Reaction mass of 2-methylpentane and Hexanol, branched and linear and diisopropyl ether

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1996

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Cross-reference

Reason / purpose for cross-reference:

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

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

Test material information

Constituent 1

Reference substance name:

Diisopropyl ether

EC Number:

203-560-6

EC Name:

Diisopropyl ether

Cas Number:

108-20-3

Molecular formula:

C6H14O

IUPAC Name:

Diisopropylether

Test material form:

other: liquid

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: refridgerated with nitrogen blanket

Test animals

Species:

rat

Strain:

other: Sprague-Dawley-derived rats [Tac:N(SD)fBR]

Sex:

male/female

Details on test animals or test system 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 mininutes 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 / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
480 ppm (2000 mg/cubic meter)
Basis:
nominal conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
3300 ppm (13800 mg/cubic meter)
Basis:
nominal conc.

Doses / concentrations 3

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 nerce, 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% respectivels) 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

1

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 discernible morphological effects at 3300 ppm; at 7100 ppm, these increased weights were accompanied by small morphologic changes).

2

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 ± 0.03^b
Cholesterol (mg/dL)	71 ± 10	74 ± 13	77 ± 17	77 ± 9	95 ± 22^d
SDH (IU/L)	11 ± 5	16 ± 7	13 ± 6	9 ± 3^c	9 ± 3^c
Lymphocytes^a	92 ± 3	92 ± 4	90 ± 6	90 ± 4	87 ± 6^b
Monocytes^a	1 ± 2	1 ± 2	2 ± 2	2 ± 2	3 ± 2^b
Females
Potassium (mmol/L)	4.96 ± 0.35	4.68 ± 0.24	4.58 ± 0.41	4.51 ± 0.37^b	4.45 ± 0.40^b
Lymphocytes^a	92 ± 3	88 ± 5	86 ± 6^b	85 ± 7^b	86 ± 3^b

^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:

Subchronic exposure to 3300 ppm diisopropyl ether lead to only minor weight changes in liver and kidneys. Subchronic exposure to 7100 ppm diisopropyl ether lead to increased weight changes, accompanied by small morphological changes. Diisopropyl ether demonstrated relatively low toxicity for the endpoints evaluated. Therefore, the NOAEC was set to 3300 ppm and the NOEC to 480 ppm.

Executive summary:

Read across from diisopropylether (DIPE) is justified on the basis that NOAECs reported for the constituents of this UVCB range from 3500 mg/m³ for hexanol to 31680 mg/m³ for propylene dimers, which can be considered indicative of low toxicity for this UVCB substance. DIPE comprises approximately 50% of the UVCB substance and is, therefore, the main constituent. The C6 alkanes/alkenes are, with 20%, the second largest constituent. The NOAECs of both DIPE and C6 alkanes/alkenes, together accounting for approximately 70% of the UVCB substance, are in the same order of magnitude (3300 ppm and 3000 ppm, respectively).

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 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.