Trifluoro(trifluoromethyl)oxirane

Endpoint summary

Description of key information

Inhalation: OECD 422; rats. NOEC = 50 ppm (340 mg/m3), based on brain, lung, and spleen weight and histopathological effects at ≥250 ppm. Reliability = 1

Key value for chemical safety assessment

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 labeling and/or risk assessment.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

Study included FOB and motor activity endpoints.

Qualifier:

according to guideline

Guideline:

other: US EPA OPPTS 870.3650

Deviations:

yes

Remarks:

Study included FOB and motor activity endpoints.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Approximately 66 days
- Weight at study initiation: Males: 170.0–235.2 g; Females: 169.1–213.1 g
- Fasting period before study: No
- Housing:
Pretest/Premating: Individually (except during cohabitation of mating pairs) in stainless steel, wire-mesh cages suspended above cage boards; sexes on separate racks.
Cohabitation: Mating pairs (females placed in males’ cages) on a 1:1 basis in stainless steel, wire-mesh cages suspended above cage boards.
Days 0–19 of gestation: Individually in stainless steel, wire-mesh cages suspended above cage boards; sexes on separate racks.
Day 19 of gestation - Day 4 of lactation: Females assumed pregnant were housed individually in polycarbonate pans with bedding material. Females presumed nonpregnant were housed in the same manner as pregnant females approximately 6 days after the mating pairs were separated.
- Diet (e.g. ad libitum): PMI® Nutrition International, LLC Certified Rodent LabDiet® 5002 (pelleted); ad libitum
- Water (e.g. ad libitum): Tap water from United Water Delaware; ad libitum
- Acclimation period: Pretest Period: 14 days; Quarantine: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18–26°C
- Humidity (%): 30%–70%
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12-hours light/12 hours dark

Route of administration:

inhalation: gas

Type of inhalation exposure:

whole body

Vehicle:

other: filtered and conditioned air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: All exposure chambers were constructed of stainless steel and glass (NYU style) with a nominal internal volume of 350 L. A tangential feed at the chamber inlet promoted uniform chamber distribution of the test atmosphere.
- Method of holding animals in test chamber: During exposure, animals were individually placed in stainless steel wire-mesh modules (one/module) and exposed, whole-body, inside the exposure chamber, except during the mating period when animals were housed as mating pairs in stainless steel wire-mesh modules and exposed in the same manner.
- Source and rate of air: filtered and conditioned air
- Method of conditioning air: filtered and conditioned air
- System of generating vapour: Chamber atmospheres were generated by dilution of the test vapour in air. The test substance was metered into the test chamber inlets using a Brooks model 0145 or 0145E mass flow controller and mixed with filtered and conditioned air, which carried the resulting atmosphere into the exposure chamber. Chamber concentrations of test substance were manually controlled by varying the test substance feed rate to the chamber.
- Temperature, humidity, pressure in air chamber: 19–24°C; 41–82%; pressure not reported
- Air flow rate: 70–114 L/min
- Air change rate: at least 10 air changes per hour
- Treatment of exhaust air: to fume hood

TEST ATMOSPHERE
- Brief description of analytical method used: During each exposure, the atmospheric concentration of the test substance was determined by
gas chromatography at approximately 30-minute intervals in the test chambers. A gas chromatograph equipped with an automated gas sample valve and a flame ionization detector was used.
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The mean concentrations (± standard error of the mean) of the test substance were 50 ± 0.098 and 250 ± 0.16 ppm in chambers targeted at 50 and 250 ppm, respectively. The mean concentration ± standard error of the mean) for the high concentration chamber was 580 ± 30 ppm. The reason for the relatively high standard error of the mean for the high concentration chamber was because the concentration was targeted to 1000 ppm for the first 6 exposures, and then targeted to 500 ppm for the remainder of the study. Overall daily mean concentrations ranged from 49-52 ppm for the 50 ppm chamber, 250 ppm for the 250 ppm chamber, and 500–1000 ppm for the 1000/500 ppm chamber. The calculated nominal concentrations were generally similar to the actual measured concentrations, with mean nominal concentrations of 58, 264, 564, and 1085 ppm for the 50, 250, 500, and 1000 ppm targeted concentrations, respectively. These data demonstrate that the exposure system and analyses were operating as expected.

Duration of treatment / exposure:

Exposures for males and females were conducted for the 14 day premating period. Subsequently, males and non-pregnant females were exposed through to the terminal sacrifice. Exposures for females with evidence of mating were conducted during the cohabitation period (up to 2 weeks in duration) and during gestation days 0–19. Gestating P1 females were not exposed after gestation day 19.

Premating period - 14 days exposure
Cohabitation - Up to 14 days exposure

Postcohabitation Exposure:
Males - Up to test days 36–37
Females with no evidence of mating - 19 days (approximate)
Gestation - (GD 0-19)
Lactation - No exposure (LD 0-4)

Frequency of treatment:

All exposures were conducted for 6 hours/day, 5 days/week during the premating period. Beginning at the mating (cohabitation) period, exposures were conducted for 6 hours/day, 7 days per week. Lactating dams were not exposed.

Remarks:

Doses / Concentrations:
0, 50, 250, 1000/500 ppm (the high level concentration was reduced to 500 ppm on test day 9 [exposure #7])
Basis:
nominal conc.

No. of animals per sex per dose:

12 animals/sex

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The test substance is slightly toxic in the rat by the inhalation route with a 2.5-hour ALC of 2000 ppm. The only signs of toxicity observed in rats exposed at 400 ppm in a 2-week inhalation study (4 hr/day; 5 days/wk) were inactivity and slightly deepened respiration; pathological examination revealed no test substance-related changes. No effects on body weight or food consumption were observed nor were there any clinical signs of toxicity in rats exposed at 100 or 400 ppm for 18-weeks (6 hr/day; 5 days/wk). Significant changes in organ/body weight ratios (spleen, kidneys and adrenals in males; liver in females) occurred at 400 ppm. Based on these results, the concentrations selected for the current study are 0, 50, 250, and 1000 ppm. However, due to excessive toxicity, the 1000 ppm concentration was reduced to 500 ppm on test day 9.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once during pretest (baseline), and weekly thereafter.

BODY WEIGHT: Yes
- Time schedule for examinations:
Quarantine: Twice
Pretest: Weekly
Cohabitation: Weekly
Post-cohabitation: Weekly for males and females without evidence of copulation
Gestation: Days 0, 7, 14, 21G
Lactation: Days 0, 4L
Sacrifice: All animals

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Premating: Weekly
Cohabitation: None
Post-cohabitation: None for males and females without evidence of copulation
Gestation: Days 0, 7, 14, 21G
Lactation: Days 0, 4L

FOOD EFFICIENCY:
- Calculation of Food Consumption:
Feeders were weighed at the beginning and end of the interval. The final weight and amount of spillage (greater than 5 g) were subtracted from initial feeder weight.
- Calculation of Mean Daily Food Efficiency: Calculated from food consumption and body weight data.

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: first 5 surviving male and female animals on test day 17
- Anaesthetic used for blood collection: Yes (carbon dioxide)
- Animals fasted: Yes
- How many animals: 5 each sex
- Parameters checked in table [No.1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: first 5 surviving male and female animals on test day 17
- Animals fasted: Yes
- How many animals: 5 each sex
- Parameters checked in table [No.2] were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes, (refer to Section 7.9.1, DI.K1.Neuro.R.D-20964 for additional details).
- Time schedule for examinations: Prior to initiation of test substance administration and prior to the end of the premating period
- Dose groups that were examined: all
- Battery of functions tested: Abbreviated Functional Observational Battery (FOB), Motor Activity Evaluation

OTHER: For additional details regarding observations and examinations of reproductive parameters refer to Section 7.8.1 (DI.K1.1Gen.Screen.RD/REPRO/DEV.R.D-20964.KD) and 7.8.2 (DI.K1.DEV.Screen.RD/REPRO/DEV.R.D-20964.KD)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table No. 3)
HISTOPATHOLOGY: Yes (see table No. 3)

SACRIFICE SCHEDULE:
Males - After siring litters (approximately 28 days of exposure)
Dams with Litters - Day 4L
Females without evidence of mating - Approximately 26 days after end of cohabitation
F1 litters - PND 4

Gross examination was performed on all adult rats. Final body and organ weights were recorded for all rats at the scheduled sacrifice. Organ weights and body weights were not determined for decedents (2 males and 2 females).

Gross Pathology - All animals
Organ Weights - All animals
Histopathology - Control and high-dose groups (target organs and gross lesions in intermediate-dose groups)
Offspring Day 4L Pups - External examination

Other examinations:

OTHER: For additional details regarding observations and examinations of reproductive parameters refer to Section 7.8.1 (DI.K1.1Gen.Screen.RD/REPRO/DEV.R.D-20964.KD) and 7.8.2 (DI.K1.DEV.Screen.RD/REPRO/DEV.R.D-20964.KD)

Statistics:

Male and female data were evaluated separately. For litter parameters, the proportion of affected pups per litter or the litter mean were used as the experimental unit for statistical evaluation. The level of significance selected was p < 0.05.
see Table 4 Materials and Methods tables below

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):

effects observed, treatment-related

Food efficiency:

effects observed, treatment-related

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

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:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY:
Adverse, test substance-related clinical signs of toxicity were observed post exposure in 1000 ppm males, which included aggressive behavior, rapid breathing, hyperactivity, and hypersensitivity. In addition, during the detailed clinical observations conducted prior to exposure at the time of weighing, hyper-reactivity was also observed in 1000 ppm males.

Adverse, test substance-related clinical signs of toxicity were observed post exposure in 1000 ppm females, which included abnormal gait, aggressive behavior, dehydration, hunched over posture, hyperactivity, hyper-reactivity, hypersensitivity, and lethargy. In addition, during the detailed clinical observations conducted prior to exposure at the time of weighing, abnormal gait hunched over posture, and hyper-reactivity were also observed in 1000 ppm females.

The concentration of the test substance was reduced to 500 ppm on test day 9, and the clinical signs of toxicity noted above were no longer observed after test day 11. Test substance-related mortality occurred in 2 males exposed to 1000 ppm on test days 3–4. Test substance-related mortality occurred in 2 females exposed to 1000 ppm on test days 4 and 8. As a result of the mortality in both males and females, the exposure concentration was reduced on test day 9 to 500 ppm for the remainder of the study. Test substance-related mortality did not occur at concentrations of 500 ppm or below. The cause of death for 3 of the decedents was cerebrocortical necrosis. The cause of death for the other male decedent was not determined. However, since this rat had autolysis of the brain tissue, it is likely that a lesser degree of cerebrocortical necrosis was also responsible for this death.

BODY WEIGHT AND WEIGHT GAIN:
Adverse, test substance-related, statistically significant decreases in body weight and weight gain occurred in males exposed to 250 ppm and above. Body weight for 1000/500 ppm males was significantly lower for test days 7–35, and was 10% lower compared to the control value on test day 35. Mean values for weight gain in 1000/500 ppm males were significantly lower compared to the control value for the interval of test days 0–7, 0–14, 0–35, and were 31% lower compared to the control value for the interval of test days 0–35. Weight gain in 250 ppm males was significantly lower for the intervals of 14–35 and 0–35, and were 19.6% lower compared to the control value for days 0–35. The lower body weight and weight gain for 250 and 1000/500 ppm males correlated with a decreased food consumption, and was considered to be test substance related.

Adverse, test substance-related, statistically significant decreases in body weight and weight gain occurred in 1000/500 ppm females. Body weight for 1000/500 ppm females was significantly lower compared to the control value on test day 7. Females in the 1000/500 ppm group lost weight (13.2 g) during test days 0–7 compared to a weight gain of 9.5 g in the control group. However, after the exposure concentration was reduced to 500 ppm, the 1000/500 ppm females recovered, gaining more weight relative to the control group. Weight gain over the interval of test days 0–14 was 59% lower than the control as a result of the weight loss during test days 0–7. There were no statistically significant differences on weight or weight gain in 50 or 250 ppm females.

FOOD CONSUMPTION AND FOOD EFFICIENCY:
Adverse, test substance-related, statistically significant decreases in food consumption and food efficiency occurred in males exposed to 250 ppm and above. Food consumption and food efficiency for 1000/500 ppm males were significantly lower for test days 0–7 and 0–14. Males in the 250 ppm group also had significantly lower food consumption over test days 0–7 and 0-14. The decreased food consumption and food efficiency in 250 ppm and above males correlated with the decreased body weight and weight gain, and was considered to be adverse. There were no adverse or statistically significant effects on food consumption or food efficiency for 50 ppm males.

Adverse, test substance-related, statistically significant decreases in food efficiency occurred in 1000/500 ppm females. Food efficiency for 1000/500 ppm females was significantly lower compared to the control value on test days 0–7 and 0–14. However, significantly greater food consumption was observed during test days 7–14, reflecting recovery after the exposure concentration was reduced to 500 ppm. There were no test substance-related effects or statistically significant differences on food consumption or food efficiency in 250 ppm or below females.
HAEMATOLOGY:
Minimal to mild, statistically significant decreases in red blood cell count (RBC) and hemoglobin concentration (HGB) were present in male (86% and 88% of control, respectively) and female rats (77% and 87% of control, respectively) exposed to 1000/500 ppm. In addition, hematocrit (HCT) was minimally decreased in females (94% of control) at this concentration. In both male and female rats, these decreases in red cell mass parameters (RBC, HGB, and HCT) were associated with an appropriate regenerative response based on mild increases in absolute reticulocyte counts (ARET). As expected with increased reticulocyte counts, mean cell volume (MCV) and red cell distribution width (RDW), were increased (RDW in females only) and mean corpuscular hemoglobin concentration (MCHC) was decreased in these groups. Reticulocytes have greater cell volume and lower hemoglobin concentration compared to mature red blood cells. The changes in red cell mass and associated parameters in males and females exposed to 1000/500 ppm were considered to be test substance related and adverse.

Similar but more minimal decreases in red cell mass and some related parameters were present in male and female rats in the 250 ppm group (variable statistical significance). However, at this concentration, decreases were less than 10% compared to controls and did not elicit a clear regenerative response. Therefore, changes in red cell mass at 250 ppm were considered nonadverse.

Mild decreases white blood cell count (WBC), due primarily to a decrease in lymphocyte count (ALYM), were present in female rats in the 250 and 1000/500 ppm groups (statistically significant for both parameters at both concentrations). Mean WBC was decreased to 72% and 60% of controls in the 250 and 1000/500 ppm groups, respectively. Mean ALYM was decreased to 69% and 57% of controls in the 250 and 1000/500 ppm groups, respectively. Mean absolute counts for eosinophils (AEOS), monocytes (AMON), Basophils (ABAS), and atypical leukocytes (ALUC which are mostly activated lymphocytes) were also decreased in these groups (variable statistical significance). The pattern of changes in white blood cell parameters is consistent with changes associated with stress, although no histopathological changes indicative of stress, such as lymphoid necrosis of thymus, were seen in female rats. These decreases in WBC and ALYM in females were considered to be treatment related and potentially adverse.

Decreased white blood cell and lymphocyte counts, as well other white blood cell types, were present in treated male groups. However, the decreases were not statistically significant (except for eosinophils in males exposed to 1000/500 ppm) and did not occur in a dose-related manner. Therefore, these changes were likely unrelated to treatment.

Coagulation - The following statistically significant change in a group mean coagulation parameter was considered to be unrelated to treatment and non-adverse because it did not occur in a doserelated pattern: Minimally prolonged prothrombin time (PT) (103% of control, respectively) in male rats dosed with 50 ppm.

CLINICAL CHEMISTRY:
Glucose (GLUC) was minimally decreased in male rats exposed to 250 or 1000/500 ppm (86% and 77% of control, respectively). In these groups, 1/5 and 3/5 individual glucose values were below the sex and age-matched historical 95% reference interval (76–121 mg/dL). Although individual rats in these groups did not display clinical signs of hypoglycemia, the change was considered to be treatment related and potentially adverse.

The following statistically significant changes in group mean clinical chemistry parameters at test day 17 were potentially related to treatment but were considered to be non-adverse:

• Alanine aminotransferase (ALT) was minimally increased in male and female rats exposed to 1000/500 ppm (122% and 131% of control, respectively; statistically significant in males only). These minimal increases in ALT were not associated with exposure-related changes in related parameters (sorbitol dehydrogenase, [SDH]; aspartate aminotransferase [AST]) or with correlative histopathological changes in the liver, and were therefore considered to be non-adverse.

• Alkaline phosphatase (ALKP) and bilirubin (BILI) were minimally increased in male rats exposed to 1000/500 ppm (128% and 185% of control, respectively). Increases in ALKP and BILI may occur in liver cholestasis. Non-hepatic mechanisms may also be associated with increases in these parameters (e.g., enzyme induction for ALKP and hemolysis for BILI). Irrespective of the underlying mechanism, increases in these parameters were very small, were not associated with correlative histopathological changes and similar changes did not occur in female rats at this exposure level. Therefore, the increase in ALKP and BILI were considered to be non-adverse.

• Chloride (Cl) was minimally increased in male rats exposed to 250 or 1000/500 ppm (104% of control in both groups). These increases may have been treatment related. However, the increases were small and individual Cl values from the control rats and those exposed to the test material were within the sex and age-matched historical 95% reference interval (96.7-113.6 mmol/L). Therefore, the minimal increases in chloride were considered to be non-adverse.

• The following statistically significant changes in group mean clinical chemistry parameters did not occur in a dose-related manner and were therefore considered to be unrelated to exposure to the test material:
• Sodium was minimally increased (102% of control) in males exposed to 250 ppm.
• Alkaline phosphatase was minimally decreased (66% of control) in female rats exposed to 250 ppm.
• Potassium was minimally decreased (90% of control) in female rats exposed to 250 ppm.

NEUROBEHAVIOUR:
Yes, (refer to Section 7.9.1, DI.K1.Neuro.D-20964 for additional details).

ORGAN WEIGHTS:
Brain - In P1 adult males and females, mean absolute brain weights were increased 7% and 4%, respectively, in the 1000/500 ppm exposure group, as compared to control values. Both increases were statistically significant. These increases were despite an 11% and 6% decrease in mean final body weights, respectively, as compared to controls. The increased brain weights correlated with the microscopic finding of vacuolar degeneration of neurons and fibers in the brains of high-exposure rats. It is likely that the vacuolar degeneration was associated with an increase in brain fluid (i.e., edema) which resulted in the increased brain weights. The increases in mean relative weights at lower exposures were a reflection of decreased final body weights and not increased brain weight.

Lungs - Mean absolute and relative (% body weight) lung weights were increased 33% and 50% in males and 10% and 18% in females, respectively, in the 1000/500 ppm exposure group, as compared to control values. Mean absolute and relative lung weights were also slightly increased (6% and. 14%, respectively) in males in the 250 ppm exposure group. All increases, except for the 6%, were statistically significant. The increased lung weights correlated with the microscopic finding of increased alveolar histiocytosis at the same exposure levels.

Spleen - In P1 adult males, mean absolute spleen weights were increased 20% in both the 250 and 1000/500 ppm exposure groups, as compared to control values. Mean relative spleen weights (% body weight) were increased 29% and 35% at 250 ppm and 1000/500 ppm, respectively. In P1 adult females, spleen weight parameters were similar in the exposed and control groups. In males, the increased spleen weights correlated with the microscopic finding of increased extramedullary hematopoiesis in male rats exposed to ≥ 250 ppm. The lack of a corresponding spleen weight increase in the high-exposure female rats was possibly related to the greater background incidence of increased extramedullary hematopoiesis (EMH) in females.

Thymus - In P1 adult males , mean absolute and relative (% body weight) thymus weights were decreased 33% and 25% in the 1000/500 ppm exposure group, respectively, as compared to control values. Female thymus weights were similar in all groups. There were no microscopic effects on the thymus that correlated with this organ weight effect.

Refer to Table below for additional information.

HISTOPATHOLOGY: NON-NEOPLASTIC:
Brain - Neuronal necrosis and/or vacuolar degeneration of neuronal fibers was observed in 8/12 and 10/12 males and 8/12 and 10/12 females exposed to 250 ppm and 1000/500 ppm of the test substance, respectively. The most severe lesions were observed in 3 of the 4 rats that died during the first 8 days on study, when they were being exposed to 1000 ppm of the test substance. Severe, locally extensive, neuronal degeneration/necrosis of the frontoparietal cerebral cortex was present in 1 male and 2 females and was responsible for their deaths. The other decedent in this study had autolysis of the brain tissue which probably obscured a less severe, albeit fatal, lesion. Moderate (grade 3 of 4) to severe (grade 4 of 4) vacuolar degeneration of fibers in the caudate putamen was also present in 2 of the decedents with severe cortical necrosis. Lesions of the cerebral cortex and the caudate putamen were only observed in rats in the highest exposure group (1000/500 ppm). Multifocal vacuolar degeneration of neurons and fibers in the thalamus and base of the cerebellum was observed in most rats exposed to ≥250 ppm. This finding was graded minimal (grade 1 of 4) in all affected 250 ppm rats and minimal to mild (grade 2 of 4) in affected 1000/500 ppm rats. Clusters of vacuolated neurons and fibers were more easily identified in sections stained with luxol fast blue myelin stain (LFB) than with hematoxylin and eosin (H&E).

Lungs - Alveolar histiocytosis was increased in males exposed to ≥250 ppm and females exposed to 1000/500 ppm of the test substance. The histiocytosis was characterized by the aggregation of pulmonary alveolar macrophages within the subpleural alveoli. The histiocytosis was graded minimal in the 250 ppm rats and minimal to mild in the 1000/500 ppm rats.

Spleen - Increased EMH was observed in the spleens of males exposed to ≥ 250 ppm and females exposed to 1000/500 ppm of the test substance. The increased EMH was predominantly erythrocytic and was graded minimal to mild.

Bone Marrow - An increased incidence of erythrocytic hyperplasia was observed in the bone marrow of high exposure females. The hyperplasia was graded minimal in all cases and correlated with the increased EMH observed in the spleen.

Dose descriptor:

NOAEL

Effect level:

50 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: based on the histopathological and clinical pathology effects, body weight changes, and changes in nutritional parameters observed at 250 ppm and above

Critical effects observed:

not specified

Mean Absolute and Relative (% body weight) Brain, Lungs, Spleen, and Thymus Weights in P1 Male and Female Rats
Concentration (ppm):	0	50	250	500^a	0	50	200	500^a
Final Body Weight	476.3	448.9	445.8^b	424.1^b	340.6	318.4	312.3	318.6
Brain	2.080	2.111	2.144	2.218^b	1.960	1.941	1.971	2.038^b
% body wt	0.438	0.472^b	0.483^b	0.524^b	0.578	0.614	0.637^b	0.644^b
Lungs	1.923	1.880	2.039	2.556^b	1.482	1.437	1.464	1.632^b
% body wt	0.403	0.421	0.459^b	0.605^b	0.435	0.453	0.471	0.514^b
Spleen	0.778	0.719	0.937^b	0.934^b	0.785	0.634	0.638	0.756
% body wt	0.163	0.160	0.211^c	0.220^c	0.226	0.200	0.204	0.238^c
Thymus	0.445	0.392	0.355	0.300^b	0.263	0.239	0.258	0.253
% body wt	0.093	0.087	0.080	0.070^b	0.078	0.076	0.082	0.078
^aGroup 4 exposure was 1000 ppm before being reduced to 500 ppm on day 9, for the remainder of the study. Underlined values were interpreted to be test substance-related weight effects. ^bStatistically significant (Dunnett/Tamhane-Dunnett parametric comparison to control). ^cStatistically significant (Dunn’s non-parametric comparison to control).

 

Incidences of Test Substance-related Microscopic Findings in P1 Male and Female Rats
Sex:	Male				Female
Group:	1	2	3	4	1	2	3	4
Concentration (ppm):	0	50	250	500^a	0	50	250	500^a
Number of Rats	12	12	12	12	12	12	12	12
Brain
Degeneration/Necrosis	0	0	8	10	0	0	8	10
Lungs
Histiocytosis, alveolar	1	1	5	10	0	0	0	8
Spleen
EMH, increased	0	0	9	9	5	4	4	10
Bone Marrow^b
Hyperplasia, erythro.	0	0	0	0	1	1	1	4
Underlined values were interpreted to be test substance-related increases in microscopic findings. EMH = extramedullary hematopoiesis; erythro. = erythrocytic ^aGroup 4 exposure was 1000 ppm before being reduced to 500 ppm on day 8, for the remainder of the study. ^bIn males, the number of rats with bone marrow examined was only 5, 1, 2, and 7 in groups 1, 2, 3, and 4, respectively.

 

Conclusions:

This study and the conclusions which are drawn from it fulfill the quality criteria (validity, reliability, repeatability).
NOAEL = 50 ppm

Executive summary:

A combined repeated dose toxicity study with reproduction/developmental toxicity screening test was conducted with the test substance. Crl:CD(SD) rats (12/sex/concentration) were exposed whole body to 0, 50, 250, or 1000/500 ppm. Due to excessive toxicity, the 1000 ppm concentration was reduced to 500 ppm beginning on exposure 7 (test day 9). Exposures for males and females were conducted for 6 hours per day, 5 days per week from the initiation of the study through the 14 day premating period. Subsequently, males and non-pregnant females were exposed 7 days a week through the terminal sacrifice. Exposures for females with evidence of mating were conducted for 6 hours per day, 7 days per week during the cohabitation period (up to 2 weeks in duration) and during gestation days 0–19. Gestating P1 females were not exposed after gestation day 19. An abbreviated neurobehavioral evaluation consisting of a functional observational battery and motor activity was conducted in P1 rats (12/sex/group) once during pretest and prior to cohabitation. Clinical pathology parameters were measured in P1 rats (5/sex/group) at the end of the premating period (hematology, clinical chemistry) and at terminal sacrifice (coagulation). All P1 rats were given a gross pathological examination and selected tissues were weighed and collected from all adult rats.

Test substance-related mortality, clinical signs of toxicity, reductions in body weight, weight gain, food consumption and/or food efficiency occurred in P1 males and females exposed to 1000/500 ppm of the test substance, and in 250 ppm P1 males. Adverse test-substance effects on chemical pathology parameters occurred in P1 rats exposed to 250 ppm and above. Red cell mass parameters were decreased in P1 males and females exposed to 1000/500 ppm. In addition, white blood cell count parameters were decreased in 250 ppm and above P1 females. Glucose was increased in P1 males at 250 ppm and above.

Adverse, test substance-related changes in organ weights occurred in P1 males at 250 ppm and above, and in 1000/500 ppm P1 females. Increased brain weights occurred in 1000/500 ppm males and females and correlated with the observation of neuronal necrosis and/or vacuolar degeneration of neuronal fibers in the brain. Males and females exposed to 250 ppm and above had similar brain lesions without changes in brain weight parameters. Lung weights were increased in 1000/500 ppm females and in 250 ppm and above males. The changes in lung weight correlated with the observation of increased alveolar histiocytosis in 250 ppm and above males, and 1000/500 ppm females. Absolute and relative spleen weights were increased in males exposed to 250 ppm and above, and correlated with the observation of increased extramedullary hematopoiesis. Absolute and relative thymus weights were decreased in 1000/500 ppm males, however there was no morphological correlate for this change. In addition, 1000/500 ppm females had erythrocytic hyperplasia of the bone marrow. There were no test substance-related morphological changes in males or females exposed to 50 ppm.

Under the conditions of the study, the No-Observed-Adverse-Effect Level (NOAEL) for systemic toxicity was 50 ppm in P1 males and females based on the histopathological and clinical pathology effects, body weight changes, and changes in nutritional parameters observed at 250 ppm and above.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

340 mg/m³

Study duration:

subchronic

Species:

rat

Additional information

Male and female rats were exposed via whole-body inhalation to concentrations of 50, 250, 1000/500 ppm of the test substance for 6 hours per day, 5 days per week in an OECD 422 Repeat Dose/Developmental/Reproductive Screening Study. Due to excessive toxicity, the 1000 ppm concentration was reduced to 500 ppm beginning on exposure 7 (test day 9). Test substance-related effects on mortality, clinical sings, body weight, food consumption, food efficiency and/or clinical pathology parameters were observed at ≥250 ppm. Increased brain weights correlated with neuronal necrosis and/or vascular degeneration of neuronal fibers in the brain were observed at 1000/500 ppm. Similar brain lesions without changes in brain weight were observed at 250 ppm. Increased lung weights which correlated with increased alveolar histiocytosis were observed at ≥250 ppm in males and at 1000/500 ppm in females. Absolute and relative spleen weights which correlated with increased extramedullary hematopoiesis were observed in males at ≥250 ppm. Decreased absolute and relative thymus weight without morphological changes were observed in males at 1000/500 ppm, and erythrocytic hyperplasia of the bone marrow was observed in females at 1000/500 ppm. The NOEC was 50 ppm.

Rats, rabbits, and dogs were exposed to the test substance via inhalation at concentrations of 100 and 400 ppm for 6 hours per day, 5 days per week, for 90 days. No mortality was observed in any species. Urine excretion was reduced in female rats at 400 ppm. Changes; in organ to body weight ratios of spleen, kidney, and adrenals was observed in male rats at 400 ppm and in the liver in females at 400 ppm. Respiratory tract irritation was observed in rabbits and dogs at 400 ppm, and decreased spermatogenesis was observed in dogs at 400 ppm. Secondary alterations were produced in the kidney of all three species were observed, but these were possibly related to changes in electrolyte balance that may have been influenced by respiratory difficulties.

Repeated dose toxicity: inhalation - systemic effects (target organ) neurologic: brain (multiple sections)

Justification for classification or non-classification

Brain effects (weight and histological changes) were observed in rat repeated inhalation study at ≥250 ppm (1.7 mg/l). The rat 28-day repeated inhalation LOAEL of 1.7 mg/l is outside of the R48 classification guidance value of 0.75 mg/l (Paragraph 3.2.3; 3 x 0.25 mg/l), and no classification is required under EU Directive 67/548/EEC. Based on the LOAEL of 250 ppm, STOT RE Cat 2 classification is appropriate for the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.