal Observations in the Sprague-Dawley Crl:CD® (SD) IGS BR Rat
Normal Ranges for Pre-Natal Study Visceral Fetal Findings in the Sprague-Dawley Crl:CD® (SD) IGS BR Rat
Normal Ranges for Pre-Natal Study Skeletal Fetal Findings in the Sprague-Dawley Crl:CD® (SD) IGS BR Rat

Clinical signs:

no effects observed

Description (incidence and severity):

Throughout the dosing phase of the study, there were no clinical signs at any dose level considered to be related to the systemic toxicity of the test item.

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths during the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At the start of dosing (over Days 5 to 6 of gestation), group mean body weight gains between day 5 to 6, at all dose levels were lower than controls with the females from the 1000 mg/kg bw/day dose group exhibiting an actual group mean body weight loss of 1.0 g. A dose-relationship was apparent between the 500 and 1000 mg/kg bw/day dose groups with the intergroup differences at these dose levels achieving statistical significance (p<0.01 and p<0.001, respectively, when compared with control values). Subsequent improvement body weight gain was evident at all dose levels such that with periodic body weight gains were generally comparable with controls.

Due to the initial effect on body weight development, however, group mean cumulative body weight gains for the 1000 mg/kg bw/day females remained slightly lower than controls throughout the dosing period with the differences being statistically significant up to Day 14 of gestation (p<0.05 or p<0.001). At 500 mg/kg bw/day, the mean cumulative body weight gains also remained lower than controls up to Day 11 of gestation attaining statistical significance over Days 7 to 8 (p<0.001). Overall body weight gain for the 1000 mg/kg bw/day females was approximately 6% lower than controls whilst the corresponding values for the 250 or 500 mg/kg bw/day dose groups were similar to controls.

At necropsy in all dose groups all dose levels, group mean gravid uterus weights were largely comparable with controls; however, the mean body weight and body weight gain for the 1000 mg/kg bw/day females, when adjusted for the contribution of the gravid uterus weight, were slightly lower than controls with the latter achieving statistical significance (p<0.05 ).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At the start of dosing (over Days 5 to 8 of gestation), group mean food consumption for the 1000 mg/kg bw/day females was marginally but statistically significantly lower than controls (p<0.01). Subsequent improvement was evident such that the corresponding values for all test item-treated dose groups were similar to controls.

Food efficiency:

not specified

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

no effects observed

Description (incidence and severity):

Daily visual inspection of water bottles did not reveal any obvious intergroup differences.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic necropsy of adult females on Day 20 of gestation did not indicate any effect of treatment at 250, 500 or 1000 mg/kg bw/day.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Remarks on result:

other: 1000 mg/kg bw/day was the highest dose tested.

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg bw/day, the group mean male and female fetal weights were marginally but statistically significantly lower than controls (controls: males 4.1 g, females 3.9 g; high dose group: males 3.9 g, females 3.7 g p<0.05 and p<0.01, respectively), which resulted in a marginally lower combined fetal weight (p<0.05 Conrols: 4.0 g high dose group 3.8 g). The corresponding values for the remaining test item-treated dose groups were comparable with controls. Group mean litter or placental weights also remained unaffected by treatment with the test item up to a dose level of 1000 mg/kg bw/day when compared with controls.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): At 1000 mg/kg bw/day, the group mean male and female fetal weights were marginally but statistically significantly lower than controls (controls: males 4.1 g, females 3.9 g; high dose group: males 3.9 g, females 3.7), which resulted in a marginally lower combined fetal weight. The corresponding values for the remaining test item-treated dose groups were comparable with controls. Group mean litter or placental weights also remained unaffected by treatment with the test item up to a dose level of 1000 mg/kg bw/day when compared with controls.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

There was no statistically detrimental effect significant effect of maternal treatment on litter data as assessed by the mean number of implantations, in-utero offspring survival (as assessed by the mean numbers of early or late resorptions), live litter size, and post-implantation losses at 250, 500 or 1000 mg/kg bw/day. The mean live litter size for the 1000 mg/kg bw/day was marginally higher than controls, but without attaining statistical significance (controls: 13.6; low dose group 13.8, mid dose group 14.0 and high dose group 14.3).

Changes in sex ratio:

effects observed, non-treatment-related

Description (incidence and severity):

Mean number of live male fetuses and sex ratio (% male) for the 250 and 1000 mg/kg bw/day dose groups were statistically significantly higher than controls (p<0.05). There was, however, no dose-dependence and this finding was considered to be due to atypically low values for some control litters.

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

Neither the type, incidence nor distribution of findings apparent during external, visceral or skeletal examination of fetuses on Day 20 of gestation indicated any obvious effect of maternal treatment on fetal development at 250, 500 or 1000 mg/kg bw/day.

Skeletal malformations:

no effects observed

Description (incidence and severity):

Skeletal examination of fetuses/litters from all dose groups revealed statistically significantly lower values for incomplete ossification of sacral (neural) arch in relation to controls. As there was no dose-relationship and the mean values for the 250 and 1000 mg/kg bw/day dose groups remained within the historical control data ranges, this observation was considered to be normal biological variation. Another statistically significant skeletal anomaly included a reduction in group mean value of metatarsal - not ossified for fetuses/litters from the 500 mg/kg bw/day dose group when compared with controls (p<0.01 ). In the absence of a similar observation in the 1000 mg/kg bw/day dose group, this finding was considered likely to be incidental.

Visceral malformations:

no effects observed

Other effects:

no effects observed

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: 1000 mg/kg bw/day was the highest dose tested.

Developmental effects observed:

no

Mortality:

There were no unscheduled deaths during the study.

Clinical Observations:

There were no clinical signs considered to be related to the systemic toxicity of the test item.

Body Weight:

At the first day of dosing group mean body weight gains between day 5 to 6, at all dose levels were lower than controls. Females in the 1000 mg/kg bw/day dose group exhibited an actual group mean body weight loss of 1.0 g. Thereafter, improvement was evident, however, group mean cumulative body weight gains for the 1000 mg/kg bw/day females remained slightly lower than controls throughout the dosing period due to the initial effect on body weight. Group mean cumulative body weight gains for females treated with 500 mg/kg bw/day also remained slightly lower than controls up to Day 11 of gestation. Overall body weight gain for the 1000 mg/kg bw/day females was only approximately 6% lower than controls and, when adjusted for the contribution of gravid uterus, overall body weight and body weight gain for these females remained slightly lower than controls. Taking into account the overall effect on body weight and body weight gain together with the clear evidence of recovery, these observations were deemed to be not adverse.

Food Consumption:

There was a marginal decrease in food consumption at the start of dosing (over Days 5 to 8 of gestation) for females given the test item at a dose level of 1000 mg/kg bw/day. Recovery was evident and this observation was considered to be of no toxicological significance. There was no effect of treatment on food intake at 250 or 500 mg/kg bw/day.

Water Consumption:

Daily visual inspection of water bottles did not reveal any obvious intergroup differences.

Post Mortem Studies:

There were no treatment-related macroscopic findings at terminal necropsy.

Litter Data and Litter Placental and Fetal Weights:

There was no detrimental effect of maternal treatment with the test item at any dose level on the mean number of implantations, in-utero offspring survival, live litter size, sex ratio and post-implantation losses. At 1000 mg/kg bw/day, group mean fetal weights were marginally lower than controls. Taking into consideration the lack of any effect of treatment with the test item on fetal structural development, the slightly increased litter size (14.3 compared to control 13.6) and unaffected total litter weight (53.3 compared to control 52.2) the observations on the fetal weight an 1000 mg/kg bw/day are not regarded to be adverse.

Fetal Examination:

Neither the type, incidence nor distribution of findings apparent during external, visceral or skeletal examination of fetuses on Day 20 of gestation indicated any obvious effect of maternal treatment on fetal development at 250, 500 or 1000 mg/kg bw/day.

Conclusion:

‘No Observed Adverse Effect Level’ (NOAEL) for maternal toxicity is 1000 mg/kg bw/day (highest dose tested).

‘No Observed Adverse Effect Level’ (NOAEL) for reproductive and developmental toxicity is 1000 mg/kg bw/day (highest dose tested).

Conclusions:

‘No Observed Adverse Effect Level’ (NOAEL) for maternal toxicity is 1000 mg/kg bw/day (highest dose tested).
‘No Observed Adverse Effect Level’ (NOAEL) for reproductive and developmental toxicity is 1000 mg/kg bw/day (highest dose tested).

Executive summary:

The test item was administered by gavage to three groups each of twenty-four time mated Sprague-Dawley Crl:CD® (SD) IGS BR strain rats, between Days 5 and 19 of gestation inclusive at dose levels of 250, 500, and 1000 mg/kg bw/day. A further group of twenty-four time mated females was exposed to the vehicle only (Arachis oil) to serve as a control.

Clinical signs, body weight change, food and water consumptions were monitored during the study.

All females were terminated on Day 20 of gestation and subjected to gross necropsy including examination of the uterine contents. The number of corpora lutea, number, position and type of implantation, placental weight, fetal weight, sex and external and internal macroscopic appearance were recorded. Half of each litter were examined for detailed skeletal development and the remaining half were subjected to detailed visceral examination.

Results:

Mortality:

There were no unscheduled deaths during the study.

Clinical Observations:

There were no clinical signs considered to be related to the systemic toxicity of the test item.

Body Weight:

At the first day of dosing group mean body weight gains between day 5 to 6, at all dose levels were lower than controls. Females in the 1000 mg/kg bw/day dose group exhibited an actual group mean body weight loss of 1.0 g. Thereafter, improvement was evident, however, group mean cumulative body weight gains for the 1000 mg/kg bw/day females remained slightly lower than controls throughout the dosing period due to the initial effect on body weight. Group mean cumulative body weight gains for females treated with 500 mg/kg bw/day also remained slightly lower than controls up to Day 11 of gestation. Overall body weight gain for the 1000 mg/kg bw/day females was only approximately 6% lower than controls and, when adjusted for the contribution of gravid uterus, overall body weight and body weight gain for these females remained slightly lower than controls. Taking into account the overall effect on body weight and body weight gain together with the clear evidence of recovery, these observations were deemed to be not adverse.

Food Consumption:

There was a marginal decrease in food consumption at the start of dosing (over Days 5 to 8 of gestation) for females given the test item at a dose level of 1000 mg/kg bw/day. Recovery was evident and this observation was considered to be of no toxicological significance. There was no effect of treatment on food intake at 250 or 500 mg/kg bw/day.

Water Consumption:

Daily visual inspection of water bottles did not reveal any obvious intergroup differences.

Post Mortem Studies:

There were no treatment-related macroscopic findings at terminal necropsy.

Litter Data and Litter Placental and Fetal Weights:

There was no detrimental effect of maternal treatment with the test item at any dose level on the mean number of implantations, in-utero offspring survival, live litter size, sex ratio and post-implantation losses. At 1000 mg/kg bw/day, group mean fetal weights were marginally lower than controls. Taking into consideration the lack of any effect of treatment with the test item on fetal structural development, the slightly increased litter size (14.3 compared to control 13.6) and unaffected total litter weight (53.3 compared to control 52.2) the observations on the fetal weight an 1000 mg/kg bw/day are not regarded to be adverse.

Fetal Examination:

Neither the type, incidence nor distribution of findings apparent during external, visceral or skeletal examination of fetuses on Day 20 of gestation indicated any obvious effect of maternal treatment on fetal development at 250, 500 or 1000 mg/kg bw/day.

Conclusion:

‘No Observed Adverse Effect Level’ (NOAEL) for maternal toxicity is 1000 mg/kg bw/day (highest dose tested).

‘No Observed Adverse Effect Level’ (NOAEL) for reproductive and developmental toxicity is 1000 mg/kg bw/day (highest dose tested).

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP guideline study

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

3,9-Di-3-cyclohexen-1-yl-2,4,8,10-tetraoxaspiro[5.5]undecane (CAS No. 6600-31-3) was administered by gavage to three groups each of twenty-four time mated Sprague-Dawley Crl:CD® (SD) IGS BR strain rats, between Days 5 and 19 of gestation inclusive at dose levels of 250, 500, and 1000 mg/kg bw/day. A further group of twenty-four time mated females was exposed to the vehicle only (Arachis oil) to serve as a control.

Clinical signs, body weight change, food and water consumptions were monitored during the study.

All females were terminated on Day 20 of gestation and subjected to gross necropsy including examination of the uterine contents. The number of corpora lutea, number, position and type of implantation, placental weight, fetal weight, sex and external and internal macroscopic appearance were recorded. Half of each litter were examined for detailed skeletal development and the remaining half were subjected to detailed visceral examination.

Results:

Mortality:

There were no unscheduled deaths during the study.

Clinical Observations:

There were no clinical signs considered to be related to the systemic toxicity of the test item.

Body Weight:

At the first day of dosing group mean body weight gains between day 5 to 6, at all dose levels were lower than controls. Females in the 1000 mg/kg bw/day dose group exhibited an actual group mean body weight loss of 1.0 g. Thereafter, improvement was evident, however, group mean cumulative body weight gains for the 1000 mg/kg bw/day females remained slightly lower than controls throughout the dosing period due to the initial effect on body weight. Group mean cumulative body weight gains for females treated with 500 mg/kg bw/day also remained slightly lower than controls up to Day 11 of gestation. Overall body weight gain for the 1000 mg/kg bw/day females was only approximately 6% lower than controls and, when adjusted for the contribution of gravid uterus, overall body weight and body weight gain for these females remained slightly lower than controls. Taking into account the overall effect on body weight and body weight gain together with the clear evidence of recovery, these observations were deemed to be not adverse.

Food Consumption:

There was a marginal decrease in food consumption at the start of dosing (over Days 5 to 8 of gestation) for females given the test item at a dose level of 1000 mg/kg bw/day. Recovery was evident and this observation was considered to be of no toxicological significance. There was no effect of treatment on food intake at 250 or 500 mg/kg bw/day.

Water Consumption:

Daily visual inspection of water bottles did not reveal any obvious intergroup differences.

Post Mortem Studies:

There were no treatment-related macroscopic findings at terminal necropsy.

Litter Data and Litter Placental and Fetal Weights:

There was no detrimental effect of maternal treatment with the test item at any dose level on the mean number of implantations, in-utero offspring survival, live litter size, sex ratio and post-implantation losses. At 1000 mg/kg bw/day, group mean fetal weights were marginally lower than controls. Taking into consideration the lack of any effect of treatment with the test item on fetal structural development, the slightly increased litter size (14.3 compared to control 13.6) and unaffected total litter weight (53.3 compared to control 52.2) the observations on the fetal weight an 1000 mg/kg bw/day are not regarded to be adverse.

Fetal Examination:

Neither the type, incidence nor distribution of findings apparent during external, visceral or skeletal examination of fetuses on Day 20 of gestation indicated any obvious effect of maternal treatment on fetal development at 250, 500 or 1000 mg/kg bw/day.

Conclusion:

‘No Observed Adverse Effect Level’ (NOAEL) for maternal toxicity is 1000 mg/kg bw/day (highest dose tested).

‘No Observed Adverse Effect Level’ (NOAEL) for reproductive and developmental toxicity is 1000 mg/kg bw/day (highest dose tested).

Toxicity to reproduction: other studies

Description of key information

No evidence of toxicity to reproductive organs was observed in a subacute repeated dose study as no treatment-related changes were observed for any reproductive organ investigated during macroscopic and microscopic examination. On the basis of this study no effects on fertility were expected (NOEL, rat: 1000 mg/kg bw/day).

Link to relevant study records

Reference

Endpoint:

toxicity to reproduction: other studies

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: In this guideline study according to OECD TG 407 and GLP reproductive organs of male and female rats were examined for adverse effects.

Principles of method if other than guideline:

3,9-Di-3-cyclohexen-1-yl-2,4,8,10-tetraoxaspiro[5,5]undecane was administered by gavage to 5 male and 5 female Wistar rats per dose group in daily doses of 0, 100, 300 or 1000 mg/kg body weight for 29 days. At the end of the treatment period all animals were killed and necropsied. The pathologic evaluation consisted of organ weight, gross and microscopic examination of reproductive organs, incl. testes, epididymis, prostate gland, seminal vesicle, vagina, cervix, uterus, ovary, and oviduct.

GLP compliance:

yes

Type of method:

in vivo

Species:

rat

Strain:

Wistar

Sex:

male/female

Route of administration:

oral: gavage

Vehicle:

other: Solutol HS 15®/ Ethanol/ Tap Water (4:1:5 v/v/v)

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remarks'

No histopathological changes were found in testes, epididymis, prostate gland, seminal vesicle, vagina, cervix, uterus, ovary, and oviduct

There were no test substance-related toxic changes in organs weights of testes, epididymis, prostate gland, seminal vesicle, vagina, cervix, uterus, ovary/oviduct. No histopathological changes were found in testes, epididymis, prostate gland, seminal vesicle, vagina, cervix, uterus, ovary, and oviduct and all other examined organs.

Conclusions:

On the basis of this study no effects on fertility were expected (NOEL, rat: 1000 mg/kg bw/day).

Executive summary:

Vulkazon AFS (3,9-Di-3-cyclohexen-1-yl-2,4,8,10-tetraoxaspiro[5,5]undecane) was administered by gavage to 5 male and 5 female Wistar rats per dose group in daily doses of 0, 100, 300 or 1000 mg/kg body weight for 29 days. At the end of the treatment period all animals were killed and necropsied. The pathologic evaluation consisted of organ weight, gross and microscopic examination of reproductive organs, incl. testes, epididymis, prostate gland, seminal vesicle, vagina, cervix, uterus, ovary, and oviduct.

No evidence of toxicity to reproduction was observed in a subacute repeated dose study as no treatment-related changes were observed for any reproductive organ investigated during macroscopic and microscopic examination. On the basis of this study no effects on fertility were expected (NOEL, rat: 1000 mg/kg bw/day).

Additional information

No evidence of toxicity to reproductive organs was observed in a subacute repeated dose study as no treatment-related changes were observed for any reproductive organ investigated during macroscopic and microscopic examination. On the basis of this study no effects on fertility were expected (NOEL, rat: 1000 mg/kg bw/day).
Short description of key information:
There is no fertility study with Vulkazon AFS (3,9-Di-3-cyclohexen-1-yl-2,4,8,10-tetraoxaspiro[5,5]undecane) available. No effects on reproductive organs were observed in a 28 day study in rats. The pathologic evaluation consisted of organ weight, gross and microscopic examination of reproductive organs, incl. testes, epididymis, prostate gland, seminal vesicle, vagina, cervix, uterus, ovary/oviduct. No treatment-related changes were observed for any reproductive organ investigated during macroscopic and microscopic examination of all major organs (NOAEL, rat: 1000 mg/kg bw/day; males and females).

Justification for classification or non-classification

Based on the available study for repeated dose toxicity and the prenatal developmental study toxicity study in rats a non-classification for toxicity to reproduction is justified.

In both studies a NOAEL of 1000 mg/kg bw/day was found.