2,4,8,10-Tetraoxaspiro[5.5]undecane-3,9-diethanol, .beta.3,.beta.3,.beta.9,.beta.9-tetramethyl-

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2006-05-11 to 2006-09-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Test was performed according to guideline.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

485-230-3

EC Name:

-

Cas Number:

1455-42-1

Molecular formula:

Hill formula: C15H28O6 CAS formula: C15H28O6

IUPAC Name:

2-[9-(1-hydroxy-2-methylpropan-2-yl)-2,4,8,10-tetraoxaspiro[5.5]undecan-3-yl]-2-methylpropan-1-ol

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): SPG (Spiroglycol)
- Physical state: White powder
- Analytical purity: > 98 %
- Lot/batch No.: 50908-02
- Storage condition of test material: Room temperature in the dark

Test animals

Species:

rat

Strain:

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

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd, Margate, Kent.
- Age at study initiation: Approximately five to eight weeks old.
- Weight at study initiation: At the start of treatment, males weighed 145 to 172 g and the females weighed 132 to 160 g.
- Fasting period before study: No
- Housing: The animals were housed in groups of five by sex in polypropylene grid-floor cages suspended over trays lined with absorbent paper.
- Diet (e.g. ad libitum): The animals were allowed free access to pelleted diet (Rodent 5LF2 (Certified) diet).
- Water (e.g. ad libitum): The animals were allowed free access to mains drinking water supplied from polycarbonate bottles attached to the cage.
- Acclimation period: Seven days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2 °C
- Humidity (%): 55 ± 15 %
- Air changes (per hr): At least 15 air changes per hour
- Photoperiod (hrs dark / hrs light): Twelve hours continuous light and twelve hours darkness.

IN-LIFE DATES: From: Day 1 To: Day 28

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The formulations were shown to be stable for at least 14 days, therefore formulations were prepared weekly.

DIET PREPARATION
- Rate of preparation of diet (frequency): Not applicable
- Mixing appropriate amounts with (Type of food): Not applicable
- Storage temperature of food: Not applicable

VEHICLE
- Justification for use and choice of vehicle: Not specified
- Concentration in vehicle: 0, 3.75, 37.5 and 250 mg/mL
- Amount of vehicle: 4 mL
- Lot/batch no.: Not specified
- Purity: Not specified

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

28 days

Frequency of treatment:

Daily

Doses / concentrations

Remarks:

Doses / Concentrations:
15, 150, 1000 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

5/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: No data
- Rationale for animal assignment (if not random): No data
- Rationale for selecting satellite groups: Not applicable
- Post-exposure recovery period in satellite groups: Not applicable
- Section schedule rationale (if not random): Not applicable

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: See clinical observations below.
- Cage side observations: See clinical observations below.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity, ill-health or behavioural change immediately before dosing, immediately post-dosing and one and five hours after dosing during the working week. Animals were observed immediately before dosing, immediately post dosing and one hour after dosing at weekends. All observations were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual bodyweights were recorded on Day 1 and at weekly intervals thereafter. Bodyweights were also performed prior to terminal kill.

FOOD CONSUMPTION AND COMPOUND INTAKE : Yes
- Time schedule: Food consumption was recorded for each cage group at weekly intervals throughout the study.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE : Yes
- Time schedule for examinations: Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes. Water intake during Week 3 was measured by the daily weighing of water bottles, which continued until the end of the treatment period.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the study (Day 28).
- Anaesthetic used for blood collection: Yes (identity) / No / No data
- Animals fasted: No
- How many animals: All animals from each test group and control.
- Parameters examined:
The following parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant:
Haemoglobin (Hb)
Erythrocyte count (RBC)
Haematocrit (Hct)
Erythrocyte indices - Mean corpuscular volume (MCV)
- Mean corpuscular haemoglobin (MCH)
- Mean corpuscular haemoglobin concentration (MCHC)
Total leucocyte count (WBC)
Differential leucocyte count - Neutrophils (Neut)
- Lymphocytes (Lymph)
- Monocytes (Mono)
- Eosinophils (Eos)
- Basophils (Bas)
Platelet count (PLT)
Reticolocyte count (Retic) - Cresyl blue stained slides were prepared but reticulocytes were not assessed.
Prothrombin time (CT) was assessed by "Thrombomax HS with calcium" and Activated partial thromboplastin time (APTT) was assessed by "Actin FS" using samples collected into sodium citrate (0.11 mol/L).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the study (Day 28).
- Animals fasted: No
- How many animals: All animals from each test group and control.
- Parameters examined:
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:
Urea, glucose, total protein (Tot.Prot.), albumin, albumin/Globulin (A/G) ration (by calculation), sodium (Na+), potassium (K+), chloride (Cl-), calcium, (Ca++), inorganic phosphorous (P), aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT), alkalkine phosphatase (AP), Creatinine (Creat), total cholesterol (Chol) and total bilirubin (Bili).

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Prior to the start of treatment and on Days 3, 10, 17 and 24, all animals were observed for signs of functional/behavioural toxicity. Functional performance tests were also performed on all animals during Week 4, together with an assessment of sensory reactivity to different stimuli. Observations were carried out from approximately two hours after dosing on each occasion.
- Dose groups that were examined: All animals.
- Battery of functions tested:
Behavioural assessments.
Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed:
Gait, tremors, twitches, hyper/hypothermia, skin colour, respiration, convulsions, bizarre/abnormal/stereotypic behaviour/salivation, pilo-erection, exophthalmia, lachryrmation, palpebral closure, urination, defecation, transfer arousal, tail elevation.
Functional performance tests.
Motor activity: twenty purpose-built 44 infra-red beam automated activity monitors were used to randomly assess motor activity. Animals of one sex were tested at each occasion and were randomly allocated to activity monitors. The evaluation period was one hour for each animal. The percentage of time each anima was active and mobile was recorded for the overall one hour period and also during the final 20 % of the period (considered to be the asymptotic period).
Forelimb/hind limb grip strength: An automated grip-strength meter was used. Each animal was allowed to grip the proximal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its hind paws gripped the distal metal bar. The animal was pulled by the base of the tail until its grip was broken. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal.
Sensory reactivity: Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. The following parameters were observed: Grasp response, vocalisation, toe pinch, tail pinch, finger approach, touch escape, pupil reflex, blink reflex, startle reflex.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes.
On completion of the dosing period, all animals were subjected to a full external and internal examination and any macroscopic abnormalities were recorded.
The following organs, removed from animals killed at the end of the study, were dissected free from fat and weighed before fixation:
Adrenals, brain, epididymides, heart, kidneys, liver, ovaries, spleen, testes and thymus.

HISTOPATHOLOGY: Yes.
Samples of the following tissues were removed from all animals and preserved in buffered 10 % formalin:
adrenals, aorta (thoracic), bone marrow (femur including stifle joint), bone and marrow (sternum), brain (including cerebrum, cerebellum and pons, caecum, oesophagus. ovaries, pancreas, pituitary, prostate, rectum, colon, duodenum, epididymides, eyes, gross lesions, heart, ileum, jejunum, kidneys, liver, lung (bronchi)#, lymph nodes (cervical and mesenteric), muscle (skeletal), salivary glands (submaxillary), sciatic nerve, seminal vesicles, skin (hind limb) spinal cord (cervical), spleen, stomach, testes, thymus, thyroid/parathyroid, trachaea, urinary bladder, uterus.
#lungs were inflated to approximately normal inspiratory volume with buffered 10 % formalin before immersion in fixative.
The following tissues from all control and 1000 mg/kg/day dose groups were prepared as paraffin blocks, sectioned at nominal thickness of 5 µm and stained with haematoxylin and eosin for subsequent microscopic examination. Any macroscopically observed lesions were also processed together with the liver and spleen from all 15 and 150 mg/kg/day dose group animals:
adrenals, bone and marrow (sternum), brain (including cerebrum, cerebellum and pons, caecum, ovaries, prostate, rectum, colon, duodenum, epididymides, gross lesions, heart, ileum, jejunum, kidneys, liver, lung (bronchi)#, lymph nodes (cervical and mesenteric), sciatic nerve, seminal vesicles, spinal cord (cervical), spleen, stomach, testes, thymus, thyroid/parathyroid, trachaea, urinary bladder, uterus.

Statistics:

Data were processed to give group mean values and standard deviations where appropriate.
Where appropriate, quantitative data were analysed by the Provantis Tables and Statistics Module. For each variable, the most suitable transformation of the data was found, the use of possible covariates checked and the homogeneity of means assessed using ANOVA or ANCOVA and Bartlett's test. The transformed data were analysed to find the lowest treatment level that showed a significant effect, using the 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 b a stepwise Dunnett (parametric) or Steel (non-parametric) test to determine significant differences from the control group. Finally, if required, pair-wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).
Probability values (p) were presented as follows:
p<0.01**
P<0.05*
P≥0.05 (not significant)

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
No toxicologically significant clinical observations were detected.
Isolated incidences of increased salivation were detected immediately after dosing for males from all treatment groups, and for one female treated with 1000 mg/kg/day and two females treated with 150 mg/kg/day. In addition, increased salivation was detected 1 h after dosing for one male treated with 150 mg/kg/day. This observation of increased salivation is associated with oral administration of an unpalatable test material formulation and in isolation is considered not to be indicative of systemic toxicity.

BODY WEIGHT AND WEIGHT GAIN
No treatment-related effects of group mean bodyweight development were detected during the study.

FOOD CONSUMPTION
No adverse effect on food consumption was detected when compared to controls.

FOOD EFFICIENCY
No adverse effect on food efficiency (the ratio of bodyweight gain to dietary intake) was detected when compared to controls.

WATER CONSUMPTION
Daily visual inspections and gravimetrical measurements carried out on Day 15 onwards did not reveal any overt changes in water intake.

HAEMATOLOGY
No treatment-related haematological effects were detected.

CLINICAL CHEMISTRY
No treatment-related blood chemical effects were detected.
All treated males displayed statistically significant decreases in urea (P>0.05) and bilirubin (p<0.01) levels. These were considered to be due to high control values for these two blood chemical parameters. In addition, these findings did not show a dose related response and therefore were considered incidental and unrelated to treatment. All treated females displayed a statistically significant reduction (p>0.01) in glucose levels. This finding was due to high control values, with three of the individual values for the control animals being above the normal range for animals of the strain and age used. Therefore this finding was considered incidental and unrelated to treatment. In addition, females treated with 1000 mg/kg/day displayed a minimal statistically significant increased (p<0.05) in total protein. All individual values were within the normal range for rats of the strain and age used and in isolation this finding was considered incidental and unrelated to treatment.

NEUROBEHAVIOUR
Behavioural assessments: There were no treatment-related changes in the behavioural parameters measured. All remaining inter and intra group differences in urination, defecation and transfer arousal scores were considered to be a result of normal variation for rats of the strain and age used and were of no toxicological importance.

Functional performance tests: Statistical analysis of the data revealed no significant intergroup differences.

Sensory reactivity assessments: There were no treatment-related changes in sensory reactivity. All inter and intra group differences in sensory reactivity scores were considered to be a result of normal variation for rats of the strain and age used and were of no toxicological importance. Statistical analysis of the quantitative data revealed no significant intergroup differences.

ORGAN WEIGHTS
No treatment-related effects were detected.
Females treated with 1000 mg/kg/day displayed a minimal statistically significant increase in liver weights, both absolute and relative to terminal bodyweight. In the absence of histopathological correlates to suggest damage to the liver for these females, the findings were considered not to be related to treatment. All treated females displayed statistical significant increases in adrenal and ovary weights both absolute and relative to terminal bodyweight. These increases did not show a dose-related response and so in isolation were considered incidental and unrelated to treatment.

GROSS PATHOLOGY
One female treated with 1000 mg/kg/day and one control female displayed reddened lungs; in the absence of any histopathological evidence to suggest any lung damage, this finding was considered incidental and unrelated to treatment.
One female treated with 150 mg/kg/day displayed damage to the right eye on removal. This was a physical injury incurred during the necropsy procedure and was unrelated to treatment.

HISTOPATHOLOGY:
No treatment-related microscopic changes were observed.
All morphological changes were those commonly observed in laboratory maintained rats of the age and strain employed, and there were no differences in incidence or severity between control and treatment groups that were considered to be of toxicological significance.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: There were no toxicologically significant changes in parameters measured.

2

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: There were no toxicologically significant changes in parameters measured.

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Oral administration of the test material to rats for a period of twenty-eight consecutive days at dose levels of 15, 150 and 1000 mg/kg/day produced no toxicologically significant changes in the parameters measured. The “No Observed Effect Level” (NOEL) was therefore considered to be 1000 mg/kg/day.

Executive summary:

Introduction.

The study was designed to investigate the systemic toxicity of the test material. It complies with the requirements for notification of a new chemical substance in the EC and follows the testing method described in Commission Directive 96/54/EC (Method B7) and OECD Guidelines for Testing of Chemicals No. 407 “Repeated Dose 28 Day Oral Toxicity Study in Rodents” (Adopted 27 July 1995).

 

Methods.

The test material was administered by gavage to three groups, each of five male and five female Sprague Dawley Crl:CD (SD) IGS BR strain rats for twenty-eight consecutive days, at dose levels of 15, 150 and 1000 mg/kg/day. A control group of five males and five females were dosed with vehicle alone (Arachis oil BP).

Clinical signs, functional observations, bodyweight development and food and water consumption were monitored during the study. Haematology and blood chemistry were evaluated for all animals at the end of the study.

All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues from high dose and control animals was performed.

 

Results.

Mortality: There were no unscheduled deaths during the study.

Clinical observations: No toxicologically significant clinical abnormalities were detected.

Behavioural assessment: There were no treatment-related changes in the behavioural parameters measured.

Functional performance tests: There were no treatment-related changes in the functional performance parameters measured.

Sensory reactivity assessments: There were no treatment-related changes in sensory reactivity.

Bodyweight: No adverse effect on group mean bodyweight development was detected throughout the study.

Food consumption: No adverse effect on food consumption or food efficiency (the ratio of bodyweight gain to dietary intake) was detected when compared to controls.

Water consumption: Daily visual inspections and gravimetric measurements carried out from Day 15 did not reveal any overt changes in water intake.

Haematology: No treatment-related haematological effects were detected.

Blood chemistry: No treatment-related blood chemical effects were detected.

Organ weights: No treatment-related effects were detected.

Necropsy: No treatment –related macroscopic abnormalities were detected.

Histopathology: No treatment-related microscopic changes were observed.

 

Conclusion.

Oral administration of the test material to rats for a period of twenty-eight consecutive days at dose levels of 15, 150 and 1000 mg/kg/day produced no toxicologically significant changes in parameters measured. The “No Observed Effect Level” (NOEL) was therefore considered to be 1000 mg/kg/day.