Registration Dossier

Administrative data

Description of key information

oral: The NOAEL for the structurally comparable MCPP-P OE is 120 ppm, corresponding to 10.8 mg/kg bw/day for rats.

inhalative: The NOAEC for male rats is 200mg/m3, the highest dose tested.

dermal: The NOAEL (systemic and local) for rats is 1000 mg/kg bw/day, the highest dose level investigated.

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:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
(1998)
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
Stability under test conditions: The stability of the test substance in the diet was confirmed for at least 15 days
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Strain: Hsd Cpb:WU
- Source: Harlan Laboratories BV, Kreuzelweg 53, 5961 Horst, The Netherlands
- Age at study initiation: about 5 weeks
- Weight at study initiation (mean): males 148-151 g, females 133-138 g
- Housing: in groups of 2 or 3 animals per cage in Makrolon cages Type IV
- Diet and water: ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): approx. 55
- Air changes (per hr): >/= 10
- Photoperiod (hrs dark / hrs light): 12
Route of administration:
oral: feed
Vehicle:
other: fed in diet
Details on oral exposure:
DIET PREPARATION
- Mixing appropriate amounts with (Type of food): Provimi Kliba 3883.G4.S15
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Homogeneity and stability of the test item in the diet were checked prior to study start and confirmed. Content checks performed three times during the study showed that the test item content agreed with the target concentrations and that the test item was homogeneously distributed in the diet within the defined limits.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
fed in diet, continuously
Dose / conc.:
0 ppm
Dose / conc.:
120 ppm
Dose / conc.:
600 ppm
Dose / conc.:
3 000 ppm
No. of animals per sex per dose:
10
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Dose levels were selected based on the results of a subacute pilot study (T2083100). In this study 5 males and females received the test item in dietary concentrations of 0, 1600, 3200 or 6400 ppm over 4 weeks. The dietary concentration of 1600 ppm was not tolerated without effects on the biochemical liver and blood parameters. Starting at 3200 ppm toxic effects were evident, which are expected to result in findings representing or exceeding the maximum tolerated dose (MTD) in a longer study.
Thus, for the subchronic study the dietary concentrations of 0, 120, 600 and 3000 ppm were chosen.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes, all animals
- Time schedule: Inspections on mortality and morbidity of the animals were performed twice daily. General clinical observations (in the home cage) were made daily.
DETAILED CLINICAL OBSERVATIONS: Yes, all animals
- Time schedule: Once before the start of treatment and once weekly thereafter animals were clinically examined in detail including observations in a standard arena (open field) for behavioral observations. Any clinical signs (findings) and abnormalities were recorded. Body surfaces and orifices, posture, general behavior, breathing and excretory products were assessed.
BODY WEIGHT: Yes, all animals
- Time schedule for examinations: Body weights of animals were determined before the study start and weekly thereafter up to scheduled necropsy. Furthermore, body weights were also recorded immediately before scheduled necropsies for calculation of relative organ weights.
FOOD AND WATER INTAKE : Yes, all animals
Food and water intake was determined per cage at comparable periodical intervals (e.g. weekly). These primary data were then used to calculate the group means for each period of approximately 7 days.
On the basis of these data the following parameters were calculated:
- for each interval: daily food intake per animal, mean daily food intake per animal, mean daily food intake per kg body weight;
- for the total period: measurement of mean food intake per animal and day, mean food intake per kg body weight and day;
- cumulative food intake per animal and cumulative food intake per kg body weight.
Comparable calculations were done for water intake.
OPHTHALMOSCOPIC EXAMINATION: Yes, all animals
- Time schedule for examinations: All animals were subjected to ophthalmological inspection before start of treatment and on day 85.
The pupillary reflex of both eyes was first tested in a darkened room and the anterior regions of the eye were inspected. After dilating the pupils with Mydriaticum Stulln® drops the refractive compartment of the eye as well as iris and fundus were examined using an indirect ophthalmoscope. In addition, the optical media were examined with a ZEISS photo-slit lamp.
HAEMATOLOGY: Yes, all animals
The blood samples were collected in the morning from the retro-bulbar venous plexus of fasting animals anesthetized with CO2/air. The samples for the hematological determinations were collected in tubes coated with EDTA (anticoagulant). The samples for the determinations of the thromboplastin time (HQUICK) were collected in tubes with sodium-citrate.
- Time schedule for collection of blood: day 86, 87
- Parameters investigated: Erythrocyte count, Hemoglobin concentration, Hematocrit, Mean corpuscular hemoglobin, Mean corpuscular hemoglobin concentration, Mean corpuscular volume, Reticulocyte count, Erythrocyte morphology, Thrombocyte count, Thromboplastin time (Hepato-Quick), Leukocyte count, Differential blood count.
CLINICAL CHEMISTRY: Yes, all animals
The blood samples for determination of glucose concentrations were taken from the caudal vein of fasting, non-anesthetized animals. The blood samples used for determining the other parameters in peripheral blood were collected in the morning from the retro-bulbar venous plexus of fasting animals anesthetized with CO2/air. The samples for biochemical tests were heparinized. The blood samples for glucose determinations were mixed with perchloric acid (1+10) to precipitate proteins.
- Time schedule for collection of blood: day 86, 87
- Parameters investigated: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Gamma glutamyl transferase, Glucose, Cholesterol, Triglyceride, Creatinine, Urea, Total bilirubin, Total protein, Albumin, Inorganic phosphate, Potassium, Sodium.
URINALYSIS: Yes, all animals
Urine samples were collected at room temperature during a period of 16 hours. During the urine collection periods, water was offered ad libitum but feed was not supplied.
- Time schedule for collection of urine: day 86, 87
- Parameters investigated: Density, Volume, Protein concentration, Protein excretion, Creatinine concentration, Creatinine excretion, Protein/creatinine ratio, Urea concentration, Urea excretion; qualitatively: Blood, Bilirubin, Glucose, Ketone bodies, Urobilinogen, Microscopy of sediment, pH.
NEUROBEHAVIOURAL EXAMINATION: Yes, all animals
- Time schedule for examinations: Functional observations were performed once (not blind)
Functional Observational Battery (FOB) was performed on days 79, 80, 83, 84. The following observations/examinations were performed: Body weights, home-cage observation, observations during handling, open field observations, reflex/physiological observations.
Motor and Locomotor Activity (MA) was performed on day 77-80. The activity for the entire 60-minute session (Summary Session MA and LMA) and activity during each 10-minute interval (Summary Interval MA and LMA) was examined. Motor activity was measured as the number of beam interruptions that occurred during the test session. Locomotor activity was measured by eliminating consecutive counts for a given beam. Thus, for locomotor activity, only one interruption of a given beam was counted until the animal relocated in the maze and interrupted one of the other beams. Habituation was evaluated as a decrement in activity during the test session.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, all animals
Scheduled necropsy: day 93 (males), day 97 (females)
All animals living on the date of their scheduled necropsy and all animals to be killed in moribund state were sacrificed by exsanguination under isoflurane anesthesia, necropsied and their organs and tissues subjected to thorough gross pathological examination.
At study termination the following organs of animals sacrificed scheduled were weighed before fixation: Brain, Adrenals (both), Heart, Liver, Kidneys (both), Spleen, Thymus, Testes (both), Epididymides (both), Uterus.
HISTOPATHOLOGY: Yes
The following organs, tissues or representative pieces of them were fixed in 10 % neutral buffered formalin or Davidson's solution and were histopathologically evaluated for control and all treated groups:
Abnormalities, Esophagus, Kidneys, Liver, Thyroid glands (with parathyroids), Trachea.
The following organs, tissues or representative pieces of them were fixed in 10 % neutral buffered formalin or Davidson's solution and were histopathologically evaluated for control and high dose group:
Adrenal glands, Aorta, Brain (cerebrum, cerebellum, brain stem), Epididymides, Eyes, Eyelids, Femur (joint, bone marrow), Heart, Intestine (Duodenum, Jejunum, Ileum, Cecum, Colon, Rectum), Larynx, Lungs, Lymph nodes (mandibular, mesenteric, popliteal), Optic nerves, Pancreas, Pharynx, Pituitary gland, Prostate, Salivary glands (parotid, submandibular, sublingual), Sciatic nerve, Seminal vesicles (incl. coagulating glands), Skeletal muscle (thigh), Skin (mammary region), Spinal cord (cervical, thoracic, lumbar), Spleen, Sternum with Bone Marrow, Stomach, Testes, Thymus, Trachea, Urinary bladder, Uterus.

The following organs, tissues or representative pieces of them were fixed in 10 % neutral buffered formalin or Davidson's solution:
Exorbital lacrimal glands, Harderian glands, Head (with skull cap, Nasal Cavity), Intestine (Peyer's patches), Mesentery, Tongue, Ureters, Urethra, Vagina, Zymbal's glands, Physical identifier.
Statistics:
For body weights, body weight gain and absolute organ weights: Dunnett Exact Homogeneous Test; for relative organ weights: Dunnett Exact Homogeneous Test after log. Transformation; for calculated food/water intake per animal: adjusted Mann-Whitney U-tests; for clinical pathology parameters: Dunnett Exact Homogeneous or Heterogeneous Test, the Dunnett Exact Homogeneous Test after log. Transformation or the Bonferroni/Mann-Whitney U-test.
Descriptive statistics were provided per sex, dose group and time point for all parameters that were recorded with a specified unit.
For continuous variables, the statistical test procedure was based on prior knowledge of the respective variable derived from previous studies. For normally distributed variables with equal variances across treatment groups Dunnett’s tests were performed. Heteroscedastic normally distributed variables were analyzed using appropriately adjusted Dunnett’s tests, using Satterthwaite adjustments for the degrees of freedom and taking the different variances within the groups into account. For log-normally distributed variables, Dunnett's tests were performed after log transformation of the original values. If experience with historical data indicated that the assumptions for parametric analyses are violated, Bonferroni-adjusted Mann-Whitney U-tests were employed in the analyses. For small sample sizes, the exact version of this test was used.
With respect to data collected in the functional observational battery categorical variables were analyzed with a repeated measures analysis of variance followed by a one-way analysis of variance using the SAS procedure PROC CATMOD.
Statistics of MA/LMA were be generated with an evaluation step of the SPADER (=Safety Pharmacology Automated Data Evaluation and Reporting) application.
For statistical evaluations of histopathological findings, if any, the PATHDATA program was used.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical observations revealed no toxicological relevant findings up to and including 600 ppm in males and females. At the dose of 3000 ppm, all animals showed increased feces excretion. The finding of gnawed fur on forelegs in several male animals of all groups and in some females in the low and high dose group was considered a chance finding, as it is a known spontaneous finding in rats and did not show a clear dose-dependence.
Mortality:
no mortality observed
Description (incidence):
All male and female animals survived until scheduled necropsy.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight gain was not affected at 120 and 600 ppm. Body weight gain was slightly decreased in males and moderately decreased in females at 3000 ppm, so that the body weight at the end of the study was reduced 5.4% in males and 8.1% in females.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food intake was increased in female animals starting at 120 ppm throughout the study. At the low and mid dose this translated into a slightly higher body weight and an increased body weight gain at the mid dose. So this was not considered as a toxicological relevant finding. At the high dose body weight gain was reduced so that there was a mismatch, indicating some disturbances in digestion or metabolism. In males this tendency was not observed.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, non-treatment-related
Description (incidence and severity):
Water consumption showed a transient increase in males dosed at 3000 ppm from day 15 up to day 36 of measurement, but not during the further course of treatment. Therefore, it was considered an adaptive change without toxicological relevance.
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Few animals in the control and dose groups showed waterklefts, calcification, retrolental opacity or a zone of discontinuity. All of these effects were randomly distributed without dose dependence and are therefore of no toxicological relevance.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Hematology revealed no toxicologically relevant changes at the doses of 120 and 600 ppm. At the high dose of 3000 ppm, some minor changes were found in male and female animals in red blood cell parameters and in males also in coagulation. These minor changes were not considered indicative of a toxic effect. However, as similar changes were observed in the previous 4-week toxicity study, they are considered indicative of a compound-related effect. This included a slight reduction in erythrocyte count and in hematocrit and a slight increase in mean corpuscular hemoglobin and in mean corpuscular hemoglobin concentration in both sexes. In addition, slight prolonged Hepato Quick was observed in high dose males. The higher monocytes count found in males starting at 120 ppm was not considered to be toxicologically relevant, as the values of the individual animals were within the range of reference values and since the control group showed low values in comparison to reference values, so that the difference was considered a chance finding due to the control values.

Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical chemistry did not reveal any clear-cut adverse findings. At the high dose of 3000 ppm, a decrease in glucose, a slight increase in alkaline phosphatase and a decrease in cholesterol were observed in the high dose males. In both sexes, the creatinine and protein levels were decreased. Most values were well within the range of historical controls and all differences were slight, so that these alterations were considered to be of minor relevance. Potassium was slightly increased in the high dosed males but even the control animals were in the upper range of the historical control data, therefore this finding was considered to be of minor relevance.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
Urinalysis did not reveal any findings, which are considered indicative of a toxic effect. At 600 and 3000 ppm, male animals showed a lower protein excretion and in parallel a reduction in the total amount of excreted protein and in the protein-creatinine ratio. In addition, pH was slightly more acidic than in controls. In females, urinary volume was lower and in parallel, urinary density and excretion of urea were increased and the total amount of excreted creatinine, urea and protein were lower. Most values were well in the range of historical control data, so that they are considered in the physiological range. In addition, such changes were not considered as indicative of any relevant organ toxicity.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No treatment-related effects in the functional observation battery were observed and no statistically significant effects on motor and locomotor activity were seen.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Organ weight evaluation did not reveal any changes at the dose of 120 ppm. At 600 ppm and higher, absolute and relative kidney weight was slightly increased in male animals. In female animals, only the high dose was affected. In addition, at 3000 ppm, relative and absolute liver weights were increased in females. There were further changes in relative organ weights: In high dose males, relative weights of adrenal glands, testes and epididymis were increased. In high dose females, relative weights of brain and adrenal glands were increased. These changes were considered a secondary effect to the reduced body weight since the absolute weights were non-altered. Thus, they were not considered of toxicological relevance.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Histopathology revealed no adverse effects at 120 ppm. Nephropathy of the renal P3 segment occurred in females at 600 ppm and above. Tubules in this area appeared to be dilated, flattened and filled with proteinaceous or cellular debris. Males showed minimal nuclear crowding of cortical tubules with an incidence of 1-2-3-5. The relevance of this non-adverse finding remains debatable. Hepatocellular hypertrophy and eosinophilic change were found in all males and females at 3000 ppm, few animals showed minimal single cell necrosis. Females were more affected by incidence and grading than males. Hepatocellular fat storage was decreased in incidence in 3000 ppm males. Diffuse follicular cell hypertrophy of the thyroid was observed in 4/10 females at 3000 ppm. This finding is possibly related to increase liver metabolism of thyroidal hormones and thus represents a secondary, reactive change.
Histopathological findings: neoplastic:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
120 other: ppm (corresponding to 10.8 mg/kg bw and day)
Sex:
male/female
Basis for effect level:
other: Adverse effects on the kidney (nepropathy, organ weight) at next high dose level (600 ppm)
Key result
Critical effects observed:
no
Conclusions:
The subchronic study with the structurally related MCPP-P OE indicated the kidney and liver as target organs for systemic toxicity. The NOAEL for systemic toxicity was 120 ppm, corresponding to 10.8 mg/kg bw/day for males and females.
Executive summary:

A subchronic repeated dose toxicity study with the structurally related MCPP-P OE was conducted according to OECD TG 408 and GLP. The test substance was administered orally via the diet to 10 male and 10 female Wistar rats per dose group in doses of 0, 120, 600 and 3000 ppm up to 97 days. Mortality was not observed during the course of the study. Ophthalmoscopical examinations revealed no evidence of treatment-related effects. No treatment-related effects were observed in the functional observation battery and on motor/locomotor activity. Gross pathology did not reveal any adverse effects. The dose of 600 ppm caused nephropathy in females. In addition, absolute and relative kidney weight was increased in male animals. The dose of 3000 ppm caused increased feces excretion in both sexes, increased food consumption in females, a slight to moderate decrease in body weight gain, minor changes in red blood cell parameters and coagulation, minor changes in plasma in one or both sexes, an increased absolute and relative weight of liver in females and an increased absolute and relative weight of kidneys in females. Microscopically, hepatocellular hypertrophy and eosinophilic change were found in all males and females, few animals showed minimal single cell necrosis. Hepatocellular fat storage was decreased in males. In summary, at 600 ppm and higher, adverse effects were observed on the kidney. At the high dose, liver was also affected. In addition, there are indications on alterations in feces, body weight development, food consumption, clinical chemistry and blood cell parameter. The NOAEL was 120 ppm, corresponding to 10.8 mg/kg bw/day for males and females.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
10.8 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
GLP and Guideline study (OECD TG 408), Read Across

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 January 2017 - 28 August 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Version / remarks:
2009
Deviations:
no
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- batch No.of test material: CHP0006

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
Storage conditions: Room temperature (15 to 25 °C)
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The rat is an animal model commonly utilized in this type of toxicity studies and is recommended by current testing guidelines, e.g. OECD 412. In addition, a historical database of this species and strain of rats (Sprague-Dawley CD® outbred), equally exposed by inhalation, is available in the laboratory for comparison.
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Envigo Dublin, Virginia
- Age at study initiation: approximately 9 weeks
- Weight at study initiation: 210 g to 286 g
- Fasting period before study: no
- Housing: Polycarbonate cages with a stainless steel mesh lid, nominally 2 or 3 per cage
- Diet: ad libitum: Teklad Global 16% Protein Rodent Diet (Certified), 2016C Envigo, Madison, Wisconsin
- Water: ad libitum: Potable water from the public supply (New Jersey-American Water Company, Cherry Hill, New Jersey) via an automated watering system
- Acclimation period: at least 6 days

DETAILS OF FOOD AND WATER QUALITY: There were no known contaminants in the feed and water that were expected to interfere with the results of this study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-26
- Humidity (%): 30-70
- Air changes (per hr): no information
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
>= 2.7 - <= 3.1 µm
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Flow through (ADG) snout-only exposure system

- Method of holding animals in test chamber: Animal restraint tube

- Source and rate of air: Dry air from in-house compressed air system – breathing quality

- System of generating particulates/aerosols:
Group 1 – Air control
Groups 2 to 4 – Micro atomizer manufactured by Envigo
One Micro atomizer with a fine needle for each group
Dynamic test item feed from disposable clinical syringe driven by syringe pump

- Temperature, humidity, pressure in air chamber: Monitored using Sunbeam and Springfield Apollo Humidity and Thermometer gauges as follows:
Frequency – continuous monitoring; documented at approximately 30 minute intervals
Sample location – gauges located in restraint tube on the exposure system
Sample position remained constant

- Air flow rate:
Inlet Generation Airflow:
Group 1 – 68.0 L/minute
Groups 2 to 4 – 4.0 L/minute
Dilution Airflow:
Dry air from in-house compressed air system – breathing quality
Flow – 64.0 L/minute per system (Groups 2 to 4)
A further 2.0 L/minute room airflow drawn passively into chamber via
tangential inlet port for each group (Groups 1 to 4)
Extract Airflow:
Drawn by in-house extract system
Drawn from base of exposure system
Flow – 70.0 L/minute per system
Filtered locally

- Method of particle size determination: Determined by cascade impaction
samples collected as follows:
Impactor type – Marple 290 Series (296 configuration)
Sample collection media – stainless steel substrates and glass fiber final stage filter
Sample flow – 3.0 L/minute
Sample volume – measured by wet-type gas meter
Sample frequency – minimum of 1 sample/test group/week
Sample location: Representative animal exposure position
Sample analysis: Gravimetric – Groups 2 to 4, Chemical (HPLC/UV) – Groups 2 to 4
MMAD and σg derived
Sample disposition
Substrates and final stage filters analyzed by FIA

Aerosol samples collected as follows:
-Filter type – glass fiber, held in an open face filter holder
-Sample flow – 2.0 L/minute
-Sample volume – measured by timed flowmeter
-Sample frequency – minimum of 3 samples/group/exposure
-Sample location: Representative animal exposure position
Sample analysis: Gravimetric – Groups 1 to 4; Chemical (HPLC/UV) – Groups 1 to 4
Sample disposition: Filters sent for chemical analysis by FIA – Exposure Days 1 to 4 and then weekly; filters not sent for analysis stored refrigerated (2 - 8ºC) an subsequently discarded after study completion
-Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Matrix: Filter papers; stainless steel slides
Calibration Range: 5.00 μg/mL to 100.00 μg/mL
Regression Type: Linear
Data Calculation: Peak area
Limit of Quantification: 5.00 μg/mL
Analytical Instrumentation: HPLC/UV Agilent Technologies
Type of Detection: UV
-Column Type Phenomenex Hypersil BDS C18 Supplied by Agilent
-Column Dimensions Length = 100 mm
Diameter = 4.6 mm
Particle Size = 5 µm
-Column Temperature 40°C
-Run Time 6.0 minutes
-Injection Volume 50 µL
Mobile Phase: Acetonitrile / Water (80 / 20, v/v)
Extraction Solvent /Sample Diluent: Acetonitrile / Water (70 / 30, v/v)
Duration of treatment / exposure:
4 weeks (+ 4 weeks test item-free recovery period)
Frequency of treatment:
6 hours once daily 5 days a week
Dose / conc.:
0 mg/L air
Dose / conc.:
0.02 mg/L air (nominal)
Remarks:
Exposures represent active ingredient, which represents approximately 0.013 mg/L
mecoprop-p acid equivalent
Dose / conc.:
0.05 mg/L air (nominal)
Remarks:
Exposures represent active ingredient, which represents approximately 0.033 mg/L
mecoprop-p acid equivalent
Dose / conc.:
0.2 mg/L air (nominal)
Remarks:
Exposures represent active ingredient, which represents approximately 0.130 mg/L
mecoprop-p acid equivalent
No. of animals per sex per dose:
28
Control animals:
yes, concurrent no treatment
Details on study design:
A three week preliminary inhalation investigation was conducted at target exposure levels of 0, 0.05, 0.22 and 1.0 mg/L to assess the toxicity and toxicokinetic profile of these exposure levels of MCPP-p 2-EHE to rats when administered via nose-only inhalation. The levels for the preliminary investigation were selected based on the results of non-inhalation studies of multiple phenoxy compounds where weight gain impairment and other changes to liver and kidney weights and histopathology were observed at approximately 0.3 mg/kg/day that would approximate an inhalation level of 1.0 mg/L. Only males were evaluated in this study because prior non-inhalation test results showed the male to be more sensitive to the test item than the female.

Training for dosing:
The animals on study were acclimated to the method of restraint for 3 days (10 minutes on Day 1, 30 minutes on Day 2 and 60 minutes on Day 3) within the 7 days preceding their initial test item exposure.


Animals were restrained (individual plastic exposure tubes) during the exposure administration procedures for a maximum of 420 minutes per day. This daily period of restraint included the 360 minutes of exposures during which interruptions for technical reasons were expected. This daily period of restraint also included the pre-exposure and post-exposure intervals in which the animals were placed in or removed from the plastic exposure tubes.
Positive control:
None
Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Animals were removed from their cages and examined twice pretest and weekly during the study period (after exposure on exposure days). Examinations included observations of general condition, skin and fur, eyes, nose, oral cavity, abdomen and external genitalia as well as evaluations of respiration.

BODY WEIGHT: Yes
- Time schedule for examinations: Non-fasted body weights were recorded for all animals twice pretest and twice weekly during the exposure period (prior to exposure on exposure days). During the recovery period, non-fasted
body weights were recorded weekly. Terminal, fasted body weights were obtained just prior to necropsy.

FOOD CONSUMPTION: Yes
Food consumption was measured (weighed) weekly (once every 7 days), beginning one week prior to treatment.

OPHTHALMOSCOPIC EXAMINATION: Yes
All animals were examined pretest and during the 4th week of the exposure period. Only animals which were within normal limits at the pretest examination were placed on test. Lids, lacrimal apparatus and conjunctiva were examined visually. The cornea, anterior chamber, lens, iris, vitreous humor, retina and optic disc were examined by indirect ophthalmoscopy. The pupils of each animal were dilated prior to examination using tropicamide ophthalmic solution.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the exposure period
- Anaesthetic used for blood collection: Yes, isoflurane anesthesia
- Animals fasted: Yes
- How many animals: 10 animals/group
- Parameters checked: Hemoglobin (HGB), Hematocrit (HCT), Red blood cell count (RBC), Platelet count (PLT), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Red cell distribution width (RDW), Total white blood cell count (WBC), Reticulocyte count (RETIC), Differential white blood cell count, Neutrophils (ANEU), Lymphocytes (ALYM), Eosinophils (AEOS), Basophils (ABASO), Monocytes (AMONO), Large unstained cells (ALUC), Prothrombin time (PT), Activated partial thromboplastin time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the exposure period
- Animals fasted: Yes
- How many animals: 10 animals/group
- Parameters checked: Aspartate aminotransferase (AST), Alanine aminotransferase (ALT), Alkaline phosphatase (ALKP), Blood urea nitrogen (BUN), Creatinine (CREAT), Glucose (GLU), Triglycerides (TRIG), Total protein (TP), Albumin (ALB), Total bilirubin (TBILI)
Sodium (NA+) , Potassium (K+) , Chloride (Cl-), Calcium (Ca++), Inorganic phosphorus (PHOS)

URINALYSIS: Yes
- Time schedule for collection of urine: Urine was collected into ice-chilled containers overnight (approximately 12 to 16 hours)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Not specified
- Parameters checked: pH, Protein, Glucose (GLU), Ketones (KET), Bilirubin (BIL), Occult blood (BLD), Appearance (APP), Specific gravity (Sp.G.), Volume (VOL)
When the protein result was ≥100 mg/dL, microscopic examination of the urine sediment was performed on centrifuged urine samples via manual microscopy and the following cellular
elements identified: Sperm (SPM), Red blood cells (RBC), White blood cells (WBC), Epithelial cells (EPC), Casts (CAST), Amorphous matter (AMOR), Triple phosphate (Trip/Phos), Other crystals (Oth/Cryst)

OTHER:
Body Temperature
Body temperature was recorded for 5 randomly selected animals/group shortly after the first exposure (within 1 hour) and after an exposure during the last week of exposure (within 1 hour). Body temperatures were recorded via rectal probes.

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Statistics:
If Bartlett's test for variance homogeneity was not significant at 1% level, then parametric methods were applied. If the F1 approximate test for monotonicity of exposure-response was not significant at the 1% level, Williams' test for a monotonic trend was applied. If the F1 test was significant, suggesting that the exposure-response was not monotone, Dunnett's test was performed instead. If Bartlett's test was significant at 1% level, then logarithmic and square-root transformations were tried. If Bartlett's test was still significant, then non-parametric methods were applied to mean ranks. If H1 approximate test for monotonicity was not significant at 1% level, Shirley's test for a monotonic trend was applied. If H1 test was significant, then Steel's test was performed instead.
If Jonckheere-Terpstra test was significant at the 5% level, then direction of trend was established and one-tailed step-down testing in this direction was performed. If the Jonckheere-Terpstra test was not significant at the 5% level, then the Kruskal-Wallis test was applied. If the Kruskal-Wallis test was significant at 5% level, the test-item treated groups were compared to the control using exact Wilcoxon rank sum tests, otherwise, no further comparisons were made. For pre-treatment data, a Kruskal-Wallis test was performed. If test was significant at 5% level treated groups were compared to control using exact Wilcoxon rank sum tests. Comparisons involving only two groups were performed using Wilcoxon rank sum tests.
For comparisons involving more than two groups, if Cochran-Armitage test was significant at 5% level, then the direction of the trend was established and one-tailed step-down testing in this direction was performed. If the Cochran-Armitage test was not significant at 5% level, then the χ2 test was applied. If the χ2 test was significant at 5% level, test-item treated groups were compared to control using Fisher’s Exact tests, otherwise, no further comparisons were made.
Clinical signs:
no effects observed
Description (incidence and severity):
There were no test item-related effects on clinical signs.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no test item-related effects on body weights.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no test item-related effects on food consumption.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There were no test item-related effects on ocular findings.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no MCPP-p-2-EHE-related hematology changes at any exposure level at study termination (Day 29). All differences between control and treated mean values, including those that were statistically significant, were considered not MCPP-p-2-EHE related as they lacked a exposure level relationship and/or there was general overlap between individual control and treated values.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no MCPP-p-2-EHE-related clinical chemistry changes at any exposure level at study termination (Day 29). All differences between control and treated mean values, including those that were statistically significant, were considered not MCPP-p-2-EHE related as there was general overlap between individual control and treated values.
Urinalysis findings:
no effects observed
Description (incidence and severity):
There were no MCPP-p-2-EHE-related urinalysis changes at any exposure level at study termination (Day 29).
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Terminal necropsy: All organ weight changes, including statistically significant changes in pituitary weights at 0.20 ml/L, were considered not to be test item-related because they were sporadic, independent of exposure level, small in magnitude and/or did not have macroscopic or microscopic correlates.
Recovery Necropsy: There was a statistically significant increase in pituitary weight in Group 4 due to one animal with a much higher pituitary weight. All other animals were comparable to controls and the other groups. The higher weight in one animal is considered to be incidental and not test-item related.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no test item-related macroscopic findings. All macroscopic findings occurred sporadically or at a similar incidence and severity in the control and test item exposed groups and were considered incidental and due to biological variability.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Terminal Necropsy:
Larynx
Minimal to slight squamous hyperplasia of the laryngeal respiratory epithelium occurred at ≥ 0.05 mg/L and exhibited an exposure-related increase in incidence and severity. Squamous hyperplasia was located in the anterior larynx along the medial aspect of the arytenoid cartilages. Minimal to slight epithelial alteration was present at ≥ 0.02 mg/L and was located in the anterior ventral larynx at or near the base of the epiglottis. Epithelial alteration is considered to be the initial stage of a generally concentration-dependent transformation from pre-existing epithelium to full laryngeal squamous metaplasia (see table 1 "information on results)
Thymus
Four of 10 animals in the 0.20 mg/L exposure group had minimal to moderate atrophy of the thymus which correlated with a macroscopic observation of small thymus and a trend for lower weight compared to controls. Given the absence of other indicators of stress, the biological significance of this finding is unclear. All other microscopic findings occurred sporadically or at similar incidence and severity in control and test item treated groups and were considered incidental and due to biological
variability.
Recovery Necropsy:
Following an up to 4 week recovery period, the absence of squamous hyperplasia and its precursor change (epithelial alteration) in the larynx was indicative of full recovery of both findings. Full recovery was seen after 1 week.


Histopathological findings: neoplastic:
not examined
Key result
Dose descriptor:
NOAEC
Effect level:
0.2 mg/L air
Based on:
test mat.
Sex:
male
Basis for effect level:
other: highest dose tested
Key result
Critical effects observed:
no

Table 1: Test item-related findings in the larynx

Group/sex

1M

2M

3M

4M

Exposure (mg/L)

0

0.02

0.05

0.20

 

 

 

 

 

Hyperplasia, squamous cell, squam.epith.

 

 

 

 

Minimal

0

0

0

o

Slight

0

0

1

1

Total

0

0

1

3

 

 

 

 

 

Alteration, epithelial, resp. epithelium

 

 

 

 

Minimal

0

5

7

5

Slight

0

0

0

3

Total

0

5

7

8

 

 

 

 

 

Number of tissues examined

10

10

10

10

 

Table 2: Target and mean achieved aerosol concentrations were as follows

Group

Target

Aerosol Concentration (mg/L)

               Gravimetric               Analytical

1

0 (air only)

0.000

0.000

 

 

2

0.02

0.020

0.023

 

 

3

0.05

0.054

0.047

 

 

4

0.20

0.190

0.170

 

 

No MCPP-p-2-EHE was detected in Group 1 samples. Mean achieved gravimetric concentration for Group 2 was at the target, for Group 3 was 8% above target and for Group 4 was 5% below

target. Mean achieved analytical concentration for Group 2 was 15% above target, for Groups 3 and 4 were 6% and 15% below target, respectively. On occasions, during the study, aerosol concentration individual and /or daily mean values were greater than 15% (above or below target).

 

Table 3: Particle size distribution measurements were as follows:

Group

MMAD (µm)      GSD

MMAD (µm)    GSD

 

       Gravimetric

         Analytical

2

3.0                   1.66-1.76

3.1                 1.64-1.91

3

2.5                   1.71-1.84

2.7                 1.73-1.79

4

2.7                   1.63-1.84

2.8                 1.54-1.81

The MMAD values for gravimetric and chemical analysis results showed that the delivered particle size was respirable to the rats.

 

Conclusions:
Under the conditions of this study and based on the histopathology findings, the NOAEC was considered to be the high exposure level of 200 mg/m3.
Executive summary:

Male Sprague-Dawley CD rats (28/group) were exposed by nose-only inhalation for 6 hours, once daily, 5 days per week with 0 (air only), 0.02, 0.05 or 0.20 mg/L MCPP-p-2-EHE for 4 consecutive weeks. At the end of the treatment period, 10 animals/group were euthanized and necropsied and the remaining 18 animals/group were held for an up to 4-week treatment free period. Following a 1, 2 or 4 week recovery period, 6 animals/group were euthanized and necropsied at each interval. Parameters evaluated during the study were: viability, clinical observations, ophthalmology, body weights, body temperature, food consumption, clinical pathology (end of exposure period), organ weights, macroscopic observations and microscopic pathology.

There were no unscheduled decedents. All animals survived until their scheduled termination. Inhalation exposures of MCPP-p 2-EHE to rats for 4 weeks at target exposure levels of 0.02, 0.05 and 0.20 mg/L resulted in no adverse effects on clinical signs, body weights, food consumption, clinical pathology parameters, organ weights or macroscopic pathology. Four-weeks repeat inhalation exposure of male rats to MCPP-p 2-EHE was associated with squamous hyperplasia in the larynx at ≥ 0.05 mg/L and epithelial alteration ≥ 0.02 mg/L, with complete recovery after a one week recovery period. Laryngeal squamous hyperplasia and epithelial alteration are considered to be adaptive non-specific response to chronic irritation. An expert international workshop on laryngeal squamous metaplasia concluded that laryngeal epithelial alteration of minimal to slight severity should be considered non-adverse as they generally regress following a sufficient recovery period after exposure, do not progress over time and are not associated with laryngeal dysfunction.

In conclusion, under the conditions of this study and based on the histopathology findings, the NOAEC was considered to be the high exposure level of 200 mg/m3.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
200 mg/m³
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP and similar to OECD TG 412 study

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Version / remarks:
adopted May 12, 1981
Qualifier:
according to guideline
Guideline:
EPA OPP 82-2 (Repeated Dose Dermal Toxicity -21/28 Days)
Version / remarks:
Revised Edition November 1984
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- batch No.of test material: 5007
- sample No.:1505E
- Expiration date of the lot/batch: March 1993

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 4°C in the dark




Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Dr. Karl Thomae GmbH, Biberach/Riss
- Age at study initiation: 8-10 weeks
- Weight at study initiation: 299 (284-323) g for males and 264 (246 -276) g for females
- Housing: housed singly in type DK III stainless steel wire cages
- Diet: ad libitum, ground Kliba maintenance diet rat/mouse/hamster, 343 meal
- Water: ad libitum
- Acclimation period: 9 days

DETAILS OF FOOD AND WATER QUALITY: The food and water used was assayed for chemical and microbiological contaminants.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light): 12hours/12 hours

IN-LIFE DATES: From: To: July 19, 1993 to August 26, 1993
Type of coverage:
semiocclusive
Vehicle:
unchanged (no vehicle)
Details on exposure:
TEST SITE
- Area of exposure: 50cm2, dorsal and dorsolateral parts of the trunk
- % coverage: 10%
- Type of wrap: 4 layers of absorbent gauze and an elastic dressing
- Time intervals for shavings or clipplings: at least twice once a week

REMOVAL OF TEST SUBSTANCE
- Washing: with lukewarm water
- Time after start of exposure: 6 hours

USE OF RESTRAINERS FOR PREVENTING INGESTION: no
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of the test item in olive oil DAB 10 over 26 hours was confirmed by analysis. In addition, analysis was performed to verify the correctness of the concentrations of the test substance preparations. The content of the active ingredient was determined by combining the results of Chiral HPLC analysis and packed column GC analysis. The analysis of the test substance preparations in olive oil were determined by Reversed Phase HPLC (stability analysis in olive oil) and by HPLC with UV-Detection (concentration control analysis).
Duration of treatment / exposure:
4 weeks (21 applications)
Frequency of treatment:
5 x 6h/week
Dose / conc.:
15 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5
Control animals:
other: yes, solvent control: olive oil DAB 10
Details on study design:
Mecoprop-P 2-EHE was applied 5 days per week for 6 hours to the clipped intact dorsal skin (at least 10 % of the body surface area) of Wistar rats over a period of 4 weeks (21 applications) using a semi occlusive dressing.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day, before and after exposure
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice a day on weekdays and once on weekends
DERMAL IRRITATION: Yes
- Time schedule for examinations: recorded daily, about 30 min after removal of the dressing
BODY WEIGHT: Yes
- Time schedule for examinations: once every week
FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day
HAEMATOLOGY: Yes
- Time schedule for collection of blood: taken in the morning
- Anesthetic used for blood collection: No
- Animals fasted: Yes
- How many animals: 5 animals per test group and sex
- Parameters: leucocytes, erythrocytes, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelets, clotting analysis
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: taken in the morning
- Animals fasted: Yes
- How many animals:5 animals per test group and sex (same as for hematology)
- Parameters checked: Alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, serum-gamma-glutamyltransferase, sodium, potassium, chloride, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol, and magnesium
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Statistics:
For body weight non-parametric one-way analysis was performed using the Kruskal-Wallis test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was carried out. This comparison was performed via the Mann-Withney U-test for hypothesis of equal medians. Both tests were performed two-sided. For all parameters, except the differential blood counts, a non-parametric one-way analysis was performed using the Kruskal-Wallis test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was carried out. This comparison was performed via the MANN-WITHNEY U-test for the hypothesis of equal medians. Both tests were performed two-sided.
Clinical signs:
no effects observed
Description (incidence and severity):
Incidence and types of clinical signs were not adversely affected by treatment.
Dermal irritation:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weight gain in test animals was comparable with that seen in controls.
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:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were some statistically significant inter-group differences in the results of the clinical pathology testing. These deviations are marginal, sporadic or incidental, inconsistent, when compared with the other sex, or lack dosage-relationship. Accordingly, these findings are considered to be of ne toxicological significance.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: No substance-related findings
Key result
Critical effects observed:
no

The stability of the test substance in olive oil DAB 10 over a time interval of 26 hours was verified by analysis. The concentration control analysis yielded 96.5% and 100% of the nominal content for the 1.5g/100 mL and 15g/100 mL samples, respectively.

Conclusions:
The NOAEL of MCPP-P 2-EHE for male and female rats after repeated dermal application for 4 weeks is 1000 mg/kg bw/day for systemic and local effects, the highest dose level investigated.
Executive summary:

An OECD TG 410 study under GLP conditions was performed to assess the dermal toxicity after repeated application of MCPP-P 2-EHE on rat skin over a period of 4 weeks. The unchanged test item was applied on clipped skin in concentrations of 1000, 150 or 15 mg/kg bw/day to 5 male and 5 female rats per dose. The frequency of treatment was 5 days per week for 6 hours. The test substance did not produce local irritation. No substance-related change in hematology and clinical chemistry was observed. In the clinical examinations, no substance-induced changes could be observed in all test groups. No treatment-related significant difference in absolute or relative weight parameters and no treatment-related gross lesions or microscopic findings were observed. The NOAEL of MCPP-P 2-EHE in this study is 1000 mg/kg bw/day for systemic and local effects for male and female rats.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP and Guideline study (OECD TG 410)

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
5.6 mg/cm²
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP and Guideline study (OECD TG 410)

Additional information

oral

A subchronic repeated dose toxicity study with the structurally related MCPP-P OE was conducted according to OECD TG 408 and GLP. The test substance was administered orally via the diet to 10 male and 10 female Wistar rats per dose group in doses of 0, 120, 600 and 3000 ppm up to 97 days. Mortality was not observed during the course of the study. Ophthalmoscopical examinations revealed no evidence of treatment-related effects. No treatment-related effects were observed in the functional observation battery and on motor/locomotor activity. Gross pathology did not reveal any adverse effects. The dose of 600 ppm caused nephropathy in females. In addition, absolute and relative kidney weight was increased in male animals. The dose of 3000 ppm caused increased feces excretion in both sexes, increased food consumption in females, a slight to moderate decrease in body weight gain, minor changes in red blood cell parameters and coagulation, minor changes in plasma in one or both sexes, an increased absolute and relative weight of liver in females and an increased absolute and relative weight of kidneys in females. Microscopically, hepatocellular hypertrophy and eosinophilic change were found in all males and females, few animals showed minimal single cell necrosis. Hepatocellular fat storage was decreased in males. In summary, at 600 ppm and higher, adverse effects were observed on the kidney. At the high dose, liver was also affected. In addition, there are indications on alterations in feces, body weight development, food consumption, clinical chemistry and blood cell parameter. The NOAEL was 120 ppm, corresponding to 10.8 mg/kg bw/day for males and females (AT06591;2013)

inhalative

Male Sprague-Dawley CD rats (28/group) were exposed by nose-only inhalation for 6 hours, once daily, 5 days per week with 0 (air only), 0.02, 0.05 or 0.20 mg/L MCPP-p-2-EHE for 4 consecutive weeks. At the end of the treatment period, 10 animals/group were euthanized and necropsied and the remaining 18 animals/group were held for an up to 4-week treatment free period. Following a 1, 2 or 4 week recovery period, 6 animals/group were euthanized and necropsied at each interval. Parameters evaluated during the study were: viability, clinical observations, ophthalmology, body weights, body temperature, food consumption, clinical pathology (end of exposure period), organ weights, macroscopic observations and microscopic pathology.

There were no unscheduled decedents. All animals survived until their scheduled termination. Inhalation exposures of MCPP-p 2-EHE to rats for 4 weeks at target exposure levels of 0.02, 0.05 and 0.20 mg/L resulted in no adverse effects on clinical signs, body weights, food consumption, clinical pathology parameters, organ weights or macroscopic pathology. Four-weeks repeat inhalation exposure of male rats to MCPP-p 2-EHE was associated with squamous hyperplasia in the larynx at ≥ 0.05 mg/L and epithelial alteration ≥ 0.02 mg/L, with complete recovery after a one week recovery period. Laryngeal squamous hyperplasia and epithelial alteration are considered to be adaptive non-specific response to chronic irritation. An expert international workshop on laryngeal squamous metaplasia concluded that laryngeal epithelial alteration of minimal to slight severity should be considered non-adverse as they generally regress following a sufficient recovery period after exposure, do not progress over time and are not associated with laryngeal dysfunction.

In conclusion, under the conditions of this study and based on the histopathology findings, the NOAEC was considered to be the high exposure level of 200 mg/m3.

In a supportive dose-range finding study Sprague-Dawley CD outbred rats were exposed to 0, 0.05, 0.22 or 1.0 mg/L of MCPP-p-2-EHE via nose-only inhalation, for 6 hours/day, for 5 days/week for 3 consecutive weeks. At the end of the treatment period, 5 animals/group were euthanized and necropsied. Following a 1 week recovery period, the remaining 5 animals/group in Groups 1 and 4 were euthanized and necropsied. Blood and urine samples for toxicokinetic analysis were obtained from two of the Group 4 animals/timepoint on Days 2 and 3 (24 and 48 hours after start of Day 1 exposure) and from 2 animals/group on Day 22 (24 hours after start of Day 21 exposure). Parameters evaluated during the study were: viability/survival, clinical observations, body weights, food consumption, clinical pathology (Days 22 and 29), organ weights, macroscopic observations and microscopic pathology.

The MMAD value (19-2.2 µm) showed the aerosol was respirable to the rats. No mortality or test substance-related clinical signs were observed following inhalation exposures of MCPP-p-2-EHE up to 1.0 mg/L.

Inhalation exposures of MCPP-p-2-EHE to rats for 3 weeks resulted in a slight decrease of questionable toxicological significance (lacking any dose-response) in body weight gain that was seen at the 0.22 and 1.0 mg/L exposure level when compared to control. Partial recovery was seen at 1.0 mg/mL following a 1 week recovery period. Test article-related clinical chemistry findings were limited to minimal to slight, statistically significant, dose-related and test article related decreases in cholesterol at ≥0.22 mg/L (-21% to -42% vs. controls). Microscopically, minimal, panlobular, hypertrophy of hepatocytes was observed at 1.0 mg/L, which correlated with an increase in liver weight. Dose-related minimal to slight squamous metaplasia was observed in the larynx at all exposure levels. Minimal hemorrhage was also noted in the larynx at the 0.22 and 1.0 mg/L exposure levels. Minimal foci of increased alveolar macrophages occurred at a higher incidence in the lung at the 0.22 and 1.0 mg/L exposure level.

All microscopic findings were considered non-adverse since they were of minimal and/or of low severity, completely recovered were not distinguishable across groups after recovery.

In conclusion, under the conditions of this study, the NOAEC was considered to be the high exposure level of 1.0 mg/L.

dermal

An OECD TG 410 study under GLP conditions was performed to assess the dermal toxicity after repeated application of MCPP-P 2-EHE on rat skin over a period of 4 weeks. The unchanged test item was applied on clipped skin in concentrations of 1000, 150 or 15 mg/kg bw/day to 5 male and 5 female rats per dose. The frequency of treatment was 5 days per week for 6 hours. The test substance did not produce local irritation. No substance-related change in hematology and clinical chemistry was observed. In the clinical examinations, no substance-induced changes could be observed in all test groups. No treatment-related significant difference in absolute or relative weight parameters and no treatment-related gross lesions or microscopic findings were observed. The NOAEL of MCPP-P 2-EHE in this study is 1000 mg/kg bw/day for systemic and local effects for male and female rats (MCPP-P Task Force 37H0383/91128; 1995).

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. The substance is not considered to be classified for repeated dose toxicity under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.

For MCPP-P OE the 90-day repeated dose study revealed for males slight but statistically significant increased kidney weights (abs.: 12,9 %, rel.: 16,2 %, compared to controls) at 600 ppm (corresponding to approx. 54 mg/kg bw/d; Conversion factor 0.09; EFSA Journal 2012, 10 (3): 2579). Regarding histopathology of kidneys no relevant adverse effect was concluded for males. Changes in organ weights with no evidence of adverse organ dysfunction are explicitly mentioned as example for effects that are considered not to support classification as STOT RE according to Regulation (EC) No 1272/2008. In the same study no effect on kidney weights was found for female animals at 600 ppm. However, at this dose level a histopathological adverse finding “nepropathy in P3-segment” (Tubules appeared to be dilated, flattened and filled with proteinaceous or celular debris) was reported. This finding was dose-related with minimal to moderate severity (600 ppm: 2 animals Grade 1, 8 animals Grade 2; 3000 ppm: 9 animals Grade 2, 1 animal Grade 3). This effect corresponds to the toxikokinetic evidence that the substance is excreted predominantly via urine. No clinical findings or further toxicologically relevant effects were reported at 600 ppm, which means that the female kidney effect defines the threshold. In the high dose group (3000 ppm; corresponding to approx. 270 mg/kg bw/day; Conversion factor 0.09; EFSA Journal 2012, 10 (3): 2579), at doses higher than the GHS guidance values for STOT RE classification, additionally slight changes with regard to haematology/hemostaseology (minor changes in red blood cell parameters and coagulation) and effects on liver (increased liver weights for females, hepatocellular hypertrophy and eosinophilia for both sexes, singular liver cell necrosis for few animals of both sexes, decreased hepatocellular lipid storage for males) were observed. The general study conclusion is that MCPP-P OE shows systemic toxicity especially in liver and kidney. Regarding the examples of relevant toxic effects according to GHS 3.9.2.7.3 no significant functional change in the kidney is anticipated at the dose level of STOT RE2 or lower. No severe organ damage is noted and the morphological changes observed do not provide clear evidence of a marked organ dysfunction. No mortalities, no consistent changes in clinical biochemistry, hematology, or urinalysis parameters are observed; no indication of necrosis, fibrosis or granuloma formation and no evidence of appreciable cell death are revealed from the study.

Taken together the effects on the female kidney, although being adverse, did not show such a grade of severity, that a primary severe organotoxicity can be deduced. Classification as STOT RE according to GHS thus is not appropriate.