trans-dichloroethylene

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

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:

publication

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

study was 14 weeks

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

trans-dichloroethylene

EC Number:

205-860-2

EC Name:

trans-dichloroethylene

Cas Number:

156-60-5

Molecular formula:

C2H2Cl2

IUPAC Name:

(1E)-1,2-dichloroethene

Details on test material:

- Purity: ≥ 99%

Test animals

Species:

rat

Strain:

other: F344/N

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 7 weeks
- Weight at study initiation: 127-129 g (males); 110-111 g (females)
- Fasting period before study: Not reported
- Housing: 5 rats/cage
- Diet (e.g. ad libitum): NIH-07 open formula pelleted diet, ad libitum
- Water (e.g. ad libitum): Tap water, ad libitum
- Acclimation period: 14 or 15 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 72±3° F
- Humidity (%): 50% ± 15%
- Air changes (per hr): at least 10/hour
- Photoperiod (hrs dark / hrs light): 12 hours/day

Administration / exposure

Route of administration:

oral: feed

Vehicle:

other: food-grade, modified corn starch (CAPSUL®) and reagent-grade sucrose (80:20)

Details on oral exposure:

The dose formulations were prepared at least every 2 weeks by mixing microencapsulated test substance with feed; placebo microcapsules were added to maintain a starch matrix concentration of 6% in the diet. Formulations were stored in doubled plastic bags, protected from light, at 5°C for up to 4 weeks.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity and stability studies of 0.5% test substance formulations were conducted by the analytical chemistry laboratory using GC. Homogeneity was confirmed. The trans-1,2-dichloroethylene formulation prepared with lot 343-1A had losses of 22.0% for samples stored for 7 days in the dark and 15.5% for samples stored for 1 day at room temperature, open to air and light; dose formulations prepared with lots 343-10TA, -11TA, and -12TA (not used in the current studies) and lot 343-1B-A were stable for 4 days when stored at room temperature, open to air and light. Additional analyses performed with GC with a 0.5% dose formulation prepared with lot 343-1B-A confirmed stability for 7 days for samples stored in a rat cage, open to air and light, at up to 50% humidity.
The study laboratory conducted homogeneity studies of 0.7% and 11.5% test substance formulations with GC. Prior to the 14-week studies, the study laboratory also performed homogeneity studies of 3125 and 50000 ppm dose formulations and stability studies of a 3125 ppm dose formulation with GC. Homogeneity of all formulations was confirmed. Stability was confirmed for 28 days for dose formulations stored in doubled plastic bags at up to 5° C, the storage conditions used during the studies. Periodic analyses of the dose formulations were conducted by the study laboratory using GC. All dose formulations that were not within 10% of the target concentrations were reblended and reanalyzed; all dose formulations analyzed and used for dosing were within 10% of the target concentrations.

Duration of treatment / exposure:

14 consecutive weeks

Frequency of treatment:

continuous in feed

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
3125, 6250, 12500, 25000, or 50000 ppm
Basis:
nominal in diet

Doses / concentrations 2

Remarks:

Doses / Concentrations:
Males: 190, 380, 770, 1540, and 3210 mg/kg
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
Females: 190, 395, 780, 1580, and 3245 mg/kg
Basis:
actual ingested

No. of animals per sex per dose:

10/sex/dose for core study
10/sex/dose for clinical pathology testing only and were not necropsied.

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The exposure concentrations selected for use in the 14-week studies were based on the results of pilot studies in which no findings of overt toxicity were observed at exposure concentrations up to 50000 ppm (5% in feed). The substitution of greater than 5% of the feed interferes with the availability of some essential vitamins and minerals and causes nutritional imbalances.
- Rationale for animal assignment (if not random):

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: initially, weekly, at the end of the study

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes; weekly by day
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

FOOD EFFICIENCY: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: days 5 and 21 and from core study rats and at the end of the studies for hematology and clinical chemistry
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: all
- Parameters checked: haematocrit; haemoglobin concentration; erythrocyte, reticulocyte, and platelet counts; erythrocyte and platelet morphology; mean cell volume; mean cell haemoglobin; mean cell haemoglobin concentration; and leukocyte count and differentials

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: days 5 and 21 and from core study rats and at the end of the studies for hematology and clinical chemistry
- Animals fasted: No data
- How many animals: all
- Parameters checked: urea nitrogen, creatinine, total protein, albumin, cholesterol, alanine aminotransferase, alkaline phosphatase, creatine kinase, sorbitol dehydrogenase, 5N-nucleotidase, and bile acids

URINALYSIS: No data

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Weeks 4 and 13
- Dose groups that were examined: 0, 0 (vehichle), 12500, 25000, 50000 ppm
- Battery of functions tested: body position, activity level, coordination of movement, gait, general behavior, head flick, head searching, compulsive biting or licking, backward walking, self-mutilation, circling, convulsions, tremors, lacrimation or chromodacryorrhea, salivation, piloerection, pupillary dilation or constriction, unusual respiration, diarrhea, excessive or diminished urination, and vocalization

OTHER: At the end of the studies, sperm samples were collected from all male animals in the 0, 0(vehicle), 12500, 25000, and 50000 ppm groups for sperm motility evaluations (spermatid heads per testis and per gram testis, spermatid counts, and epididymal spermatozoal motility and concentration). The left cauda, left epididymis, and left testis were weighed. Vaginal samples were collected for 12 consecutive days before the end of the studies from all females in the 0, 0(vehicle), 12500, 25000, and 50000 ppm groups for vaginal cytology evaluations. The percentage of time spent in the various estrous cycle stages and estrous cycle length were evaluated.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes; Necropsies were performed on all animals. Organs weighed were heart, right kidney, liver, lung, right testis, and thymus.
HISTOPATHOLOGY: Yes; Complete histopathology was performed on all rats in the 0, 0 (vehicle) and 50000 ppm groups. In addition to gross lesions and tissue masses, the following tissues were examined: adrenal gland, bone with marrow, brain, clitoral gland, esophagus, heart, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, liver, lung, lymph nodes (mandibular and mesenteric), mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, skin, spleen, stomach (forestomach and glandular), testis with epididymis and seminal vesicle, thymus, thyroid gland, trachea, urinary bladder, uterus, and Zymbal’s gland.

Statistics:

The Fisher exact test was used to determine significance of incidences of lesions

Two approaches were employed to assess the significance of pairwise comparisons between exposed and control groups in the analysis of continuous variables. Organ and body weight data, which have approximately normal distributions, were analyzed with the parametric multiple comparison procedures of Dunnett and Williams. Haematology, clinical chemistry, spermatid, and epididymal spermatozoal data, which have typically skewed distributions, were analyzed using the nonparametric multiple comparison methods of Shirley and Dunn. Jonckheere’s test was used to assess the significance of the dose-related trends and to determine whether a trend-sensitive test (Williams’ or Shirley’s test) was more appropriate for pairwise comparisons than a test that does not assume a monotonic dose-related trend (Dunnett’s or Dunn’s test). Prior to statistical analysis, extreme values identified by the outlier test of Dixon and Massey were examinedl, and implausible values were eliminated from the analysis. Average severity values were analyzed for significance with the Mann-Whitney U test. Because vaginal cytology data are proportions (the proportion of the observation period that an animal was in a given estrous stage), an arcsine transformation was used to bring the data into closer conformance with a normality assumption. Treatment effects were investigated by applying a multivariate analysis of variance to the transformed data to test for simultaneous equality of measurements across exposure concentrations.

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 specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not specified

Behaviour (functional findings):

no effects observed

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:

BODY WEIGHT AND WEIGHT GAIN: The final mean body weight and body weight gain of males in the 50000 ppm group were significantly less than those of the vehicle controls

HAEMATOLOGY: On day 21 and at week 14, there were decreases in the circulating erythroid mass in exposed males and females, as evidenced by decreases in haematocrit values, haemoglobin concentrations, and erythrocyte counts. On day 21, these erythron effects occurred most consistently in the 25000 and 50000 ppm groups; at week 14, these effects were also observed in males exposed to 6250 or 12500 ppm. At both time points, the decrease in the erythron mass was of minimal severity, and, generally, the suppression was approximately 5% or less compared to the vehicle control values.

CLINICAL CHEMISTRY: Females exposed to 12500 ppm or greater had significantly decreased serum alkaline phosphatase activities compared to the vehicle controls on day 21. These decreases were minimal in severity, were no greater than approximately 13%, and were transient, with alkaline phosphatase activities in the affected groups returning to vehicle control levels by week 14. On day 21 and at week 14, there was minimal suppression of serum 5N-nucleotidase activities in males and females in the 50000 ppm groups. There were sporadic differences in clinical chemistry parameters at various time points that generally did not demonstrate an exposure concentration relationship or were inconsistent between males and females; these differences were not considered to be toxicologically relevant.

ORGAN WEIGHTS: The liver weights of female rats exposed to 6250 ppm or greater were significantly greater than those of the vehicle controls. Males in the 25000 and 50000 ppm groups had significantly lower absolute kidney weights than the vehicle control group.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

>= 3 210 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: highest dose tested

2

Dose descriptor:

NOAEL

Effect level:

>= 3 245 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: highest dose tested

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

NOAEL (male): ≥3210 mg/kg (highest dose tested)
NOAEL (female): ≥3245 mg/kg (highest dose tested)
This study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability).

Executive summary:

In the 14-week feed study, groups of 10 male and 10 female rats were fed diets containing microcapsules with a chemical load of the test item. Dietary concentrations of 3125, 6250, 12500, 25000, and 50000 ppm were selected. These dietary concentrations resulted in average daily doses of 190, 380, 770, 1540, and 3210 mg/kg for males and 190, 395, 780, 1580, and 3245 mg/kg for females. Additional groups of 10 male and 10 female rats served as untreated and vehicle controls.

There were no exposure-related deaths of rats. Mean body weights of male rats in the 50000 ppm groups were significantly less than those of the vehicle controls. On day 21 and at week 14, there were mild decreases in haematocrit values, haemoglobin concentrations, and erythrocyte counts in groups of male and female rats in the 25000 and 50000 ppm groups. At week 14, these effects were seen in male rats exposed to 6250 and 12500 ppm. There were no exposure-related alterations in clinical chemistry parameters in rats. The liver weights of female rats exposed to 6250 ppm or greater were significantly greater than those of the vehicle controls. The absolute kidney weights of male rats exposed to 25000 or 50000 ppm were significantly decreased. No gross or microscopic lesions were observed that could be attributed to test item exposure. Very little toxicity was associated with ingestion of microencapsulated test substance up to the dose levels of 3210 and 3245 mg/kg in male and female rats, respectively. Histopathology and clinical chemistry data, combined with body and organ weight data, revealed that the maximum tolerated dose was not reached in this study.