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Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 August 2012 - 11 January 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant study conducted in accordance with OECD guidelines.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.8 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
not specified
Details on test material:
- Name of test material (as cited in study report): Bis(2-ethoxyethyl)ether EC 203-963-7 (DEDG)
- Substance type: Organic
- Physical state: Colourless liquid
- Analytical purity: 99.95%
- Lot/batch No.: 2E09WA
- Expiration date of the lot/batch: 31 December 2012
- Storage condition of test material: In a refrigerator (2 to 8 degC)

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 53 to 60 days
- Weight at study initiation: 259 to 322 g for males and 190 to 250 g for females
- Fasting period before study:
- Housing: Animals were housed inside a restricted access rodent facility. The facility was designed and operated to minimise the entry of external biological and chemical agents and to minimise the transference of such agents between rooms. Before the study the room was cleaned and disinfected.
The animals were housed five of one sex per cage. The cages were made of a polycarbonate body with a stainless steel mesh lid. Softwood bark-free fibre was used as bedding and was sterilised by autoclaving and changed at appropriate intervals each week. Cages, food
hoppers and water bottles were changed at appropriate intervals.
- Diet (e.g. ad libitum): The animals were allowed free access to a standard rodent diet (Rat and Mouse No. 1
Maintenance Diet), except overnight before routine blood sampling and during exposure. This diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
- Water (e.g. ad libitum): Potable water taken from the public supply was freely available via polycarbonate bottles fitted with sipper tubes, except during exposure.
- Acclimation period: 11 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 23
- Humidity (%): 40-70
- Air changes (per hr): Each animal room was supplied with filtered fresh air, which was passed to atmosphere and not re-circulated.
- Photoperiod (hrs dark / hrs light):12/12

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Each exposure system consisted of a stainless steel concentric jet atomiser and a snout only ADG exposure chamber. Different doses of DEDG were achieved by varying the concentrations of the test article in the system whilst keeping the duration of exposure constant.
- System of generating particulates/aerosols: Aerosol produced by a stainless steel concentric jet atomiser A narrow bore needle was used
The atomiser was mounted vertically down into the top of the prechamber
- Temperature: The chamber temperatures were similar for each group on each day of the study. The observed temperatures for all groups remained within the normally accepted range for inhalation exposure of rats, as per OECD Guidelines for the Testing of Chemicals Number 412 (22±3°C)
- Air flow rate: Inlet: From in-house compressed air system – breathing quality
Atomiser – 8 L/minute
Diluent – 10L/min
- Extract Airflow:
Drawn by in-house vacuum system
Filtered locally
Flow – 20 L/minute




Duration of treatment / exposure:
4 weeks
Frequency of treatment:
6 hours a day, 5 days a week
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0.25 mg/L
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0.8 mg/L
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
2.5 mg/L
Basis:
nominal conc.
No. of animals per sex per dose:
10
Control animals:
yes
Details on study design:
- Dose selection rationale: The exposure concentrations (0, 0.25, 0.80 or 2.5 mg/L) were selected following completion of a one week inhalation dose range finding study in rats (Huntingdon Life Sciences Study Number: OOE0008), in which no effects were noted at a target exposure concentration of
2.5 mg/L.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
Pre-exposure observation
During exposure however, observation severely restricted due to tube restraint
As each animal is returned to its home cage
As late as possible in the working day

In addition, a more detailed weekly physical examination was performed on each animal to monitor general health.
During the acclimatisation period, observations of the animals and their cages were recorded at least once per day.

BODY WEIGHT: Yes
- Time schedule for examinations: Twice weekly throughout the treatment and on the day of necropsy

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data but the weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded for the week before treatment started (Week -1), and each week throughout the
treatment period.

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: No data

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: During Week 4 of treatment (before dosing), blood samples were obtained from all animals
after overnight withdrawal of food. Animals were held under light general anaesthesia induced by isoflurane and blood samples were withdrawn from the sublingual vein.
- Anaesthetic used for blood collection: Yes (identity) / No / No data
- Animals fasted: Yes (overnight)
- How many animals: all study animals

CLINICAL CHEMISTRY: Yes
At the same time and using the same animals as for peripheral haematology, further blood samples (nominally 0.7 mL) were collected into tubes containing lithium heparin as anticoagulant. All tubes were mechanically agitated for at least two minutes and the sample subsequently centrifuged at 2000 g for 10 minutes in order to separate the plasma. After separation, the plasma was examined in respect of:
Using a Roche P Modular Analyser:
Alkaline phosphatase (ALP)
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Gamma-glutamyl transpeptidase (gGT)
Total bilirubin (Bili)
Urea
Creatinine (Creat)
Glucose (Gluc)
Total cholesterol (Chol)
Triglycerides (Trig)
Sodium (Na)
Potassium (K)
Chloride (Cl)
Calcium (Ca)
Inorganic phosphorus (Phos)
Total protein (Total Prot)
Albumin (Alb)


URINALYSIS: No data

NEUROBEHAVIOURAL EXAMINATION: No data


Other examinations:
sperm analysis and sperm morphology

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
blood chemistry
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Details on results:
CLINICAL SIGNS AND MORTALITY
There were no unscheduled deaths on this study.
There were no treatment-related clinical signs observed during the weekly detailed physical examination.

BODY WEIGHT AND WEIGHT GAIN
Bodyweight gain of males exposed to 2.49 mg/L was lower than the concurrent controls over the 4 weeks of treatment (Days 1-29, 0.85X Control). Review of the individual twice weekly bodyweights, however, did not show any consistent effects and as there were no similar effects on the females and the difference was not statistically significant; the lower bodyweight gain in males is considered unrelated to DEDG.
There were no effects of DEDG in males exposed to 0.257 or 0.686 mg/L or any females given DEDG.

FOOD CONSUMPTION
There were no effects of DEDG on food consumption.

HAEMATOLOGY, BONE MARROW
There were no treatment-related effects on the cellularity and composition of the marrow or the myeloid to erythroid ratio.

BLOOD CHEMISTRY
Group mean cholesterol and triglyceride levels were higher than the concurrent controls in all treated male groups. A similar effect was not apparent in females and there was no dose response. There were no corroborating pathological changes to explain these higher levels,
and as there were no similar effects in females and no dose response, these intergroup differences are considered to be of no toxicological significance.
There were no treatment-related effects on the other blood chemistry parameters investigated.

ORGAN WEIGHTS
Group mean adrenal weights (adjusted for bodyweight) of males exposed to 2.49 mg/L were statistically significantly higher than the concurrent control. There was no similar effect in females. Pathological changes in the adrenals correlated with the increased weights, but the adrenal changes were considered likely to be related to the stress of restraint during the inhalation procedure, unlikely to be related to treatment and not adverse.

Group mean liver weight (adjusted for bodyweight) of males exposed to 2.49 mg/L was statistically significantly higher than the concurrent control, but as there was intergroup variability in this parameter, no changes noted for females, and no corroborating pathological changes in the liver this change is considered to be unrelated to treatment.
Other intergroup differences seen were small and largely inconsistent between groups and sexes. Therefore, these were considered to be due to individual variation in results and not to be related to treatment

GROSS PATHOLOGY
The macroscopic examination performed after 4 weeks of treatment revealed no intergroup differences of note.
The nature and incidence of all findings were consistent with the commonly seen background of macroscopic changes in Sprague Dawley rats at these laboratories

HISTOPATHOLOGY: NON-NEOPLASTIC
No findings were seen that were clearly related to treatment with DEDG.
Minimal or slight generalised hypertrophy of the zona fasciculata was seen in three male animals exposed to 2.49 mg/L. This finding correlated with the higher bodyweight-adjusted group mean adrenal weight, compared with male controls, for males exposed to 2.49 mg/L.

In the right testis, seminiferous tubular vacuolation and or cellular debris, and in the right epididymis, degenerative spermatogenic cells and or cellular debris in the ducts, were seen at higher incidences in control males than in males exposed to 2.49 mg/L. Therefore, these
findings were not related to treatment, but were considered possibly related to the stress associated with the inhalation procedure (Dodd, 1999; Lee, 1993) and correlated with the atypical values reported at sperm analysis for control animals.



Effect levels

Dose descriptor:
NOAEL
Effect level:
2.49
Based on:
test mat.
Sex:
male/female

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

There was a very low level of DEDG detected at analysis of the samples collected from the control group. In 18/23 occasions the identified peak was below the limit of detection, and in the other 5 samples was very low (approximately 25% that of the low dose levels). It was established that there was a low level of DEDG vapour within the animal room, which could have contaminated the external surfaces of the sampler and possibly absorbed into the trapping solvent used in analysis. Animals of the control group were exposed to clean external fresh air in a sealed system and were considered to have no direct contact with this low level of contamination, and this is considered to have no impact on the study integrity.

Applicant's summary and conclusion

Conclusions:
DEDG was administered to Sprague Dawley rats by snout only inhalation exposure for 6 hours a day, 5 days a week for 4 weeks at estimated exposure concentrations of 0.257, 0.686 or 2.49 mg/L.
No adverse treatment related effects were apparent in any of the parameters investigated and DEDG was well tolerated over the 4 week treatment period.
The No Observed Adverse Effect Level (NOAEL) was 2.49 mg/L.