Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1993
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study without detailed documentation

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report Date:
1993

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Version / remarks:
1981 followed; reliability scoring based on 2009 guideline.
Deviations:
yes
Remarks:
-see below
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Isopropyl chloride, IPC.
- Physical state: Solvent.
- Analytical purity: 97%.
- Storage condition of test material: Stored in glass bottles at approximately 4 °C, no apparent changes in the test substance during storage were observed.

Test animals

Species:
rat
Strain:
other: Sprague-Dawley rats (Crl:CDBR) SPF
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Wilmington, MA, U.S.A.
- Age at study initiation: 37 days at the start of the inhalation period.
- Weight at study initiation: mean body weight: 130 g (RSD: 5.7%) for males; 111 g (RSD: 5.7%) for females
- Fasting period before study: Not reported.
- Housing: 2 rats were housed together per wire mesh cage.
- Diet (e.g. ad libitum): A fortified, autoclaved pellet diet ("Maus/Ratte/Haster-Haltung" fortified diet), ad libitum.
- Water (e.g. ad libitum): Heat-treated (70°C) tap water, supplied from water bottles with sipper tubes, ad libitum.
- Acclimation period: 7 days.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21±0.5
- Humidity (%): 57± 6.1
- Air changes (per hr): 12 to 15
- Photoperiod (hrs dark / hrs light): 12/12

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 (vapour test)
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Chamber volume: 0.8L, 20 to 40 rats per chamber (individually held in 0.8L sub-chambers). See Attached file 1 (Figure 1) for the setup for test atmosphere generation and exposure.
- Method of holding animals in test chamber: Whole body exposure chamber (no details reported)
- Source and rate of air: Not reported.
- Method of conditioning air: Not reported.
- System of generating particulates/aerosols: The liquid test substance was vaporized at defined rates (at approximately 35°C).
- Temperature, humidity, pressure in air chamber:
Temperature: Temperature of the incoming air was 21.7±1.1°C and in the exposure chamber was 24.5±0.5°C.
Humidity: Relative humidity of the incoming air was 50.3±4.4% and within the chamber was 52.6±3.7%
Pressure: Not reported.
- Air flow rate: 160 L/minute through the exposure chamber
- Air change rate: 12 to 15 times/hour
- Method of particle size determination: Not applicable (vapour test)
- Treatment of exhaust air: Not reported.


TEST ATMOSPHERE
- Brief description of analytical method used: The concentration of the test substance in the exposure chambers was monitored continuously during exposure by a flame ionization detector (FID). The FID was calibrated by “off-line” CGC analysis of samples collected using adsorption tubes. Differences in the distribution of the test substance in the test atmosphere within the exposure chambers were less than 0.1%.
- Samples taken from breathing zone: Not reported.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
See below Table 1 Analysis of test substance and test atmosphere
Duration of treatment / exposure:
6 hours per day
Frequency of treatment:
7 days per week for 90 days
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
250, 500, and 1000 ppm
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
255, 492, and 996 ppm
Basis:
analytical conc.
No. of animals per sex per dose:
10 per sex per dose
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale: For the high concentration (1000 ppm), toxic effects were seen in a previous 90-day inhalation study (DOW Chemical Company, 1958). For 250 ppm, no toxic effects were reported after subchronic inhalation (Gage, 1970).
- Rationale for animal assignment (if not random): Random
- Rationale for selecting satellite groups: Random
- Post-exposure recovery period in satellite groups: 30 days (sham-exposed and 1000 ppm groups)
- Section schedule rationale (if not random): Not reported.
Positive control:
Positive control was not included.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
- Cage side observations checked: mortality, non-systemic observation of general condition and behavior

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: 1 time/week, according to check list, which included indicators of neurotoxicity (no details provided)

BODY WEIGHT: Yes
- Time schedule for examinations: Once weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

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 (however, results on food consumption relative to body weight commented within the results section of the study report)

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: 2 times (before start and at the end of the inhalation period
- Dose groups that were examined: 1st time: all rats; 2dn time: 5 male and female rats per group and all satellite groups.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Days 84 and 85
- Anaesthetic used for blood collection: Yes (diethylether)
- Animals fasted: No data
- How many animals: 5 male and 5 female preselected rats per group
- Parameters checked: hematocrit, hemoglobin concentration, number of erythrocytes, average volume of individual erythrocytes, average hemoglobin content per erythrocyte, mean corpuscular hemoglobin in the erythrocyte, number of leukocytes, differential blood cell count, number of thrombocytes.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Immediately prior to dissection
- Animals fasted: No
- How many animals: 5 male and 5 female preselected rats per group
- Parameters checked: calcium, inorganic phosphorus, chloride, sodium, potassium, alanine aminotransferase activity, gamma-glutamyltransferase activity, albumin, glucose, urea, creatinine, bilirubin, protein, triglyceride


URINALYSIS: Yes
- Time schedule for collection of urine: Day 78
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No data
- Parameters checked: volume, color, transparency, and specific gravity, pH, protein and glucose, nitrite presence, ketone body, bilirubin and urobilinogen, hemoglobin, erythrocytes, organized and unorganized components


NEUROBEHAVIOURAL EXAMINATION: No data
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (See Attached file 2)
HISTOPATHOLOGY: Yes (See Attached file 2)
Other examinations:
Larynx morphometry was conducted.
Statistics:
Continuous and ordinal data were analyzed according to the following statistics:
-descriptive statistics with mean, relative standard deviation, standard error, and number of values for body weight, hematology, clinical biochemistry, urine analysis, organ weights, and larynx morphometry;
-all the aforementioned statistical parameters, but standard deviation instead of standard error were used for IPC concentration, relative humidity, and temperature in exposure chambers;
-test of equality of means for continuous data by analysis of variance followed by Duncan test for body weight, hematology, clinical biochemistry, some urine analysis parameters, organ weights, and larynx morphometry;
-test of equality of samples for ordinal data by a generalized Cochran-Mantel-Haenszel test for some urine analysis parameters;
-test of equality of incidences for most histopathological kidney findings (intracytoplasmic eosinophilic granular inclusions in male rats, large intracytoplasmic eosinophilic inclusions in male rats, and dilated collecting ducts in female rats) of the sham-exposed group and the high concentration group by Fischer’s exact test.

All statistical tests were carried out at a nominal level of significance alpha=0.05 (two-tailed).

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):
effects observed, treatment-related
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
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 examined
Details on results:
250 (low-concentration), 500 (medium-concentration), and 1000 (high-concentration) ppm

CLINICAL SIGNS AND MORTALITY
One female rat (high-concentration group rat number 819) was found dead in exposure week 4. This was most probably due to technical reasons. The subsequent pathological examination did not reveal the cause of death.

There were no signs of neurotoxicity. Minor findings such as focal hair loss and focal skin lesions were observed in several rats of all groups. They may be related to the implantation of transponders used for animal identification.


BODY WEIGHT AND WEIGHT GAIN
The body weight of the male and female rats in all groups increased continuously during the inhalation period.

For the male rats, a comparison of the test substance-exposed groups to the sham-exposed group showed that the body weight of the rats in the high-concentration group was statistically significantly lower (8%) at the end of the inhalation period. This differences was not seen for the satellite groups during the inhalation and post-inhalation periods. While it is not clear in the data presented in the report, the study author indicated that at the start of the inhalation period there was statistically significant difference of 5% between the high test substance-exposed groups and the sham-exposed group despite randomization. For the absolute body weight increase compared to Day 1 as well as the relative body weight in crease compared to Day 1, no statistically significant differences were seen at the end of the inhalation period.

For the female rats, no test substance-related effect on body weight development was observed.

A slight test substance-related decrease in the body weight gain of the male rats in the high-concentration group cannot be excluded.

FOOD CONSUMPTION
For the male rats, during the entire course of the inhalation period, the food consumption per rat was slightly lower (approximately 8%) in the high-concentration group compared to the sham-exposed group. The food consumption relative to body weight showed no differences between the groups. During the post-inhalation period, no differences between the 2 satellite groups were observed for both end points.

For the female rats, no test substance-related effect on food consumption was observed.
A slight test substance-related decrease in food consumption for the male rats in the high-concentration group cannot be excluded.

FOOD EFFICIENCY
Refer to discussion on food consumption.

OPHTHALMOSCOPIC EXAMINATION
At the end of the inhalation period, no test substance-related morphological changes of the eyelid with conjunctiva, cornea, iris, lens, vitreous, and retina were observed. In 3 rats, age-related changes (remnants of hyaloid artery in vitreous) were observed.

HAEMATOLOGY
For the red blood picture: hematocrit, hemoglobin concentration, number of erythrocytes, average volume of individual erythrocytes (MCV), average hemoglobin content per erythrocyte (MCH), and mean corpuscular hemoglobin concentration in the erythrocyte (MCHC), no statistically significant differences were seen between the sham- and test substance-exposed groups. For each parameter, the differences in mean values for all groups were less than 10%.

For the white blood picture: number of leukocytes and thrombocytes, the mean values for the number of leukocytes for the male rats in all test substance-exposed groups showed an increase (up to 48%) over the sham-exposed group. This increase tended to be dose-dependent. However, it was not statistically significant, and in addition, in the histopathologically evaluated organs and tissues there were no findings that correlated to this increase. This finding is not considered to be test substance-related. The mean values for the female rats in the test substance-exposed groups showed no consistent increase for this parameter.

For the differential blood cell count, no statistically significant differences between the test substance and sham-exposed groups were seen. For the number of thrombocytes the same holds true.

The results of the hematological determinations for the sham-exposed rats were in accordance with the values given by the breeder for untreated Sprague-Dawley rats (Meingassner and Schmook, 1990).

In conclusion, for the hematological parameters determined, no test substance-related effects were observed.

CLINICAL CHEMISTRY
For the serum electrolyte concentrations determined, i.e., concentrations of calcium, inorganic phosphorus, chloride, sodium, and potassium, no statistically significant differences between the sham-exposed and test substance-exposed rats were seen. No dose-related effects were seen.

For the serum enzyme activities determined, i.e., alanine aminotransferase, aspartate aminotransferase, and gamma-glutamyltransferase, no test substance-related effects were seen. The following results not related to the test substance were observed: For alanine aminotransferase activity, a statistically significant difference was seen for male rats in the medium-concentration group compared to the sham-exposed group (approximately 29% higher). The difference can be attributed to the abnormally high activity of this enzyme (approximately 50% higher than normal) determined in a single rat of this group. For gamma- glutamyltransferase, it is known that the activity of this enzyme in serum of untreated rats is close to non-detectable (Meeks, 1989). This was the case in all groups in the present study, which resulted in high relative standard deviations (12 to 83%). Differences between the groups for this enzyme activity were not statistically significant and not considered to be biologically relevant.

The determination of glucose, urea, creatinine, bilirubin, and protein concentrations as well as of the amount of albumin after electrophoretic separation showed no significant differences between the sham-exposed and test substance-exposed rats. No dose-related trends were seen.

The results of the clinical chemistry determinations for the sham-exposed groups were in accordance with the values given by the breeder for untreated rats (Meingassner and Schmook, 1990).

In summary, no test substance-related effects for the clinical chemistry parameters were observed.

URINALYSIS
For all urine analysis parameters, no statistically significant differences were observed between sham-exposed and test substance-exposed rats with 1 exception which is considered to be a chance finding: in the medium test substance-exposed group there was a lower incidence of female rats with transitional epithelial cells in urine. No dose-related changes were observed.

In conclusion, no test substance-related effects for urinalysis parameters were observed.

ORGAN WEIGHTS
The absolute organ weights of the male rats of the high test substance-concentration groups were lower compared to the sham-exposed group, statistically significant differences being observed for the right and left kidneys. For the organ weights relative to body weight, these differences were not observed. The lower absolute organ weights of the male rats in the high-concentration group can be attributed to the lower body weight gain observed for this group. The absolute and relative weights of the adrenals were in general lower for the test substance-exposed groups compared to the sham-exposed group. As the variability of the individual results is high (RSD: 10 to 37%) and as there were no histopathological changes observed for the adrenals, this finding is not considered to be test substance-related.

For the female rats, no statistically significant differences between sham-exposed and test substance-exposed groups and no dose-related changes were observed for the organ weights.

In conclusion, no test substance-related effects for organ weights were observed.

GROSS PATHOLOGY
The following findings not related to test substance exposure were observed: enlarged mandibular and cervical lymph nodes with red dots (probably petechiae), enlarged spleen, thymus and lungs with red dots (probably petechiae), lungs with red dots (probably petechiae)/speckled/mottled, kidney with scarry depression, eschar on skin, and dilation of uterus horn. As these findings were observed in most cases also in the sham-exposed groups with an almost similar incidence, they are considered to be chance findings.

HISTOPATHOLOGY: NON-NEOPLASTIC
In the kidneys of male rats of the high test substance-concentration group, intracytoplasmic eosinophilic granular inclusions in some proximal convoluted tubules were observed at the end of the inhalation period; the incidence of rats with this finding was slightly higher (3/10) compared to the sham-exposed group (1/10). This finding was associated with or without dilated proximal convoluted basophilic tubules with eosinophilic material in the lumen. The severity of these granular inclusions were trace to minimal and affected only small groups of these tubules focally. The slightly higher incidence is not statistically significant. This finding was not observed in any of the rats at the end of the post-inhalation period.

In the renal cortical tubules of male rats of the high-concentration group, a second type of intracytoplasmic inclusion which appeared large, pale, and ovoid was observed at the end of the inhalation period; the incidence of rats with this focal finding in this group (4/10) was higher compared to the sham-exposed group (1/10). The slightly higher incidence is not statistically significant. This finding was associated with or without eosinophilic material in the lumen. The same finding was also observed in male rats of the low-concentration group (1/10). The second type of inclusion was present in male rats of the sham-exposed group (2/5) and of the high-concentration group (5/10) at the end of the post-inhalation period.

In the kidneys of female rats of the high-concentration group, an increased incidence (4/10) compared to the sham-exposed group (2/10) of dilated collecting ducts was observed at the end of the inhalation period. This slightly higher incidence is not statistically significant. This finding was not seen at the end of the post-inhalation period.

The eosinophilic granular inclusions observed are not considered to be test substance-related in view of the low severity and incidence as well as the lack of statistical significance. The large, pale, and ovoid intracytoplasmic inclusions are considered to be of no toxicological importance, because they are normally seen in untreated male control rats.

The dilated collecting ducts of the kidneys seen in female rats in the control and in the high test substance-concentration groups are considered to be incidental findings.

In the trachea of male and female rats, multifocal loss of epithelium was observed in all groups including the sham-exposed groups at the end of the inhalation period. It was also observed in both male and female rats of the sham-exposed groups as well as in female rats of the high-concentration group at the end of the post-inhalation period. This finding is considered to be aretefactual as no inflammatory reaction was seen.

In summary, there were no test substance-related findings observed in the liver, kidneys, brain, peripheral nerve, and in all other organs.


HISTORICAL CONTROL DATA (if applicable)
See comments above.

OTHER FINDINGS
For the male rats, a statistically significant increase (approximately 15%) in laryngeal epithelial thickness at the ventral depression was determined in the medium- and high-concentration groups compared to the sham-exposed group at the end of the inhalation period. At the ventral lumen, a statistically significant increase (approximately 15%) was determined in all 3 test substance concentration groups.

For the female rats, a statistically significant increase (10%) was determined at the ventral lumen only in the medium concentration group at the end of the inhalation period. It is considered to be a chance finding.

These statistically significant increases were within the range of the values obtained for the sham exposed rats on Days 90 and 120. In addition, all results obtained in the present study for this parameter lie well within the range of historical data for sham-exposed rats (INBIFO, unpublished data). Furthermore, there was no correlation with histopathological findings.

The slight increase in laryngeal epithelial thickness is not considered to be test substance-related.

Effect levels

open allclose all
Dose descriptor:
NOAEC
Effect level:
1 000 ppm (nominal)
Sex:
male/female
Basis for effect level:
other: A slight decrease in food consumption and body weight (approximately 8%) was observed at this dose level, but in the absence of any other test article-related findings, these changes are not considered adverse following a review of the data.
Dose descriptor:
NOEC
Effect level:
500 ppm (nominal)
Sex:
male/female
Basis for effect level:
other: No definitive toxic effects of test substance exposure at 500 ppm.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

No test substance-related mortality or changes in the general condition and behavior of the rats and in particular no signs of neurotoxicity were observed. For the ophthalmological, hematological, clinical chemistry, and urine parameters, there were no test substance-related findings nor were any revealed by the gross pathological examinations, organ weight determinations, histopathological examinations (including liver and kidneys), and determination of laryngeal epithelial thickness. A slight decrease in food consumption and in body weight gain was observed only for the male rats of the high test substance concentration group. An effect of the test substance with regard to these findings cannot be excluded. However, the decrease in body weight appears to be solely related to a decrease in food consumption as there were no changes in food efficiency reported.

Applicant's summary and conclusion