Ethyl 2-methyl-1,3-dioxolane-2-acetate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 February 2013 to 6 June 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Ethyl 2-methyl-1,3-dioxolane-2-acetate

EC Number:

229-114-0

EC Name:

Ethyl 2-methyl-1,3-dioxolane-2-acetate

Cas Number:

6413-10-1

Molecular formula:

C8H14O4

IUPAC Name:

ethyl 2-(2-methyl-1,3-dioxolan-2-yl)acetate

Details on test material:

- Name of test material (as cited in study report): Methyl Dioxolan
- Appearance: colourless liquid
- Storage condition of test material: room temperature protected from light

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Crl CD®(SD) IGS BR
- Age at study initiation: 10 weeks old (males); 9 weeks old (females)
- Weight at study initiation: 323 - 409 g (males); 204 - 277 g (females)
- Housing: Individually housed (except during mating) in polycarbonate cages with stainless steel lids and containing autoclaved sawdust.
- Diet: ad libitum
- Water: filtered tap water (0.22 µm filter), ad libitum.
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 °C
- Humidity (%): 50 ± 20 %
- Air changes (per hr): approximately 12 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

drinking water treated by reverse osmosis

Details on oral exposure:

PREPARATION AND ADMINISTRATION OF DOSING SOLUTIONS
Test material was formulated in vehicle at the required concentrations. The quantity of dose formulation administered to each animal was adjusted according to the most recently recorded body weight.
A constant dose volume of 10 mL/kg/day was used.
Control animals (group 1) received vehicle only.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of test material in the dose formulations was analysed by GC-FID.

- Instrumentation: Varian Gas Chromatography system with FID detector
- Chromatographic conditions:
Column: HP-ULTRA (Agilent) 50 m x 0.32 mm (0.52 µm)
Mobile phase: hydrogen
Sensitivity: 12
Column temperature: from 70°C to 300°C at 10°C/min
Constant flow rate: 2 mL/min
Split rate: 10
Software: Empower 2 (Waters)
Detector temperature: 300°C
Injector temperature: 250°C
Injected volume: 2 µL
Detector gas flows: 30 mL/min for make up; 30 mL/min for hydrogen and 300 µL/min for air
Retention time: 8.4 min (test material); 8.8 min (internal standard)

Standard solutions of test material were prepared. An internal standard (1,4 diisopropylbenzene) was used to correct the injection variability.

- Preparation of Standard Solutions
Stock solutions of test material in diluent were prepared at 0.2, 0.02 and 0.0015 mg/mL.

- Preparation of Internal Standard
A solution of 1,4-diisopropylbenzene in methanol was prepared at 10 µg/mL.

ANALYTICAL METHOD
1 mL of dose formulation was diluted with diluent to reach the nominal concentration for injection. The diluted samples were analysed by GC-FID, bracketed by standard solutions and quantified by the mean response factors calculated for the standard solutions. The internal standard was used to correct the injection variability.

ANALYSIS OF DOSE FORMULATIONS
The test material concentrations in the administered dose formulations analysed in weeks 1, 4 and 6 remained within an acceptable range of -4.4 to +8.69 % when compared to the nominal values. Test material was not detected in the control samples.

Duration of treatment / exposure:

Males received dose formulations for 2 weeks before pairing, during the 2 week pairing period, until sacrifice (at least 5 weeks in total). Females received dose formulations for 2 weeks before mating, during the 2 week pairing period, during gestation, and during lactation until day 5 post partum inclusive (or until sacrifice).

Frequency of treatment:

Daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0 (vehicle control), 100, 300, 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

10 males and 10 females per dose.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: the selected dose levels were based on results of previous studies 2 acute oral studies where the LD50 values were > 5000 mg/kg and a 2-week preliminary toxicity study, by the oral route, in rats in which no animals died at dose levels up to 1000 mg/kg bw/day, however, some clinical signs were noted.
- Rationale for animal assignment: during the acclimation period, the required number of animals was selected according to body weight and/or clinical condition. The animals were allocated to groups (by sex) according to a computerised stratification procedure based on body weight, so that the average body weight of each group was similar.
Therefore, 1000 mg/kg/day was selected as the high dose-level for this study. Mid and low dose-levels were selected in order to cover approximately 3-fold intervals.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: mortality and morbidity was checked once daily before the treatment period and at least twice a day during the treatment period. General clinical observations were performed once a day after treatment at approximately the same time.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the beginning of the treatment period and weekly thereafter, observations were performed on all animals outside the home cage in a standard arena.
- Observations included (but were not limited to): changes in fur, skin, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes (e.g. excessive grooming, repetitive circling) or bizarre behaviour (e.g. self-mutilation, walking backwards) were also recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: males were weighed on the first day of treatment and weekly thereafter until sacrifice. Females were weighed on the first day of treatment then weekly until mated (or until sacrifice) and on days 0, 7, 14 and 20 post coital and days 1 and 5 post partum.
Bodyweight gain was calculated for selected intervals and for the overall study.

FOOD CONSUMPTION: Yes
- Food consumption by each male was measured once a week, over a 7-day period, from the first day of treatment until the start of the pairing period. The measures coincided with body weight measurements.
The quantity of food consumed by each female was measured once a week, over a 7-day period, from the first day of treatment until the start of the pairing period, during pregnancy at the intervals days 0-7, 7-14 and 14-20 p.c. and during lactation for interval days 1-5 post partum.
During the pairing period, the food consumption was measured for neither males nor females.
Food intake per animal and per day was calculated by noting the difference between the food given and that remaining in the food-hopper at the next filling.

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of sacrifice
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (overnight)
- How many animals: the first five males and the first five females which delivered from each group
- Parameters examined included: erythrocytes, mean cell volume, packed cell volume, haemoglobin, mean cell haemoglobin concentration, mean cell haemoglobin, thrombocytes, leucocytes, differential white cell count with cell morphology, neutrophils, eosinophils, basophils, lymphocytes and large unstained cells, monocytes, reticulocytes, prothrombin time, fibrinogen and activated partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of sacrifice
- Animals fasted: Yes (overnight)
- How many animals: the first five males and the first five females which delivered from each group
- Parameters examined included: sodium, potassium, chloride, calcium, inorganic phosphorus, glucose, urea, creatinine, total bilirubin, total cholesterol, triglycerides, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, total proteins, albumin, albumin/globulin ratio, bile acids.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once at the end of the treatment period (for females this was performed on day 5 post partum, after the sacrifice of the pups).
- Dose groups that were examined: the first five males and the first five females which delivered from each group.
- Reactivity tests included: touch response, forelimb grip strength, pupillary reflex, visual stimulus response, auditory startle reflex, tail pinch response, righting reflex, landing foot splay and rectal temperature.
- Motor activity assessment included: horizontal movements and rearing, measured once by automated infra-red sensor equipment over a 60-minute period.
- Detailed observations inculded: "touch escape" or ease of removal from the cage. During handling: fur appearance, salivation, lacrimation, piloerection, exophthalmos, reactivity to handling, pupil size (presence of myosis or mydriasis). In the standard arena (2-minute recording): grooming, palpebral closure, defecation, urination, tremors, twitches, tonic and clonic convulsions, gait, arousal (hypo- and hyper-activity), posture, stereotypy, behaviour, breathing, ataxia and hypotonia.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
A complete macroscopic examination was performed. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, the thoracic, abdominal and pelvic cavities/muscles with their associated organs and tissues and the neck with its associated organs and tissues. All gross lesions were recorded and preserved.

ORGAN WEIGHTS: Yes
The wet weights of the following were recorded: adrenals, brain, epidiymides, heart, kidneys, liver, ovaries, prostate, seminal vesicles, spleen, testes, thymus, thyroids with parathyroids and uterus.
The ratio of organ weight to body weight (recorded immediately before sacrifice) was calculated.

HISTOPATHOLOGY: Yes
All tissues listed below were from the first five sacrificed as scheduled males and the first five females which delivered and were sacrificed on day 6 post partum of the control and high groups (groups 1 and 4) and for the female sacrificed prematurely:
adrenals, brain, cecum, colon, duodenum, epididymides, esophagus, gut-associated lymphoid tissue, heart, ileum, jejunum, kidneys, liver, lungs with bronchi, lymph nodes, ovaries, prostate, rectum, sciatic nerves, seminal vesicles, spinal cord, spleen, sternum with marrow bone, stomach with forestomach, testes, thymus, thyroids with parathyroids, trachea, urinary bladder, uterus and vagina.
Microscopic examination was also performed on all lesions of all the animals of the low- and intermediate-dose groups sacrificed on completion of the treatment period.

Other examinations:

Pup examinations and reproductive parameters were performed as part of the reproductive and developmental toxicity screen.

Statistics:

Citox software was used to perform the statistical analyses of haematology and blood biochemistry data. The other data were compared by one-way analysis of variances and Dunnett test (mean values being considered as normally distributed, variances being considered as homogenous) or by Fischer exact probability test (proportions). PathData software (version 6.2d2) was used to perform the statistical analysis of organ weight data (level of significance: 0.05 or 0.01).

PathData software (version 6.2d2) was used to perform the statistical analysis of organ weight data (level of significance: 0.05 or 0.01).

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:

not examined

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

not examined

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:

not specified

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no unscheduled deaths that could be attributed to treatment during the study.
In the 100 mg/kg/day group, one female was prematurely sacrificed on day 0 post coitum due to its poor clinical conditions (piloerection, round back, swollen on left forelimb and chromorhinorrhea). At necropsy, a pouch was observed in the subcutaneous tissue of the left axillary region with a brown thick content which correlated histologically with acute inflammation. Inflammation with hemorrhage was seen in the larynx and particularly in surrounding tissues with edema and hemorrhage. There was no histological correlation with the perforation of the larynx noted at necropsy. Inflammation in tissues adjacent to the thymus was also observed. Additional changes consisted of a moderate hemorrhage in the left adrenal which correlated with a red discoloration at necropsy. Moderate increased apoptotic cell numbers was seen in the thymus along with decreased periarteriolar lymphoid sheaths (PALS) in the spleen. These observations in the lymphoid tissue were considered to be non specific, probably stress related. All these changes were suggestive of a gavage trauma due to the technical procedures (gavage error).
Ptyalism (hypersalivation) was recorded in males receiving 300 mg/kg/day (2/10 males) and in most males and females dosed at 1000 mg/kg/day during the premating period (9/10 males and 5/10 females) and in all females during the gestation and lactation period. This finding was considered to be related to the test material and a sign of discomfort but not an adverse effect.
A treatment-related effect was considered to be unlikely for the few other clinical signs as they are commonly observed in this species and strain or observed with no dose-level relationship.

BODY WEIGHT AND WEIGHT GAIN
There were no treatment-related effects in mean body weights and mean body weight changes.

FOOD CONSUMPTION
There were no biologically significant effects on mean food consumption associated with treatment with the test material.

HAEMATOLOGY
There were no treatment-related findings on haematology parameters.

CLINICAL CHEMISTRY
There were no treatment-related findings on blood biochemistry parameters.

NEUROBEHAVIOUR
There were no treatment-related findings recorded during the Functional Observation Battery tests. Furthermore, there were no differences between the test material-treated and control groups in terms of motor activity (horizontal movements and rearing).

ORGAN WEIGHTS
There were no changes in the mean organ weights which were suggestive of a test material-related effect. The small differences observed between groups were considered to reflect normal variations and were not considered to be due to treatment with the test material.

GROSS PATHOLOGY
There were no changes at necropsy which were indicative of a test material-related effect. Any changes observed were considered to be part of the normal background commonly seen in rats.

HISTOPATHOLOGY
There were no changes at histopathological examination which were suggestive of a test material-related effect.
In the mandibular lymph nodes, presence of sinusal red blood cells was seen at a higher incidence in females given 1000 mg/kg/day than in controls. As this observation is commonly seen in rats, was observed unilaterally in 3/5 treated females and in the absence of a similar trend in males, any relationship with the test material was excluded.
Tubular basophilia was seen in the kidneys of 2/5 females given 1000 mg/kg/day but not in controls. As this observation was focal and unilateral, in the absence of a similar trend in males, any relationship with the test material was excluded.
Other changes observed were considered to be part of the normal background commonly seen in rats and without relationship with the test material.
Careful examination of testes, epididymides and ovaries did not reveal any treatment related changes.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment-related effects were observed.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1: Mean Body Weight and Body Weight Change

Sex	Male				Female
Dose level (mg/kg/day)	0	100	300	1000	0	100	300	1000
Pre-mating
Day 1	372	371	372	370	236	232	235	236
Day 15	413	414	421	416	255	257	254	258
Day 36	449	465	470	464
Days 1-15	+41	+43	+49	+46	+19	+25	+19	+22
Days 15-36	+36	+52	+48	+48
Days 1-36	+77	+94	+98	+93
Gestation
Day 0 p.c.					258	262	264	262
Day 20 p.c.					410	419	414	423
Days 0-20 p.c.					+152	+158	+150	+161
Lactation
Day 1 p.p.					309	318	317	316
Day 5 p.p.					329	337	343	337
Days 1-5 p.p.					+20	+19	+26	+21

p.c = post coitum; p.p. = post partum

Applicant's summary and conclusion

Conclusions:

Under the conditions of the study the No Observed Adverse Effect Level (NOAEL) was determined to be 1000 mg/kg bw/day based on the absence of treatment related findings at this dose level.

Executive summary:

The repeated dose toxicity of the test material was determined in a study which was conducted under GLP conditions and in accordance with the standardised guideline OECD 422.

During the study groups of 10 male and 10 female animals received daily doses of test material at dose levels of 0 (vehicle control), 100, 300 or 1000 mg/kg bw/day for 2 weeks before mating, during mating and, for the females, throughout gestation until day 5 post partum. During the study animals were observed for mortality and clinical effects. Bodyweights and food consumption were recorded and functional observational battery, blood biochemistry and haematology parameters were examined. At necropsy, gross pathological examinations were performed on all animals. Organ weights were recorded and a histopathological examination was performed on five males and five females in each group.

There were no unscheduled deaths that could be attributed to treatment during the study. In the 100 mg/kg/day group, one female was prematurely sacrificed on day 0 post coitum due to its poor clinical conditions which were considered to be due to a gavage trauma. There were no treatment-related effects on body weight, food consumption, functional observation battery, blood biochemistry or haematology parameters. Furthermore, there were no treatment-related findings noted at necropsy.

Therefore, under the conditions of the study the No Observed Adverse Effect Level (NOAEL) was determined to be 1000 mg/kg bw/day, the highest dose tested.