Reaction mass of isopropyl dihydrogen phosphate and phosphoric acid and propan-2-ol

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1991

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: publication with acceptable reporting

Data source

Reference

Reference Type:

publication

Title:

Isopropanol 13-week vapor inhalation study in rats and mice with neurotoxicity evaluation in rats

Author:

Burleigh-Flayer HD, Gill MW, Strother DE, Masten LW, McKee RH, Tyler TR, Gardiner T

Year:

1994

Bibliographic source:

Fundam. Appl. Toxicol. 23:421-428

Report date:

1994

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

no

GLP compliance:

not specified

Test material

Test material information

Constituent 1

Reference substance name:

Propan-2-ol

EC Number:

200-661-7

EC Name:

Propan-2-ol

Cas Number:

67-63-0

Molecular formula:

C3H8O

IUPAC Name:

propan-2-ol

Details on test material:

The substance was identified by chemical name and CAS number.

Test animals

Species:

other: rat and mouse

Strain:

other: Fischer 344 rats and CD®-1 mice

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Source: Fischer 344 rats from Harlan Sprague-Dawley, Inc. (Indianapolis, IN); CD®-1 mice from Charles River Breeding Laboratories, Inc. (Portage, MI).
- Age at study initiation: rats: 8-12 weeks; mice: 8-10 weeks
- Weight at study initiation: male rats: 140-165 g; female rats: 112-130 g; male mice: 25-37 g; female mice: 19-26 g
- Fasting period before study: not reported
- Housing: individually in stainless steel, wire-mesh cages (15 cm x 22 cm x 18 cm for rats; 22.5 cm x 9.5 cm x 12.5 cm for mice); during exposure, animals were individually housed, separated by sex and exposure group, in stainless steel, wire-mesh cages (14.5 cm x 9.5 cm x 17.5 cm or 17.5 cm x 12 cm x 18 cm for rats, and 7.5 cm x 9.5 cm x 17.5 cm for mice).
- Diet (e.g. ad libitum): powdered food (Certified Rodent Chow 5002, Ralston Purina Co., St. Louis, MO) ad libitum
- Water (e.g. ad libitum): water (Municipal Authority of Westmoreland County, Greensburg, PA) ad libitum
- Acclimation period: animals were acclimated to the exposure chambers (air-only exposure) on 2 days prior to the initiation of the exposure regimen


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-24 °C
- Humidity (%): 52-56 %
- Air changes (per hr): 13.5 per hr (in inhalation chamber)
- Photoperiod (hrs dark / hrs light): 12 hrs dark/12 hrs light

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

other: no vehicle

Details on inhalation exposure:

The volume of each chamber was approximately 1330 liters, and the airflow was approximately 300 liters/minute (13.5 air changes per hour).
Temperature and relative humidity measurements were recorded at least 12 times per exposure.

Animals were acclimated to the exposure chambers (air-only exposure) for 2 days prior to the initiation of the exposure regimen. Target isopropanol vapor concentrations of 0 (control), 100, 500, 1500, and 5000 ppm were selected for this study. The rats (55 days of age) and mice (56 days of age) were exposed for 6 hours per day, 5 days per week for 13 weeks. During the 14th week, male and female rats (excluding those animals designated for neuroanatomic pathology evaluation) received 2 and 3 consecutive days of exposure, respectively. The 10 female rats of the 500, 1500, and 5000 ppm group designated for neuroanatomic pathology evaluation were exposed for 1 day during the 14th week; the male rats of the 500, 1500, and 5000 ppm group designated for neuroanatomic pathology evaluation were not exposed during the 14th week. Male and female mice received 4 and 5 consecutive days of exposure during the 14th week, respectively. Control (air-only exposure) animals were handled in an identical manner as the isopropanol-exposed animals.

Liquid isopropanol was metered from a piston pump (Fluid Metering, Inc., Oyster Bay, NY) into a glass evaporator similar in design to that described by Snellings and Dodd (1990). Piston pumps were equipped with a 1/8'' piston (G-6 FMI pump), 1/4" piston (C-6 FMI pump), 3/8" piston (C-6 FMI pump) and 1/4" piston (G-20 FMI pump) for the 100, 500, 1500 and 5000 ppm exposure chambers, respectively. The temperature in the evaporator was maintained at a level sufficient to vaporize the test substance

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chamber concentrations of isopropanol vapor were analyzed by flame ionization gas chromatography. At least 2 samples per hour were obtained from each exposure chamber and also the control chamber.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

6 hours per day, 5 days per week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 ppm (nominal)

Doses / concentrations 2

Dose / conc.:

500 ppm (nominal)

Doses / concentrations 3

Dose / conc.:

1 500 ppm (nominal)

Doses / concentrations 4

Dose / conc.:

5 000 ppm (nominal)

No. of animals per sex per dose:

All exposure groups (except for the 100 ppm group) consisted of 10 rats/sex. An additional 15 rats/sex were assigned to the 0, 500, 1500, and 5000 ppm groups for assessment of neurobehavioral function.

Control animals:

yes, sham-exposed

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: during exposures, observations were recorded on a group basis. Preceding and following each exposure, animals were examined individually, and observations were recorded for each animal exhibiting overt clinical signs


BODY WEIGHT: Yes
- Time schedule for examinations: All rats and mice were weighed on Week 0 (prior to the start of exposures). These values were used as the preexposure reference weights and were subtracted from each subsequent weight to determine the change in body weight. Animals were weighed weekly during the study, at neurobehavioral evaluations, and immediately preceding sacrifice.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Weekly. For rats, food consumption was determined over a 7-day period. For mice, food consumption was measured over a 6-day period rather than a 7-day period due to excessive food spillage during the first 24 hours after the containers were filled with fresh food

WATER CONSUMPTION: Yes
- Time schedule for examinations: Weekly.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: During the 6th week of the study in 10 rats/sex/group. At sacrifice in 10 rats/sex/group and 10 mice/sex/group.
- Anaesthetic used for blood collection: Yes (methoxyflurane)
- Animals fasted: Yes
- How many animals: 10 rats/sex/group during the 6th week; 10 rats/sex/group and 10 mice/sex/group at sacrifice

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at sacrifice
- Animals fasted: Yes
- How many animals: 10 rats/sex/group and 10 mice/sex/group

NEUROBEHAVIOURAL EXAMINATION: Yes (rats only)
- Time schedule for examinations: 10/15 rats selected were evaluated with the functional observational battery (FOB) prior to the first exposure and on the weekend following Weeks 1, 2, 4, 9, and 13. Motor activity evaluations were conducted on all 15 rats per sex selected from the 0, 500, 1500, and 5000 ppm groups prior to the first exposure and on the weekend following Weeks 4, 9, and 13.
- Dose groups that were examined: 15 rats per sex from each group

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Organ Weights: The brain, liver, lungs, kidneys, adrenals, testes (males), and ovaries (females) from all surviving animals (except animals designated for neuroanatomic pathology evaluation) were weighed at sacrifice.

Statistics:

The data for continuous, parametric variables were intercompared for the exposure and control groups by use of Levene's test for homogeneity of variances, by analysis of variance, and by t-tests. The t-tests were used, if the analysis of variance was significant, to delineate which groups differed from the control group. If Levene's test indicated homogeneous variances, the groups were compared by an analysis of variance for equal variances followed, when appropriate, by pooled variance t-tests. If Levene's test indicated heterogeneous variances, the groups were compared by an analysis of variance for unequal variance followed, when appropriate, by separate variance t-tests. Frequency data, such as microscopic diagnoses, were compared using Fisher's exact test. All statistical tests, except the frequency comparisons, were performed using BMDP Statistical Software (Dixon, 1985). The frequency data tests are described in Biometry (Sokal and Rohlf, 1969). The probability value of p < 0.05 (two-tailed) was used as the critical level of significance for all tests.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not examined

Ophthalmological findings:

effects observed, treatment-related

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Effect levels

Key result

Dose descriptor:

NOAEC

Effect level:

5 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Remarks on result:

other: Based on elevated liver weight and hyaline droplets in kidneys. However, these effects are unspecific and a clear NOAEC is not derivable

Target system / organ toxicity

Key result

Critical effects observed:

not specified

Organ:

kidney

liver

Any other information on results incl. tables

- Clinical signs:

In rats, clinical signs observed following exposures included swollen periocular tissue (females) at the highest dose and perinasal encrustation (males) at 500 ppm and above.

- Behaviour:

Narcosis was observed in a few animals of both species during exposure to 5000 ppm and possibly 1500 ppm as well. However, the animals became tolerant to the narcotic effects of isopropanol after week 2. No neurobehavioral changes were observed in any parameters of the functional observational battery. However, increased motor activity was noted at week 9 of exposure in female rats of the 5000 ppm group.

- Body weight/body weight gain:

After an initial drop in body weight gain in the first week of exposure at the high dose (5000 ppm), rats in the 1500 and 5000 ppm groups had significant increases in body weight gain and/or body weight throughout most of the exposure period. But only the 5000 ppm group had greater than 10% body weight gain compared to controls. Increases in body weight and body weight gain greater than 10% were also noted in female mice in the 5000 ppm group.

- Haematology/clinical chemistry:

Consistent clinical pathology changes included an increase in mean corpuscular volume (rats; female mice) and mean corpuscular hemoglobin (male rats; female mice) at the 5000 ppm exposure level. Other changes noted include a slight anemia in rats at week 6 only and a slight dehydration in female mice at the end of the study.

- Organ weight:

Relative liver weight in rats was elevated no more than 8% in the 5000 ppm groups. However, a 10 and 21% increase in relative liver weight was observed in female mice at 1500 and 5000 ppm, respectively.

Macroscopy:

No gross lesions were observed in any organs.

- Histopathology:

The only microscopic change observed was hyaline droplets within kidneys of all male rats. This change was not clearly concentration related, although it was most pronounced in the 5000 ppm group.

Applicant's summary and conclusion

Conclusions:

Based on the observed effects after inhalation exposure to 110, 500, 1500 or 5000 ppm isopropanol for 13 weeks no clear NOAEC is derivable. However, since effects on liver and kidneys of unclear relevance are reported the NOAEC might be set at 5000 ppm.

Executive summary:

Repeated exposure to isopropanol for 13 weeks produced effects at the highest concentration (5000 ppm) and a kidney change of unknown biological signifiance. Clinical signs of toxicity on the central nervous system (including ataxia, narcosis, ) are acute effects and not relevant for limit value determination for repeated dose systemic effects. Increases in absolute body weight and body weight gain, and changes in hematology parameters in animals exposed to 1500 and 5000 ppm of isopropanol, increased relative liver weight in rats exposed to 5000 ppm, as well as increased motor activity for female rats in the 5000 ppm group have been observed.