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Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Equivalent to Guideline study This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labelling and/or risk assessment.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1986
Report Date:
1986

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 452 (Chronic Toxicity Studies)
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Purity: 99.98%

Test animals

Species:
rat
Strain:
other: Crl:CD(R)(SD)BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Labs, Kingston, New York
- Age at study initiation: weanling rats
- Weight at study initiation: not reported
- Fasting period before study: none
- Housing: stainless steel, wire-mesh cages. Housed 3/cage upon arrival
- Diet (e.g. ad libitum): Purina Laboratory Chow Checkers #5001 (PLCC) available ad libitum except during exposures
- Water (e.g. ad libitum): ad libitum except during exposures
- Acclimation period: 12 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24±2°C
- Humidity (%): 50% ±10%
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): not reported

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
other: air
Details on inhalation exposure:
Four similarly constructed non test substance reactive inhalation chambers were dedicated to this study. They were operated in a single pass mode, slight negative pressure and cleaned at the end of each exposure day. Chamber concentrations of the test substance were regulated by controlling the flow rate of test substance vapors into each chamber.

Test substance vapors were generated by warming cylinders containing liquefied test substance in a 21-27°C water bath. The vapors were metered into the intake manifold at the top of the chamber. Filtered, conditioned air also entered the top of the chamber, swept the test material into the respective exposure chambers and was exhausted out the bottom of the chambers. Chamber concentrations of the test substance were regulated by controlling the flow rate of test substance vapors into each exposure chamber. Filtered air, alone, was metered in a similar manner into the control chamber. Total flow of air in the control and test substance chambers was maintained at approximately 800 L/min.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chamber atmospheres were analyzed by a GC every half hour during the 6 hour daily exposure period.
Duration of treatment / exposure:
2 years
Frequency of treatment:
6 hours a day/ five days a week (excluding holidays)
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 0.2, 1.0, 2.5 %
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0, 0.21, 1.02, 2.47 %
Basis:
analytical conc.
No. of animals per sex per dose:
100
Control animals:
yes
Details on study design:
The study was designed to evaluate the potential chronic toxicity and oncogenicity of the test substance in male and female rats when exposed by inhalation. This route was chosen because it most appropriately simulates potential human exposure.

Examinations

Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Moribund, dead rats, abnormal behavior and appearance / twice a day.

BODY WEIGHT: Rats weighed weekly- first 3 months/ twice a month for the remainder of the exposure.

FOOD CONSUMPTION: No

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: 3, 6, 9, 12, 18 months after the initiation of the study, evaluations were conducted. See Table 1 for parameters examined

CLINICAL CHEMISTRY: 3, 6, 9, 12, 18 months after the initiation of the study, evaluations were conducted. See Table 2 for parameters examined

URINALYSIS: 3, 6, 9, 12, 18 months after the initiation of the study, evaluations were conducted. See Table 3 for parameters examined
Sacrifice and pathology:
GROSS PATHOLOGY: evaluations on 10 rats/sex/exposure group after 6, 12, 18 months and all rats alive after 2 years. See Table 4
HISTOPATHOLOGY: evaluations on 10 rats/sex/exposure group after 6, 12, 18 months and all rats alive after 2 years. See Table 4
Other examinations:
Male and female rats were sampled between 606 and 653 days on test for the presence of antibodies to Kilham Rat Virus (KRV). This sampling was performed because KRV was detected in rats from a separate study that shared the same hallway. Antibodies to KRV were detected in 12 of 24 study rats that were sampled. The 12 antibody-positive rats were distributed evenly across all groups. Since the virus was present in all groups and since the adverse effects of the virus are exhibited primarily in reproductive efficiency in young rats, the interpretation of results from this study were not affected by the presence of KRV.
Statistics:
A one-way analysis of variance and the least significant difference test (LSD) were used to test statistical differences between groups in body weight and weight gain. Organ weight data were evaluated by using a one-way analysis of variance and Bartlett's test. Pair-wise comparisons were made by using the LSD and/or Dunnett's test. Clinical laboratory data were evaluated by a partially nested and crossed analysis of variance and by the LSD test.

The incidences of clinical observations and gross and histopathological lesions were compared to control group incidences by the Fisher's Exact test. A Bonferroni correction was applied to the comparison of clinical observations. Survival probability was generated by the Kaplan-Meier procedure and analyzed by using the Tarone test.

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):
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:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
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
Histopathological findings: neoplastic:
no effects observed
Details on results:
Body weight and Survival:
Body weights were greater and survival rates were less than the control group for male rats in the intermediate and high-exposure group.

Clinical Observations:
The incidences of stained wet inguinal/perineal area in male rats in the groups exposed to test substance vapors were greater than the control group. Since increases were observed in male rats in all exposure groups and since these increases were not dose-related, the significance of this observation was not clear. Decreases in the incidences of swollen testes and in the incidences of swollen paws in male rats were not considered to be compound related. The incidences of torn ears for both male and female rats in the intermediate and high-exposure groups were greater than in the corresponding control groups. Torn ears may have been the consequence of tearing the ear punches used to identify the rats in this study. The double punching of one ear in high-exposure group rats, and the single punching of both ears in the intermediate exposure group may have lead to an increase in the incidence of torn ears.

Clinical laboratory measurements:
Compound-related hematologic or clinical chemical effects were not observed for male rats exposed to DME vapors for 2 years. The statistically significant decreases in erythrocyte count and hematocrit observed in male rats in the intermediate and high-exposure groups at the 6-month sampling time were interpreted to be evidence of in vitro hemolysis that was not compound-related. This conclusion was based on concurrent increases in the MCH and MCHC. Other statistically significant changes in hematologic or clinical chemical parameters were within the range of expected biological variation and were interpreted not to be of biological significance.

At 3 months on test, there was evidence of a minimal compound-related hemolytic effect (decreases in erythrocyte and hematocrit) together with evidence of in vitro hemolysis (increased MCHC) for females in the high-exposure group. The decreases in erythrocyte count and hematocrit that were observed in the intermediate- and high-exposure groups at the 6 month sampling were interpreted not to be compound related but due to in vitro hemolysis. This interpretation was based on concomitant increases in MCH and MCHC values. Other statistically significant changes for female rats in hematologic or clinical chemical parameters that were measured during the study were within the range of expected biological variation and were interpreted not to be biologically significant or compound related.

The observed hemolytic effects seen in males and females were interpreted to be transient effects that were not representative of the long-term effects of the test substance.

Organ Weights:
The only consistent organ weight change was an increase in the absolute spleen weights of male rats in the high exposure group at the 6- and 12-month sacrifices. The changes in relative liver weights in female rats were not consistent; at the 12-month sacrifice, relative liver weights in the intermediate and high-exposure groups were less than the control group, and at the 18-month sacrifice, relative liver weight was greater than the control group and occurred only in the high-exposure group. All other changes in absolute or relative organ weights were considered transient or within the range of biological variation and therefore were not compound related.

Histopathology: Non-neoplastic
The only test substance-related histologic effect was slight splenic congestion in male rats in the high-exposure group observed at the 6-month sacrifice. This splenic observation was consistent with the increased spleen weight observed at 6 months.

No consistent histologic lesion was observed that would explain the decreased survival rate of male rats in the intermediate and high-exposure groups. The greater body weights in these groups could have been a contributing factor in the increased mortality. Although the test substance is known to have anesthetic properties that could lead to deceased physical activity and a consequent increase in body weight, clinical observations indicated normal physical activity of the rats in all groups. Therefore, increases in body weight along with the decrease in survival may be circumstantial and unrelated as to cause and effect.

Histopathology: Neoplastic
Results are reported in Section 7.7

Effect levels

Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
2.5 other: %
Sex:
male/female

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

The authors report a NOAEL for the study of 0.2% based on an increase in body weight and a decrease in survival in male rats exposed to 1.0 and 2.5% test substance vapors and on hemolytic effects noted in male rats exposed to 2.5% test substance vapors for six months. However, body weight gain (male only) was occasionally higher than controls in the early phases of the study, but this finding was not consistent throughout the study. Further, the observation in increased body weight was not seen in any other of the subchronic or repeated inhalation studies involving the test substance. While there was a statistically significant decrease in survivability (males only) at the termination of the 2-yr study, no impact on survivability was observed throughout the chronic toxicity portion of the study (12 months). There were no pathological, clinical or clinical chemistry effects observed that would support a conclusion that there was a substance-related reduction in survival. The study authors did not consider the possibility that the statistical difference in survivability at the 2-yr point is due to the high survival rate of the controls in this study, and that comparison with historical control values may have led to the dismissal of the effects. Finally, the observed hemolytic effects seen in males and females were interpreted to be transient effects that were not representative of the long-term effects of the test substance.

Based on careful consideration of the chronic portion of this study and the supporting information of several subchronic studies involving multiple species, the repeated inhalation NOAEL is determined to be 2.5% (47106 mg/m3). 

Applicant's summary and conclusion

Conclusions:
The NOAEL = 2.5%

The study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability).
Executive summary:

Groups of 100 male and female rats were exposed for 6 hours/day, 5 days/week (excluding holidays) for up to two years to atmospheres containing either 0 (control), 0.2, 1.0, or 2.5% test substance vapors. Clinical chemical and hematological measurements were performed on 10 rats/sex/exposure group after approximately 3, 6, 9, 12, and 18 months of exposure. Gross and histopathological evaluations were conducted on 10 rats/sex/exposure group after 6, 12, or 18 months of exposure, on all rats alive after 24 months of exposure, and on all rats found dead or in a moribund condition, tissue integrity permitting.

Body weights were greater and survival rates were less than the control group for male rats in the intermediate- and high-exposure groups. No clear association could be made between body weight increases and decreased survival even though these changes were concurrent observations in the same exposure groups. No histological lesion was found that would explain the decrease in survival rate.

A compound-related hemolytic effect was observed in male rats in the high-exposure group at six months on test. This effect was characterized by a decrease in erythrocyte count, increase in spleen weight, histological evidence of splenic congestion, along with normal bone marrow histology. A decrease in erythrocyte count was also observed in female rats in the high-exposure group at three months that was considered compound related. These changes were interpreted to be transient effects that were not representative to the long term effects of the test substance.

The authors report a NOAEL for the study of 0.2% based on an increase in body weight and a decrease in survival in male rats exposed to 1.0 and 2.5% test substance vapors and on hemolytic effects noted in male rats exposed to 2.5% test substance vapors for six months. However, body weight gain (male only) was occasionally higher than controls in the early phases of the study, but this finding was not consistent throughout the study. Further, the observation in increased body weight was not seen in any other of the subchronic or repeated inhalation studies involving the test substance. While there was a statistically significant decrease in survivability (males only) at the termination of the 2-yr study, no impact on survivability was observed throughout the chronic toxicity portion of the study (12 months). There were no pathological, clinical or clinical chemistry effects observed that would support a conclusion that there was a substance-related reduction in survival. The study authors did not consider the possibility that the statistical difference in survivability at the 2-yr point is due to the high survival rate of the controls in this study, and that comparison with historical control values may have led to the dismissal of the effects. Finally, the observed hemolytic effects seen in males and females were interpreted to be transient effects that were not representative of the long-term effects of the test substance.

Based on careful consideration of the chronic portion of this study and the supporting information of several subchronic studies involving multiple species, the repeated inhalation NOAEL is determined to be 2.5% (47106 mg/m3).