Registration Dossier

Administrative data

Description of key information

A 14, 28, and 90-day repeated dose inhalation study were available to fill this endpoint. The main adverse effect observed in these studies was multifocal mononuclear cell infiltration of the heart. This effect was observed in the 14-day study at 7500 and 20000 ppm. In the 28-day study this effect was absent up to the highest dose tested: 10000 ppm, but in 90-day study this effect was observed in the 10000 and 15000 ppm dose groups. At the lowest concentration (4000 ppm) tested in the 90-day test, one rat was observed with multifocal mononuclear cell infiltrate localized to the apex. However, this was considered to be a spontaneous lesion. Therefore, the overall NOAEL was determined to be 4,000 ppm (21,240 mg/m3).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

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other: Study conducted in compliance with GLP and OECD guideline, available as unpublished report, no restrictions, fully adequate for assessment
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
GLP compliance:
yes (incl. certificate)
Remarks:
TNO Triskelion, Utrechtseweg 48, P.O. Box 360, 3700 AJ Zeist, The Netherlands
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Deutschland, Sulzfeld, Germany
- Age at study initiation: 7 weeks
- Mean weight at study initiation: 295 g for males and 199 g for females
- Fasting period before study: No
- Housing: Macrolon cages with a bedding of wood shavings (Lignocel, Type ¾) and strips of paper (Enviro-dri) as environmental enrichment. Initially, all animals were housed five per cage, separated by sex. However, from 2 July 2010 onwards, because of their increased weight, male animals were housed 2 or 3 animals per cage.
- Diet: Rat and Mouse No. 3 Breeding Diet RM3 (SDS Special Diets Services, Witham, England) was available ad libitum, except during periods of exposure to test substance
- Water: Tap water was available ad libitum via polypropylene bottles, except during periods of exposure to test substance.
- Acclimation period: 8 Days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-24
- Humidity (%): 45 - 65
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
inhalation: gas
Type of inhalation exposure:
nose only
Vehicle:
air
Details on inhalation exposure:
CHAMBER DESCRIPTION
- Exposure equipment: Animals were exposed to the test atmosphere in nose-only exposure units. Each unit consisted of a cylindrical PVC column with a volume of ca. 70 litres, surrounded by a transparent hood. The test atmosphere was introduced at the bottom of the central column, and was exhausted at the top. Each column included three rodent tube sections and each rodent tube section accommodated 20 ports for animal exposure. Additional ports were used for test atmosphere sampling, measurement of oxygen concentration, temperature and relative humidity. The animals were secured in plastic animal holders (Battelle), positioned radially through the outer hood around the central column (males and females alternated). The remaining ports were closed. Only the nose of the rats protruded into the interior of the column. The units were illuminated externally by normal laboratory TL-lighting.

GENERATION OF TEST ATMOSPHERE
- The inhalation equipment was designed to expose the rats to a continuous supply of fresh test atmosphere. To generate the test atmosphere, a flow of cooled liquid test material controlled by a peristaltic pump was allowed to evaporate in a flow of humidified air (mass flow controlled). The air was supplemented with mass flow controlled oxygen for the mid and high concentrations to ensure a sufficiently high and equal oxygen concentration. Total test atmosphere flow was between 27 and 28 L/min for all groups except during the period from 7 June to 1 July 2010 (the period when also animals of a prenatal developmental inhalation toxicity study were exposed) when it was between 54 and 55 L/min and on 28 June 2010 where it was between 29 and 30 L/min. At all times the flow was higher than 1 L/min/animal with regard to the amount of animals present. The exposure unit for control animals was supplied with a mass flow controlled stream of humidified compressed air only.
- The measured concentrations were used in a PI feedback system to control the peristaltic pumps. The feed back system took into account the proportional (P) and the integrated deviations (I) of the concentrations from the set point.
- The settings of the mass flow controllers were checked each morning at the start of the generation and subsequently at regular intervals during exposure (approximately bi-hourly, i.e. three times a day).

MEASUREMENT OF TEMPERATURE AND HUMIDITY
- The temperature and the relative humidity of the test atmospheres were measured continuously and recorded every minute during exposure using a CAN transmitter with temperature and relative humidity probes.
- Mean temperature (± standard deviation) during exposure was 23.8 (± 0.4), 23.5 (± 0.5), 23.4 (± 0.5) and 23.6 (± 0.5)°C for the control, low, mid and high concentration exposure conditions, respectively. Measured minimum and maximum temperatures were 20.2 and 25.1°C, respectively. The temperature ranged between 25.0 and 25.1°C during 70 minutes on 11 June for the control group, during 23 minutes on 9 July for the low concentration group and during 10 minutes on 9 July for the high concentration group.
- Mean relative humidity (± standard deviation) during exposure was 40% (± 3), 40% (± 3), 38% (± 2) and 37% (± 3), respectively. Measured minimum and maximum relative humidity was 28 and 74%, respectively. Relative humidity was less than 30% (minimum 28%) during periods ranging from 1 to 9 minutes on 27 May, 1, 15, 20 and 21 July and 19 and 26 August for the high concentration group and on 1 July for the control group. Relative humidity was higher than 70% for group 1 on 1 June (duration 5 min, maximum 74%), for group 3 on 6 July (duration 3 min, maximum 71.5%) and for group 4 on 23 July (duration 1 min, maximum 71%) and on 5 August (duration 5 min, maximum 72.5%.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The actual concentration of the test substance in the atmospheres was measured by total carbon analysis. The test atmospheres were sampled from the exposure units at the animals’ breathing zone and were passed to total carbon analyzers. The response of the analyzers was recorded on a pc every minute using a CAN transmitter. The daily mean response was calculated by averaging values read every minute.
The total carbon analyzers were calibrated in the period 18-21 May 2010. Calibrations were done by sampling from 3 concentrations (in duplicate) in a range including the target concentration. The concentrations of the test material used to calibrate the total carbon analyzer were prepared in a sample bag by injecting known amounts (by mass) of the (cooled) test material. The calibrations were checked weekly by measuring the concentration from a freshly prepared sample bag with a concentration around the target concentration. The measured concentrations from the sample bags did not deviate more than 5% from the calculated concentration in the sample bags during the study (maximum difference 2.1%, 4.4% and -2.5% for the low, mid and high concentration, respectively). At the end of the study calibration was also checked with a sample bag with a concentration around the target concentration. Results were 0.5%, 5.7% and -0.7% deviations for the low, mid and high concentration, respectively. Because the deviation of the mid concentration was above 5%, the measurement was repeated with a newly prepared sample bag. The result was a deviation of the calculated concentration of 3.7%.
Duration of treatment / exposure:
6 hours/day
Frequency of treatment:
5 days/week (i.e., 65 exposure days over a 91-day study period)
Remarks:
Doses / Concentrations:
0, 4000, 10000 or 15000 ppm
Basis:
other: target concentrations
Remarks:
Doses / Concentrations:
0, 3987, 9974 or 14903 ppm
Basis:
analytical conc.
No. of animals per sex per dose:
10
Control animals:
yes
Details on study design:
- Dose selection rationale: Doses were selected based on results of a sub-acute (28-day) inhalation toxcity study with this test substance in rats.
- Group 1: 0 ppm, Group 2: 4000 ppm, Group 3: 10000 ppm, Group 4: 15000 ppm.
Observations and examinations performed and frequency:
CLINICAL OBSERVATIONS:
Each animal was observed daily in the morning hours by cage-side observations and, if necessary, handled to detect signs of toxicity. A group-wise observation was made halfway during each exposure day. On working days, all cages were checked again in the afternoon. At weekend days and public holidays only one check per day was carried out.

BODY WEIGHT: Yes
The body weight of each animal was recorded once during the acclimatization period (one or two days before the start of the study; nominal day -1/-2 for males and females respectively), at initiation of treatment prior to the first exposure (Nominal day 0), and weekly thereafter. In addition, animals were weighed before sacrifice for calculation of relative organ weights..

FOOD CONSUMPTION:
Food consumption of the main groups was measured per cage, over successive weekly periods (starting on Nominal day 0). The results were expressed in g per animal per day.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION:
Ophthalmoscopic examinations were made prior to the first exposure in all animals and during the last week of exposure in animals of the control and high concentration group (groups 1 and 4). Because no treatment-related changes were observed in animals of the high concentration group, the eye examinations were not extended to the animals of the intermediate concentration groups (groups 2 and 3). Eye examinations were carried out using an ophthalmoscope after induction of mydriasis by a solution of atropine sulphate.

HAEMATOLOGY:
At scheduled necropsy (Nominal day 91) blood samples of all surviving animals were taken from the abdominal aorta of (overnight) fasted rats whilst under pentobarbital anaesthesia. K3-EDTA was used as anticoagulant. In each sample the following determinations were carried out: haemoglobin, packed cell volume, red blood cell count, reticulocytes, total white blood cell count, differential white blood cell count, prothrombin time, thrombocyte count. The following parameters were calculated: mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), and mean corpuscular haemoglobin concentration (MCHC).

CLINICAL CHEMISTRY:
Clinical chemistry determinations were conducted on blood plasma of all surviving animals. Blood samples were collected in heparinized plastic tubes at the same time blood samples for haematology were collected. Plasma was prepared by centrifugation. The following measurements were made: alkaline phosphatase activity (ALP), aspartate aminotransferase activity (ASAT), alanine aminotransferase activity (ALAT), gamma glutamyl transferase activity (GGT), total protein, albumin, ratio albumin to globulin, urea, creatinine, fasting glucose, bilirubin total, cholesterol, triglycerides, phospholipids, calcium (Ca), sodium (Na), potassium (K), chloride (Cl), inorganic phosphate

URINALYSIS: Yes
On the day before necropsy, animals were transferred to metabolism cages (one animal per cage) for overnight urine collection. During urine collection the animals were deprived of food (approximately 16 hours) but not of water. The following determinations were carried out in individual samples: volume, density, appearance, dipstick measurements (pH, glucose, occult blood, ketones, protein, bilirubin, urobilinogen), microscopic examination of the sediment (red blood cells, white blood cells, epithelial cells, amorphous material, crystals, casts, bacteria, sperm cells, worm eggs).

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS NECROPSY
At the end of the exposure period, all surviving animals were killed in such a sequence that the average time of killing was approximately the same for each group. Animals were killed on Nominal day 91. The animals were killed by exsanguination from the abdominal aorta under pentobarbital anaesthesia and then examined grossly for pathological changes. The following organs were weighed (paired organs together) as soon as possible after dissection (to avoid drying): adrenals, brain, heart, kidneys, liver, spleen, testes, thymus, thyroids (with parathyroids), lungs with trachea and larynx, ovaries, uterus, epididymides

Samples of the following tissues and organs of all animals were preserved in a neutral aqueous phosphate-buffered 4 per cent solution of formaldehyde (10% solution of formalin). The lungs (after weighing) were infused with the fixative under ca. 15 cm water pressure to insure fixation. adrenals, aorta, axillary lymph nodes, brain (brain stem, cerebrum and cerebellum), caecum, colon, epididymides, eyes (with optic nerve), exorbital lachrymal glands, femur with joint, heart, kidneys, liver, lungs/trachea/larynx, lungs/trachea/larynx, mammary glands (females), cervical lymph nodes, nasopharyngeal tissue (with nasal associated lymphoid tissue and teeth), nerve-peripheral (sciatic nerve), oesophagus, olfactory bulb, ovaries, pancreas, parathyroids, pharynx, parotid salivary glands, pituitary, prostate, rectum, seminal vesicles with coagulating glands, skeletal muscle (thigh), skin (flank), small intestines (duodenum, ileum, jejunum), spinal cord (three levels), spleen, sternum with bone marrow, stomach (glandular, non-glandular), sublingual salivary glands, submaxillary salivary glands, testes, thymus, thyroid, tongue, tracheobronchial (mediastinal) lymph nodes, ureter, urethra, urinary bladder, uterus (with cervix), all relevant gross lesions.

HISTOPATHOLOGY:
- Preparation of slides: All tissues to be examined microscopically were embedded in paraffin wax, sectioned at 5 µm and stained with haematoxylin and eosin.
- Histopathological examination: The organs of the list above were examined in animals of the high concentration group (group 4) and control group (group 1). The nasal tissues were examined at 6 levels, the larynx at 3 levels, the trachea at 3 levels (including the bifurcation), and each lung lobe at 1 level. A few gross lesions and the hearts of the animals of the low and mid concentration groups were also examined microscopically.
Statistics:
- Body weight data, clinical pathology data measured on continuous or semi-continuous scales, and organ weights: data were analysed using one-way analysis of variance (Anova), after checking for homogeneity of variance (Bartlett test) and normality of data distribution (Shapiro-Wilks test). If variances were not homogeneous or data not normally distributed, the data were stepwise log or rank transformed prior to the Anova. If the Anova yields a significant effect (p<0.05), intergroup comparisons with the control group were made by Dunnett’s multiple comparison test.
- Food consumption: no statistics were applied on food intake (only two cages per sex).
- Incidences of histopathological changes: Fisher’s exact probability test.
Tests generally performed as two-sided tests with results taken as significant where the probability of the results is p<0.05 or p<0.01. Because numerous variables were subjected to statistical analysis, the overall false positive rate (Type I errors) is greater than suggested by a probability level of 0.05. Therefore, the final interpretation of results was based not only on statistical analysis but also on other considerations such as dose-response relationships and whether the results are significant in the light of other biological and pathological findings.
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:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
- The individual observations in the mornings, before the start of each day’s exposure did not indicate exposure-related clinical abnormalities. The group-wise observations during each day’s exposure did not reveal abnormalities.

BODY WEIGHT AND WEIGHT GAIN
- No significant differences in body weights between the groups were seen.

FOOD CONSUMPTION
- Food consumption was similar among the groups throughout the study period.

OPHTHALMOSCOPIC EXAMINATION
- Near the end of the exposure period no abnormalities were seen in groups 1 and 4, therefore the animals of groups 2 and 3 were not investigated.

HAEMATOLOGY
- The concentration of thrombocytes was statistically significantly decreased in the female animals of the low and mid concentration group. Since a concentration-response relation was not seen, these small differences were not considered related to the treatment. Other parameters associated with the red blood cells were not different among the groups. The relative concentration of the lymphocytes was decreased in male animals of the high concentration group. The decrease in the relative concentration of the lymphocytes and the increase of the relative concentration of the neutrophils in female animals of the low concentration group was considered not related to the treatment because of the absence of a relation with the concentration.

CLINICAL CHEMISTRY
- The concentration of ASAT and ALAT was significantly increased in plasma of the male animals of the high concentration group. The isolated small significantly decreased concentration of ASAT in male animals of the low concentration group was not considered to be treatment-related. In female animals of the mid and high concentration groups, glucose and urea were significantly increased. Potassium was significantly increased and triglyceride was slightly, but significantly decreased in female animals of the high concentration group.

URINALYSIS
- Significant differences in urinary volume and density were not seen. Measurements indicated that the amount of occult blood was significantly increased in male animals of the high concentration group. Other parameters or microscopic observations of urine did not show significant changes.

ORGAN WEIGHTS
- The relative and absolute weight of the heart was significantly decreased in male animals of the high concentration group. In female animals of that group relative weight was also significantly increased. In male animals of the high concentration group, relative liver weight was slightly, but significantly increased. The significant increase in relative kidney weight of the male animals of the low concentration group was considered an isolated incidental finding, not related to the treatment. Other significant differences in organ weight were not found.

GROSS PATHOLOGY
- Macroscopic examination at necropsy did not reveal treatment-related gross changes.

HISTOPATHOLOGY: NON-NEOPLASTIC
- Microscopic examination revealed treatment related histopathological changes in the hearts of the treated females and males. The changes comprised very slight to moderate multifocal (diffuse in one high-dose male) mononuclear cell infiltrates in the ventricular part of the heart muscle. Several animals showed very slight to slight focal mononuclear cell infiltrates in the heart, but this was not considered treatment related because this finding is a common background change and occurred also in some control animals. The multifocal inflammatory infiltrations were observed in 9/10 high-dose males, in 5/10 high-dose females, in 7/10 mid-dose males and in 1/10 low-dose males. In several cases the inflammatory changes were accompanied by vacuolisation of cardiac muscle cells. The males were clearly more affected than the females. Because of the presence of moderate multifocal mononuclear cell infiltrates with concomitant muscle cell vacuolation in the heart of one male animal of the low-concentration group, a No Adverse Effect Level could not be established. The other histopathological changes observed are common findings in rats of this age and strain. Furthermore, they were about equally distributed amongst the groups or they occurred in only one or a few animals.
Dose descriptor:
LOAEC
Effect level:
4 000 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
other: see 'Remark'
Critical effects observed:
not specified
Conclusions:
Under the conditions of the test, a NOAEL in rats exposed to the test substance for 6 hours/day, 5 days/week for a period of 90 days could not be derived. Therefore the lowest concentration was considered to be the LOAEC: 4000 ppm (21240 mg/m3).
Executive summary:

In a 90-day repeated dose toxicity study, performed according to OECD Guideline 413 and under GLP, the test substance was administered by inhalation 6 hours/day on 5 days/week at concentrations of 4000, 10000, and 15000 ppm to groups of 10 Sprague-Dawley rats per sex each. The actual concentrations, as measured by total carbon analysis, were close to the target concentrations. Daily observation of the animals did not reveal treatment-related clinical abnormalities. Treatment-related differences in body weight gain or food consumption were not seen. Ophthalmoscopic examination near the end of the exposure period did not reveal any abnormalities. Treatment-related effects in red blood cell parameters were not seen. In male animals of the high concentration group the relative concentration of lymphocytes was decreased. No other treatment-related effects were seen in any of the other haematology parameters. In the high concentration group, the concentration of ASAT and ALAT in blood plasma was significantly increased in male animals. In female animals of the high and mid concentration groups glucose and urea were increased. Female animals of the high concentration group also showed increased potassium and decreased triglyceride levels. Other treatment-related changes in clinical chemistry parameters were not seen. Occult blood in urine was significantly increased in male animals of the high concentration group. The relative and absolute weight of the heart was significantly decreased and the relative weight of the liver was significantly increased in male animals of the high concentration group. Relative weight of the heart was also significantly decreased in female animals of the high concentration group. No other treatment-related changes in absolute or relative organ weights were detected. Macroscopic examination at necropsy did not show exposure-related gross changes. Microscopic examination revealed that exposure to the test substance induced multifocal mononuclear cell infiltrates in the ventricular part of the heart muscle. It was seen in males and females of the high-concentration group and with a lower incidence in male animals of the mid and low concentration groups. Because the histopathological changes in the heart of one male animal of the low concentration group was considered to be related to the exposure, a NOAEL can not be given and the lowest concentration tested was considered to be the LOAEC: 4000 ppm (21240 mg/m3).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
21 240 mg/m³
Study duration:
subchronic
Species:
rat
Quality of whole database:
High, 2 week, 4 week and 13 week inhalation studies all performed according to OECD Guidelines and GLP are available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

A 14-day, 28-day and a 90-day inhalation toxicity study are available investigating the toxicity of the test substance after repeated exposure.

In the 14-day repeated dose toxicity study, performed according to OECD Guideline 412 and under GLP, the test substance was administered by inhalation 6 hours/day on 5 days/week at target concentrations of 2000, 7500, and 20000 ppm to groups of 5 Sprague-Dawley rats per sex each (TNO, 2008). The actual concentrations, as measured by total carbon analysis, were close to the target concentrations. At the start of the generation of the test atmospheres, the animals of the high concentration group, and to a lesser extent those of the low- and mid-concentration groups, were restless. Restlessness was no longer seen after about 15 minutes. No other clinical abnormalities were seen during the study. Body weight and food consumption were not affected by treatment.Treatment-related changes in haematology parameters were limited to an increase in prothrombin time in high concentration group females and an increase in absolute and relative number of monocytes and absolute number of neutrophils in males of the high concentration group. Clinical chemistry revealed treatment-related changes in ALAT and ASAT (increased in high concentration group males), plasma fasting glucose level (increased in mid and high concentration group females), and plasma urea concentration (increased in males and females at the high concentration). These clinical chemistry changes were not corroborated by histopathological changes.Microscopic examination showed histopathological changes in the heart of mid and high concentration group animals, characterized by multifocal mononuclear cell infiltrates. No other treatment-related histopathological changes were found in the organs examined.Based on the histopathological changes in the heart at 7500 and 20000 ppm, it can be concluded that the NOAEL in rats exposed to the test substance for 6 hours/day, 5 days/week for a period of 14 days was at 2000 ppm (the lowest concentration tested).

In a 28-day repeated dose toxicity study, performed according to OECD Guideline 412 and under GLP, the test substance was administered by inhalation 6 hours/day on 5 days/week at target concentrations of 2000, 4500, 7500, and 10000 ppm to groups of 5 Sprague-Dawley rats per sex each (TNO, 2009b). Additionally, groups of 5 animals per sex similarly exposed to clean air or 10000 ppm were kept for a recovery period of 14 days before sacrifice. The actual concentrations, as measured by total carbon analysis, were close to the target concentrations. No treatment-related clinical signs were seen. No effects were found on body weight or food consumption. Changes in haematology parameters were limited to an increase in the percentage of basophils in males exposed to 10000 ppm. Statistically significant intergroup differences in clinical chemistry parameters were limited to a decrease in creatinine in males of the 10000 ppm group, an increase in potassium in males of the 7500 and 10000 ppm groups, and an increase in cholesterol in females of the 2000 and 4500 ppm groups. At the end of the recovery period, haematology and clinical chemistry parameters showed no statistically significant differences. Urinalysis revealed no treatment-related changes. No treatment-related effects were found on organ weights (both absolute and relative) in animals of the main groups or the recovery groups. Treatment-related gross findings or histopathological changes were not observed. In the 14-day study preceding this 28-day study, multifocal mononuclear cell infiltrates were observed in the heart of rats exposed to 7500 or 20000 ppm. In the present 28-day study this lesion was not seen up to the highest concentration tested (10000 ppm). The few changes in haematology and clinical chemistry parameters found in the high concentration group (10000 ppm) are mild and reversible in a 14-day recovery period. It cannot be excluded that the slight increase in plasma postassium in males exposed to 7500 ppm is treatment related. Therefore, the NOAEL in this study was conservatively placed at 4500 ppm (23895 mg/m3).

In the 90-day repeated dose toxicity study, performed according to OECD Guideline 413 and under GLP, the test substance was administered by inhalation 6 hours/day on 5 days/week at concentrations of 4000, 10000, and 15000 ppm to groups of 10 Sprague-Dawley rats per sex each (TNO Triskelion, 2011a). The actual concentrations, as measured by total carbon analysis, were close to the target concentrations. Daily observation of the animals did not reveal treatment-related clinical abnormalities. Treatment-related differences in body weight gain or food consumption were not seen. Ophthalmoscopic examination near the end of the exposure period did not reveal any abnormalities. Treatment-related effects in red blood cell parameters were not seen. In male animals of the high concentration group the relative concentration of lymphocytes was decreased. No other treatment-related effects were seen in any of the other haematology parameters. In the high concentration group, the concentration of ASAT and ALAT in blood plasma was significantly increased in male animals. In female animals of the high and mid concentration groups glucose and urea were increased. Female animals of the high concentration group also showed increased potassium and decreased triglyceride levels. Other treatment-related changes in clinical chemistry parameters were not seen. Occult blood in urine was significantly increased in male animals of the high concentration group. The relative and absolute weight of the heart was significantly decreased and the relative weight of the liver was significantly increased in male animals of the high concentration group. Relative weight of the heart was also significantly decreased in female animals of the high concentration group. No other treatment-related changes in absolute or relative organ weights were detected. Macroscopic examination at necropsy did not show exposure-related gross changes. Microscopic examination revealed that exposure to the test substance induced multifocal mononuclear cell infiltrates in the ventricular part of the heart muscle. It was seen in males and females of the high-concentration group and with a lower incidence in male animals of the mid and low concentration groups. Because the histopathological changes in the heart of one male animal of the low concentration group was considered to be related to the exposure, a NOAEL cannot be given and the lowest concentration tested was considered to be the LOAEC: 4000 ppm (21240 mg/m3).

To investigate whether the myocardial lesion observed in one male rat (animal number B0022) of the low concentration group of the above 90 -day inhalation toxicity study should be regarded as test article-related or as a spontaneous lesion, a comprehensive review of the cardiac lesions identified this 90-day study was conducted (by Experimental Pathology Laboratories, Inc., 2013) in accordance with applicable regulatory guidance and scientific best practice (Crissman et al., 2004; Morton et al., 2006).

Microscopic sections of heart were reviewed in conjunction with the final study report. The review applied a single set of diagnostic criteria for cardiac alterations (Ruben et al., 2000) and a uniform diagnostic threshold. Briefly, the diagnostic criteria used for the spontaneous alterations were infiltration of the myocardium by mononuclear cells, focal or multifocal distribution, and most commonly in the apex (Ruben et al., 2000; Lewis, 1992).

Two distinct patterns of lesions are evident in the heart in this study. Those lesions typical of rodent spontaneous cardiomyopathy in young rats are localized primarily in the apex and/or adjacent portions of the right and left ventricles and interventricular septum. Lesions of the spontaneous type were characterized by variably sized aggregates of mononuclear cells, lymphocytes and macrophages, within interstitial spaces. Adjacent cardiac myocytes were typically unaffected. On rare occasions, the adjacent myofibers had small cytoplasmic vacuoles or an increase in eosinophilic staining.

Test article-induced changes initially appeared in the ventricular free wall, most often the left ventricle, at the heart base, or in the atria. This is consistent with observations in F344 rats exposed to a variety of cardiotoxic chemicals (Jokinen et al., 2005). The earliest characteristics of the spontaneous and induced lesions were morphologically identical as infiltrates of mononuclear cells and differed only in their distribution. Extensive lesions due to test article were more inflammatory in nature with degeneration/necrosis of adjacent cardiac myocytes. Mononuclear cells were still the predominant cell type present, but low numbers of neutrophils were sometimes present. More extensive lesions had a similar distribution pattern as the early lesions.

Using the diagnostic approach described above, the myocardial lesion noted in rat B0022 is regarded as a slight multifocal mononuclear cell infiltrate localized to the apex, which is consistent with a spontaneous lesion. This is in contrast to the report diagnosis of moderate multifocal mononuclear cell infiltrate and vacuolation associated with inflammation, which is difficult to differentiate from a test article-induced lesion. As such, this rat is not considered to have a test article-related alteration in the heart. Therefore, the NOAEL for this study is interpreted to be 4000 ppm based on the type, incidence, and severity of morphologic alterations present in the heart of exposed rats. This value is taken forward in the derivation of the DNEL.

References:

Reference of review: Experimental Pathology Laboratories, Inc. (2013). Lack of toxicologic significance of myocardial lesion in rat B0022 exposed to 4,000 ppm HCFO-1233zd.

References cited in review:

- Crissman JW, Goodman DG, Hildebrandt PK, Maronpot RR, Prater DA, Riley JH, Seaman WJ, Thake DC. 2004. Best practice guideline: Toxicologic histopathology. Toxicol Pathol. 32:126-131.

- Jokinen MP, Lieuallen WG, Johnson CL, Dunnick J, Nyska A. 2005. Characterization of spontaneous and chemically induced cardiac lesions in rodent model systems: The National Toxicology Program Experience. Cardiovasc Toxicol. 5:227-244.

- Lewis OJ. 1992. "Nonneoplastic lesions in the cardiovascular system" In : Pathobioloqy of the Aging Rat. Vol 1. Mohr U, Dungworth DL, and Capen CC (Eds). ILSI Press. Washington, DC. 301~303.

- Morton D, Kemp RK, Francke-Carrol! S, Jensen K, McCartney J, Monticello TM, Perry R, Pulido 0, Roome N, Schafer K, Sellers R, Snyder PW. 2006. Best practices for reporting pathology interpretations with GLP toxicology studies. Toxicol Pathol. 34:806 -809.

- Ruben Z, Arceo RJ, Bishop SP, Elwell MR, Kerns WD, Mesfin GM, Sandusky GE, Van Vleet JF. 2000. Non-proliferative lesions of the heart and vasculature in rats. CV-1. Guides for Toxicologic Pathology. STP/ARP/AFIP. Washington, DC.


Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
A 14-day, 28-day and a 90-day inhalation toxicity study, all performed according to OECD Guidelines and GLP, were available. The test with the longest exposure period has been selected as key study.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
No local toxicity was observed in any of the repeated exposure toxicity studies.

Repeated dose toxicity: inhalation - systemic effects (target organ) cardiovascular / hematological: heart

Justification for classification or non-classification

Based on the results of the 14, 28, and 90 -day inhalation toxicity studies, the test substance does not have to be classified for repeated inhalation toxicity in accordance with EU Directive 67/548/EEC (DSD) and EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.