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Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
1996
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study without detailed documentation.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
publication
Title:
Subchronic and developmental toxicity studies of vaporised diisopropyl ether in rats
Author:
Dalbey W & Feuston M
Year:
1996
Bibliographic source:
Journal of Toxicology and Environmental Health, 1996, 49, 29-43

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
yes
Remarks:
-Some observations/parameters were not reported for certain organs; food and water consumption not reported
GLP compliance:
no
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): diisopropyl ether, DIPE, isopropyl ether.
- Physical state: colourless liquid
- Analytical purity: commercial grade, approximately 92% purity
- Storage condition of test material: refrigerated with nitrogen blanket

Test animals

Species:
rat
Strain:
other: Sprague-Dawley-derived rats [Tac:N(SD)fBR]
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Single shipment from Taconic Farms, German-town, N.Y.
- Age at study initiation: approximately 8 weeks
- Housing: Individually housed in 1-cubic meter (H-1000) inhalation chambers; untreated control animals were housed in a separate animal room in the same caging.
- Diet: ad libitum, except during exposure; certified Purina rodent chow 5002
- Water: ad libitum, except during exposure
- Acclimation period: 2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-22 °C
- Humidity (%): 40-60%
- Photoperiod (hrs dark / hrs light): 12h/12h

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
other: unchanged (no vehicle)
Remarks on MMAD:
MMAD / GSD: Not applicable
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: three 1 cubic meter, H1000 exposure chambers
- Source and rate of air: Filtered room air combined with air stream containing diisopropyl ether generate by evaporation from a heated (46 °C) wick. The wick surrounded a stainless steel tube and the wick and tube were enclosed in a 5.08 cm glass pipe through which HEPA-filtered room air flowed at approximately 290 Ipm.
- Temperature, humidity, pressure in air chamber: 23-24 °C, 60-67%, monitored every 30 min
- Air change rate: at least 12 per hour
- Treatment of exhaust air: filtered through charcoal beds

TEST ATMOSPHERE
- Brief description of analytical method used: 50-250 µL samples injected directly to a Hewlett-Packard 5880A gas chromatograph (GC) with flame ionization detector (FID) and fused silica column (30 m, 0.53 mm ID, 1 µm film). The helium carrier flow was 5 mL/min with 27 mL/min makeup air; oven temperature at 40 °C for 10 minutes followed by a ramp at 4 °C/min to 200 °C. The GC calibration was done with known volumes vapourised in 500 mL glass sampling flask. In addition, periodic samples were taken and analysed by GC/ mass spectroscopy (MS) with mass spectrum scanned from 35 to 200 amu.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
480 ppm: 2000±200 mg/cubic meter, 480±48 ppm; Total hydrocarbons: 2100±200 mg/cubic meter (95±4 weight% diisopropyl ether)
3300 ppm: 13800±900 mg/cubic meter, 3300±215 ppm; Total hydrocarbons: 14900±1100 mg/cubic meter (92±2 weight% diisopropyl ether)
7100 ppm: 29700±1600 mg/cubic meter, 7100±383 ppm; Total hydrocarbons: 32600±2000 mg/cubic meter (91±2 weight% diisopropyl ether)

In GC/MS analysis the sample comprised approximately 86-87% diisopropyl ether, with the remaining 13-14% comprising of more than 20 alkanes, alkenes, cycloalkanes, ketones, alcohols in the range C3-C9; a trace amount of toluene was also present.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
6-hour exposure per day, 5 exposure days per week
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
480 ppm (2000 mg/cubic meter)
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
3300 ppm (13800 mg/cubic meter)
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
7100 ppm (29700 mg/cubic meter)
Basis:
nominal conc.
No. of animals per sex per dose:
14 animals per sex per dose
Control animals:
yes, concurrent no treatment
yes, sham-exposed
Details on study design:
- Dose selection rationale: Review of available literature data on toxicity. Due to safety considerations, the upper dose was selected to be approximately half the lower explosive limit of the substance.
- Untreated control remained in a separate animal room. Apart from routine animal care, body weights and clinical observations, they were not handled.
- Sham control were treated the same as the diisopropyl ether group except actual exposure.
Positive control:
No positive control was used.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily except weekends

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily except weekends

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: No

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at sacrifice
- Anaesthetic used for blood collection: yes; light ether
- Animals fasted: yes; after last exposure, fasted overnight before sampling
- How many animals: all
- Parameters checked: white blood cell (WBC), red blood cell (RBC), hemoglobin (Hb), hematocrit (Hct), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelets, and differential cell count.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at sacrifice
- Animals fasted: yes; after last exposure, fasted overnight before sampling
- How many animals: all
- Parameters checked: glucose, urea nitrogen, total protein, globulin (G), albumin (A), A:G ratio, sorbital dehydrogenase, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, creatinine, cholesterol, total bilirubin, triglycerides, uric acid, potassium (K), sodium (Na), chloride (Cl), calcium (Ca), and phosphorus (P) using Hitachi blood analyser 704.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
The animals were sacrificed over several days during the 14th week. Exposures and control animals were continued during this period.
GROSS PATHOLOGY: Yes
The following organs were examined and weighed when present: adrenals, kidney, spleen, brain, liver, testes, epididymides, ovaris, thymus, heart, prostate, and uterus. In addition, the weight of the right middle lung lobe was measured after drying at 90°C.

HISTOPATHOLOGY: Yes
The following tissues, from sham and high dose groups, were examined and preserved in buffered 10% formalin: adrenals ovaries, sternum, pancreas, brain (three sections), salivary gland, eye, optic nerve, spleen, heart, stomach, colon, testes, duodenum, thymus, kidneys, thyroid, liver, lymph nodes (tracheobronchial, anterior mediastinal, cervical), lung (left lobe), nasal turbinates, thigh muscle, skin (six sites), urinary bladder, sciatic nerve, seminal vesicals, preputial glands, and gross lesions.
The following tissues, from untreated and remaining groups, were examined and preserved in buffered 10% formalin: lungs, tracheobronchial lymph nodes, and gross lesions.
Finally, the male 14600 mg/cubic meter group also had slides prepared for liver and kidneys.
Other examinations:
Testis and associated tissues were preserved in 10% formalin for all groups.
From the two control groups and the high dose group, 10 animals were selected and the left cauda epididymis examined for sperm morphology and number. The left testis was used for weight and spermatid number.
Methodology: morphology (Wyrobek, A.J., Mutat. Res., 1983, 115, 1-72), number of sperm and spermatids (Blazak, W.F., Fundam. Appl. Toxicol., 1985, 5, 1097-1103)
Statistics:
-ANOVA and Tukey's studentized range test: Serum chemistry and counts of testicular spermatids, epididymal sperm
-Duncan's multiple range test: Hematology, body weights, organ weights

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
No mortality was observed, nor changes in any clinical signs.

BODY WEIGHT AND WEIGHT GAIN
Exposed males tended to have greater weight gain in the initial half of the study, compared to the controls. However, this was only statistically significant in weeks 6-14 for the mid-dose (3300 ppm) group compared to untreated controls. No similar trend was observed in females.

HAEMATOLOGY
Only a few parameters were statistically different, and only in one control group but not both; no observed changes were considered biologically relevant. See Table 1 for results.

CLINICAL CHEMISTRY
Only a few parameters were statistically different, and only in one control group but not both; no observed changes were considered biologically relevant. However, statistically significant elevation in comparison to both control groups was seen in the high-dose (7100 ppm) male group (this group also showed the largest liver weight increase). See Table 1 for results.

ORGAN WEIGHTS
Significant liver-weight increase was observed in both male and female animals (39% and 18% respectively) at the high-dose (7100 ppm). At the middle-dose (3300 ppm) only the increase in males (26%) was significant compared to both controls; the increase observed in females (6%) was only significant compared to sham-exposed animals.
Significant kidney-weight increase was observed in males at the high- (7100 ppm) and mid-dose (3300 ppm). The increase observed in females at the high-dose (7100 ppm) was only significant compared to sham-exposed animals.
Weight increase tended to be concentration related.

GROSS PATHOLOGY
No gross pathological changes associated with exposure to diisopropyl ether were observed.

HISTOPATHOLOGY: NON-NEOPLASTIC
Mild hypertrophy was observed in liver cells from central to mid-zonal areas for the male high-dose (7100 ppm) group. Similar changes in morphology were not observed in females, or either sex at the mid-dose (3300 ppm). It was suggested that hypertrophy was not visible with light microscopy until bulk-tissue weight changes were greater than ~30%.
In the kidney there was a mild increase in hyaline droplets observed in the proximal convoluted tubles of males in the high-dose (7100 ppm) group. No similar changes were observed in any other group.
No other histopathological changes were observed in other tissues.

HISTORICAL CONTROL DATA
Percentage of abnormal sperm: 2.8-5.6%

OTHER FINDINGS
The numbers of sperm and spermatids were not changed by exposure to diisopropyl ether compared to the control groups.
At the high-dose (7100 ppm), the number (5.3%) of abnormal sperm (altered head morphology or broken) was significantly increased compared to the control groups (2.8%). However, no specific abnormality was predominant, and the result was not considered biologically significant.

Effect levels

open allclose all
Dose descriptor:
NOAEC
Effect level:
3 300 ppm
Sex:
male/female
Basis for effect level:
other: Effect concentration identified by registrant (minor changes in liver and kidney weights without discernable morphological effects at 3300 ppm; at 7100 ppm, these increased weights were accompanied by small morphologic changes).
Dose descriptor:
NOEC
Effect level:
480 ppm
Sex:
male/female
Basis for effect level:
other: See above remark

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1. Mean parameters ( ±SD) of hematology and serum chemistry that had significant differences between exposed and control groups following subchronic exposures.

Parameters

Untreated controls

Sham-exposed

480 ppm DIPE

3300 ppm DIPE

7100 ppm DIPE

Males

Creatinine (mg/dL)

0.61 ± 0.06

0.64 ± 0.04

0.64 ± 0.04

0.67 ± 0.06

0.69 ± 0.03b

Cholesterol (mg/dL)

71 ± 10

74 ± 13

77 ± 17

77 ± 9

95 ± 22d

SDH (IU/L)

11 ± 5

16 ± 7

13 ± 6

9 ± 3c

9 ± 3c

Lymphocytesa

92 ± 3

92 ± 4

90 ± 6

90 ± 4

87 ± 6b

Monocytesa

1 ± 2

1 ± 2

2 ± 2

2 ± 2

3 ± 2b

Females

Potassium (mmol/L)

4.96 ± 0.35

4.68 ± 0.24

4.58 ± 0.41

4.51 ± 0.37b

4.45 ± 0.40b

Lymphocytesa

92 ± 3

88 ± 5

86 ± 6b

85 ± 7b

86 ± 3b

apercent of total white blood cells.

bSignificantly different from untreated controls.

cSignificantly different from sham-exposed controls.

dSignificantly different from both control groups.

Applicant's summary and conclusion

Conclusions:
Mild hypertrophy was observed at the high-dose in combination with 39% (male) and 18% (female) increased absolute liver weight. The high dose of 7100 ppm was considered to be the LOAEC effect level.
At the mid-dose, no histopathological changes were observed. Absolute liver weight gain was 26% in males only; 6% in females. Sorbitol dehydrogenase dropped from 11(5) or 16(7) IU/L in controls to 9(3) IU/L. The absolute liver weight gain seen only in males, and unaccompanied by other effects, is considered for the purposes of read-across to diethyl ether to be a suitable NOAEC.
Based on "adverse liver effects" criteria by TERA (Toxicology Excellence for Risk Assessment): presence of histopathology (moderate hypertrophy) in combination with statistically significant absolute or relative weight changes; or liver weight change >10%; or doubling of serum levels of liver enzyme activity.