trans-dichloroethylene

Endpoint summary

Description of key information

The NOAEC in a GLP guideline 90-day inhalation study was 4000 ppm (15859 mg/m3), the highest concentration tested in both male and female rats.
The NOAEL in  a GLP guideline 90-day oral feeding study was 3210 mg/kg bw/day and 3245 mg/kg bw/day in male and female rats, respectively, the highest doses tested for each sex.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

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.

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

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

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

Remarks:

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

Remarks:

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

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):

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.

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.

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

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

Critical effects observed:

not specified

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

3 210 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

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.

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

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

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

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.

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.

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.

Dose descriptor:

NOAEC

Effect level:

>= 4 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest concentration tested

Critical effects observed:

not specified

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

15 859 mg/m³

Study duration:

subchronic

Species:

rat

Additional information

The toxicity of the test substance was evaluated in male and female rats exposed to analytically-determined mean atmosphere concentrations of 0, 200, 1000, or 4000 ppm for 6 hours/day, 5 days/week over a 90-day period. There were no test substance-related adverse effects on body weight, clinical observations, body weight gain, food consumption, clinical or anatomical pathology parameters, or liver cell proliferation. The no observed-adverse-effect concentration for this study was 4000 ppm (15859 mg/m³), the highest concentration tested in both male and female rats.

In a sub-chronic feeding study, rats were continuously fed diets containing microcapsules with a chemical load resulting in dietary concentrations of 3125, 6250, 12500, 25000, or 50000 ppm for 14 consecutive weeks. 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. Histopathology and clinical chemistry data, combined with body and organ weight data revealed that the maximum tolerated dose was not reached in this study. The no- observed-adverse-effect levels for this study were 3245 and 3210 mg/kg, the highest doses tested in both male and female rats, respectively. 

The toxicity of the test substance was evaluated in multiple additional oral (gavage, drinking water, or dietary) repeat dose toxicity studies in rats and mice. There were no test substance-related adverse effects in any of the studies. The 90-day drinking water NOAELs were 387 and 452 mg/kg bw/day in male and female rats (highest dose tested), respectively; and 3114 and 2809 mg/kg bw/day in male and female mice (highest dose tested), respectively. The 14-week oral NOAELs were 8065 and 7925 mg/kg bw/day in male and female mice (highest dose tested), respectively.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
OECD guideline, GLP study

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
OECD guideline, GLP study

Justification for classification or non-classification

The NOAEC in a 90-day inhalation study in rats was 4000 ppm (15859 mg/m³), the highest concentration tested. The NOAEL in a 90-day feeding study in rats was 3210 mg/kg (males) and 3245 mg/kg (females), the highest doses tested. Therefore, the substance does not need to be classified for repeated dose toxicity according the EU Directive 67/548/EEC and EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.