Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: 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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report date:
1998

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
no
Remarks:
Conducted according to the guideline in effect at the time of study conduct.
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
trans-dichloroethylene
EC Number:
205-860-2
EC Name:
trans-dichloroethylene
Cas Number:
156-60-5
Molecular formula:
C2H2Cl2
IUPAC Name:
arsenic
Details on test material:
- Purity: 99.86%

Test animals

Species:
rat
Strain:
other: Crl:CD®(SD) BR
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 7 weeks
- Housing: Rats were housed (sexes separate) in suspended, stainless steel, wire-mesh cages. During quarantine and the majority of the pretest, rats were housed 3 per cage. Upon grouping and during the test period, rats were housed individually.
- Diet: PMI Nutrition International, Inc. Certified Rodent LabDiet® 5002, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature: 23 ± 1 °C
- Humidity: 50 ± 10%
- Photoperiod: 12-hour light/12-hour dark

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
other: Nitrogen and filtered/conditioned air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The exposure chambers (NYU style) were constructed of stainless steel and glass and had a nominal internal volume of 1.4 m3
- Method of holding animals in test chamber: wire-mesh cages
- Source and rate of air: Nitrogen and filtered/conditioned air
- System of generating vapour: Atmospheres were generated by metering liquid test substance into a heated glass Instatherm flask with either a Fluid Metering Inc. pump or a Hamilton Syringe Drive. Nitrogen, introduced into the flask, swept the test substance vapour into the air supply duct to the exposure chamber.
- Temperature, humidity, pressure in air chamber: Mean temperatures were 25°C; mean relative humidities were 51 to 57%; mean airflows were 280 to 310 L/min; pressure not reported
- Air flow rate: approximately 300 L/min
- Air change rate: least 12 air changes per hour
- Treatment of exhaust air: atmospheres were exhausted into the main plenum exhaust system and emitted into the atmosphere as allowed by permit

TEST ATMOSPHERE
- Brief description of analytical method used: The atmospheric concentration of the test substance was determined by gas chromatography at approximately 15-minute intervals during each exposure. Samples were taken starting 20 to 60 minutes after the start of exposures. Chamber atmosphere samples were directly injected into a Hewlett Packard Model 6890 Gas Chromatograph equipped with stream-selection and gas sampling valves and a flame ionization detector. All samples were chromatographed isothermally at 115°C on a 30 m, Restek 502.2, 530 µm capillary column.
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The mean analytically determined concentrations of the test substance over the 67 exposures were as targeted: 200, 1000, and 4000 ppm (2 significant figures) with standard errors of 0.48, 1.3, and 4.7, respectively. Daily mean concentrations were consistent throughout the study and were generally well within 20% of the nominal concentrations determined from the test item amounts used and the chamber airflows. Nominal concentrations for the low-, mid-, and high concentration chambers ranged from 200 - 240 ppm, 950 - 1100 ppm, and 3700- 4600 ppm, respectively. No test item was found in the control chamber.
Duration of treatment / exposure:
90 days
Frequency of treatment:
6 hours/day, 5 days/week
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 200, 1000, and 4000 ppm
Basis:
nominal conc.
No. of animals per sex per dose:
Standard toxicological evaluations: 15 rats/sex/concentration
Cell proliferation evaluation: 15 rats/sex/concentration
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Exposure concentrations were based on toxicology information available in the scientific literature and on a developmental toxicity study conducted at the test facility.

Examinations

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice a week

BODY WEIGHT: Yes
- Time schedule for examinations: twice a week

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined group was determined weekly

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: study start and at 90 days

HAEMATOLOGY: Yes
- Time schedule for collection of blood: days 48 or 49 and 93 or 94
- Anaesthetic used for blood collection: Yes, light carbon dioxide
- Animals fasted: Yes
- How many animals: 10 male and 10 female rats per group
- Parameters checked in table [No.1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: days 48 or 49 and 93 or 94
- Animals fasted: Yes
- How many animals: 10 male and 10 female rats per group
- Parameters checked in table [No.2] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: days 48 or 49 and 93 or 94
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes
- Parameters checked in table [No.2] were examined.

NEUROBEHAVIOURAL EXAMINATION: No

OTHER:
- The alerting response to an auditory stimulus was checked 3 times (approximately every 2 hours) during each exposure. Rats were also checked for the alerting response (in all but 4 exposures) immediately after exposure prior to being removed from the chambers.

- On test days 9, 99, and 135 (males), or test days 9, 98, and 134 (females) 5 rats per sex per group were sacrificed for liver cell proliferation evaluations.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table No. 3)

HISTOPATHOLOGY: Yes (see table No. 3)

Ten rats per group were sacrificed by carbon dioxide anesthesia and exsanguination and necropsied after the last exposure. After an approximately 1 -month recovery period, the remaining 5 rats per group were sacrificed and evaluated in the same way. All tissues were fixed in 10% neutral buffered formalin except for eyes, testes, and epididymides which were fixed in Bouin’s solution. All tissues from rats control (0 ppm) and high (4000 ppm) dose groups and rats found dead (FD), sacrificed in extremis (SE), or accidentally killed (AK), were microscopically examined (except male mammary gland).

For low (200 ppm) and intermediate (1000 ppm) groups, nose, pharynx/larynx, lungs, liver, kidneys, heart, prostate, testes, epididymides, seminal vesicles, female mammary gland, ovaries, uterus, and vagina were microscopically examined. Since no treatment-related lesions were found in the high dose groups, tissues from rats in the recovery groups were saved but not microscopically examined. Processed tissues were embedded in paraffin, cut at a nominal thickness of five micrometers, stained with hematoxylin and eosin (H&E), and examined with a light microscope.
Other examinations:
Samples for hepatic cell proliferation were collected after approximately 1 week (test day 9; 1-week timepoint) and 90 days (test days 98 or 99; 90-day timepoint) of test substance administration. At each timepoint, 5 previously designated rats from each group were implanted (subcutaneously) with Alzet@ osmotic pumps filled with 20 mg/mL 5-bromo-2’deoxyuridine (BrdU) dissolved in a 0.5 N sodium bicarbonate buffer. Three days after implantation, on test day 9 and 99 for males and on test days 9 and 98 for females, the rats were sacrificed by carbon dioxide anesthesia and exsanguination. The liver and duodenum were collected and fixed in neutral buffered formalin. Liver and duodenum were processed for immunohistochemical analysis of BrdU incorporation into DNA. The hepatic labeling indices were evaluated for the control and high-concentration groups. The duodenum of each rat was examined as a control to assess BrdU delivery and to ensure proper tissue processing. Labeling indices were not determined for duodenal tissue.
Statistics:
Mean body weights and body weight gains were statistically analysed with a 1-way analysis of variance (ANOVA). Pairwise comparisons between test and control groups (sexes separate) were made with the Dunnett's test. Increases in the incidence of clinical observations of toxicity were evaluated by the Cochran Armitage trend test at the discretion of the study director. For clinical laboratory data, ANOVA and Bartlett's test were calculated for each sampling time. Dunnett's test was used to compare means from the control groups and each of the groups exposed to the test item. When the results of the Bartlett's test were significant, the Kruskal-Wallis test was employed and the Mann-Whitney U test was used to compare means from the control groups and each of the groups exposed to the test item. Mean final body weights and mean absolute and relative (to body and to brain) organ weights were analyzed with ANOVA. When the value of the F-statistic for differences among groups was significant, pairwise comparisons between treated and control groups were made with Dunnett's test. Bartlett's test was used to test for homogeneity of variances. Final body weights shown in tables were used for determination of organ to body weight ratios. These body weights, determined just prior to necropsy, were not used to assess the effects of the test substance on body weights (in-life data was used for this purpose). Cell proliferation data were analysed with ANOVA. Bartlett's test was used to test for homogeneity of variances. Except for the Bartlett's test (p < 0.005), all significance was judged at p < 0.05.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
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:
no effects observed
Details on results:
HAEMATOLOGY:
No toxicologically important haematology changes occurred during this study. Some statistically significant haematology findings occurred in males and females, but they were not considered to be toxicologically important for the following reasons:

* Significantly decreased mean haemoglobin concentration and haematocrit in the 1000 and 4000 ppm male groups at the 45-day sampling time were not considered to be compound related because the mean values were not part of a relevant dose-response relationship. Also, similar changes did not occur at the 90-day sampling time.
* In the 4000 ppm male group, significantly decreased mean WBC and lymphocyte count at the 45-day sampling time and significantly decreased mean lymphocyte count at the 90-day sampling time were not considered to be toxicologically important because the magnitude of the changes was small in the context of historical controls and therefore, biologically inconsequential. Furthermore, the causal relationship between treatment and the leukocytic alterations is somewhat equivocal. In rodents, a common cause of decreased lymphocyte counts is increased release of endogenous glucocorticoids. This is a secondary effect associated with "stress" in which endogenous glucocorticoids cause redistribution of lymphocytes from the circulation into lymphoid tissue. This type of stress response has been observed in other inhalation studies at this laboratory and elsewhere.
* Significantly decreased mean monocyte count in the 4000 ppm female group at the 45-day sampling time was not considered to be toxicologically important because the magnitude of the change was small in the context of historical controls and therefore, biologically inconsequential. Also, a similar change did not occur at the 90-day sampling time.


CLINICAL CHEMISTRY:
No toxicologically important clinical chemistry changes occurred during this study. Some statistically significant clinical chemistry findings occurred in males and females, but they were not considered to be toxicologically important for the following reasons:

* Significant decreases in liver enzymes (AST in males and SDH in males and females) were not toxicologically important because decreases in serum enzyme activity are not relevant to liver injury or dysfunction. Conversely it is increases in serum enzyme activity that may indicate liver toxicity. Likewise, significantly decreased mean bilirubin concentration in females was not toxicologically important for the same reason.
* Significantly increased mean alkaline phosphatase in the 200 ppm female group at the 45-day sampling time was not compound related because the mean value was not part of a relevant dose-response relationship.
* Significant decreases in mean albumin concentration in all treated groups of males at the 45-day sampling time were not considered to be compound related because the mean values did not exhibit a relevant dose-response relationship. Also, similar changes did not occur at the 90-day sampling time.
* Significantly decreased mean albumin concentration in 200 ppm females at the 45-day sampling time and significantly increased mean globulin concentration in 200 ppm females at the 90-day sampling time were not compound related because the mean values were not part of a relevant dose-response relationship.
* Significantly increased mean glucose concentrations in 4000 ppm males at both sampling times and in 4000 ppm females at the 90-day sampling time were not considered to be toxicologically important because the magnitude of the changes was small and therefore, biologically inconsequential. These mild changes in glucose concentration may have been due to the aforementioned stress response.
* Significantly increased mean creatinine concentration in 200 ppm females at the 90-day sampling time was not compound related because the mean value was not part of a relevant dose-response relationship.
* Significantly decreased mean calcium concentration in the 4000 ppm female group at the 45-day sampling time was not considered to be toxicologically important because the magnitude of the change was small and therefore, biologically inconsequential. Also, a similar change did not occur at the 90-day sampling time.
* Significantly increased mean chloride concentrations in 4000 ppm males at both sampling times were not considered to be toxicologically important because the magnitude of the changes was small and therefore, biologically inconsequential.
* Significant increases in sodium and chloride in the 200 ppm female group were not compound related because the mean values were not part of a relevant dose-response relationship.

ORGAN WEIGHTS:
* Dunn's multiple comparison test was used to compare the mean recovery control male relative (to body weight) adrenal gland weight with the recovery 1000 ppm male relative (to body weight) adrenal gland weight. The latter's mean relative adrenal weight was statistically significantly lower than controls. This difference was considered spurious as there was no dose response and there were no adrenal weight differences between 0-day control and treated rats adrenals and no adrenal microscopic changes at that time period.

CELL PROLIFERATION: No compound-related increase in hepatocyte labeling index was detected in male and female rats at either sampling time. Therefore, the test substance did not induce cell proliferation under the conditions of this study.

Effect levels

Dose descriptor:
NOAEC
Effect level:
>= 4 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest concentration tested

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
This study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability).
Male NOAEC: 4000 ppm (highest concentration tested)
Female NOAEC: 4000 ppm (highest concentration tested)
Executive summary:

The toxicity of the test substance was evaluated in Crl:CD®(SD) BR male and female rats exposed to analytically determined mean concentrations of 0, 200, 1000, or 4000 ppm for 6 hours/day, 5 days/week over a 90-day period. 

There were no adverse, compound-related effects on body weight, clinical observations, body weight gain, food consumption, clinical or anatomical pathology parameters, or liver cell proliferation. The no observed-effect concentration for this study was 4000 ppm, the highest concentration tested in both male and female rats.