Reaction products of 4,4'-methylenebis(cyclohexylamine) and 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane

Endpoint summary

Description of key information

Based on the findings of 28 day oral repeat dose toxicity study with PACM BADGE ADDUCT No Observed Adverse Effect Level (NOAEL) of 50 mg/kg was established.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose 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 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Rat: Crl:WI(Han) (outbred, SPF-Quality)
Rationale - Recognized by international guidelines as the recommended
test system (e.g. EPA, FDA, OECD and EC).

Sex:

male/female

Details on test animals or test system and environmental conditions:

Total number of animals 30 males, 30 females. (females were nulliparous and nonpregnant).
Age at start of treatment Approximately 6 weeks.
Identification Earmark and tattoo.
Randomization By computer-generated random algorithm according to body
weight, with all animals within ± 20% of the sex mean.
Acclimatization period At least 5 days before the start of treatment under laboratory
conditions.
Health inspection At least upon receipt of the animals.

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

validated method (Test Facility Study No. 514809). Samples of formulations were analyzed
for homogeneity (highest and lowest concentration) and accuracy of preparation (all
concentrations).
The accuracy of preparation was considered acceptable if the mean measured concentrations
were 85-115% of the target concentration suspensions. Homogeneity was demonstrated if the
coefficient of variation was ≤ 10%.

Duration of treatment / exposure:

28 day

Frequency of treatment:

Daily

Dose / conc.:

10 mg/kg bw/day (nominal)

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

250 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

Treatment
Method Oral gavage, using a plastic feeding tube.
Formulations were placed on a magnetic stirrer during dosing.
A dose control system was used as additional check to verify
the dosing procedure according to Standard Operating
Procedures.
Dose volume 5 mL/kg body weight.
Actual dose volumes were calculated weekly according to the
latest body weight.
Frequency Once daily, 7 days per week, approximately the same time each
day with a maximum of 6 hours difference between the earliest
and latest dose.
Duration of treatment 28 days. Animal no. 54 was treated for 26 days.
Animals were dosed up to the day prior to necropsy of Main
group animals.
Duration of recovery 14 days.

Observations and examinations performed and frequency:

Observations
Mortality / Viability
At least twice daily. Animals showing pain, distress or discomfort, which was considered not transient in nature or was
likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/2000/7).
Clinical signs
At least once daily from start of treatment onwards, detailed clinical observations were made in all animals after dosing (no peak effect of occurrence of clinical signs was observed in the
dose range finding study (Test Facility Study No. 513042)). Once prior to start of treatment and at weekly intervals, this was also performed outside the home cage in a standard arena.
The time of onset, grade and duration of any observed signs were recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded
as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. In the data tables,
the scored grades are reported, as well as the percentage of animals affected in summary tables.

Functional Observations
During Week 4 of treatment, functional observation tests were performed on all Recovery Group 1 and 4 animals, Main Group 4 animals Nos. 21, 22 and 51 and all Main Group 2 and 3
animals. Tests were performed after dosing at no specific time point, but within a similar time period after dosing (based on the absence of a peak effect in occurrence of clinical signs in
the dose range finding study (Test Facility Study No. 513042)).
The following tests were performed (abbreviations mentioned in the respective tables indicated between brackets):
• hearing ability (HEARING), pupillary reflex (PUPIL L/R), static righting reflex (STATIC R) (Score 0 =normal/present, score 1 = abnormal/absent).
• fore- and hind-limb grip strength, recorded as the mean of three measurements per animal (Series M4-10, Mark-10 Corporation, J.J. Bos, Gouda, The Netherlands).
• locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system, Kinder Scientific LLC, Poway, USA).
Total movements and ambulations are reported.
Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the
animals, including ambulations but also smaller or more fine movements like grooming, weaving or movements of the head.
Since the abovementioned measurements did not reveal treatment-related effects, the functional observation tests were not performed at the end of the recovery phase.

Body weight
Weekly. Additionally, to monitor health status, all females of Group 4 were also weighed on Day 6.

Food consumption
Weekly.

Water consumption
Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

Sacrifice and pathology:

Necropsy
Animals surviving to the scheduled day of necropsy and all moribund animals were deeply
anaesthetized using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) and subsequently
exsanguinated and subjected to a full post mortem examination. Animals were deprived of
food overnight (with a maximum of 24 hours) prior to scheduled necropsy.
All animals assigned to the study were necropsied and descriptions of all macroscopic
abnormalities recorded.
Samples of the following tissues and organs were collected from all animals at necropsy and
fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution,
Klinipath, Duiven, The Netherlands):
Identification marks: not processed (Pancreas)
Adrenal glands Peyer's patches [jejunum, ileum] if detectable
(Aorta) Pituitary gland
Brain [cerebellum, mid-brain, cortex](7
levels)
(Preputial gland)
Caecum Prostate gland
Cervix Rectum
(Clitoral gland) (Salivary glands - mandibular, sublingual)
Colon Sciatic nerve
Duodenum Seminal vesicles including coagulating gland
Epididymides * (Skeletal muscle)
Eyes (including optic nerve [if detectable]
and harderian gland) *
(Skin)
(Female mammary gland area) Spinal cord -cervical, midthoracic, lumbar
Femur including joint Spleen
Heart Sternum with bone marrow
Ileum Stomach
Jejunum Testes *
Kidneys Thymus
(Larynx) Thyroid including parathyroid [if detectable]
(Lacrimal gland, exorbital) (Tongue)
Liver Trachea
Lung, infused with formalin Urinary bladder
Lymph nodes - mandibular, mesenteric
(Nasopharynx)
Uterus
(Oesophagus) Vagina
Ovaries All gross lesions
* Fixed in modified Davidson's solution, prepared at Charles River Den Bosch using Formaldehyde 37-40%,
Ethanol, Acetic acid - glacial (all Merck, Darmstadt, Germany) and Milli-Ro water (Millipore Corporation,
Bedford, USA). Tissues were transferred to formalin after fixation for at least 24 hours.
Tissues/organs mentioned in parentheses were not examined by the pathologist, since no
signs of toxicity were noted at macroscopic examination.
Organ Weights
The following organ weights and terminal body weight were recorded from the surviving
animals on the scheduled day of necropsy:
Adrenal glands Spleen
Brain Testes
Epididymides Thymus
Heart Uterus (including cervix)
Kidneys Prostate
Liver Seminal vesicles including coagulating glands
Ovaries Thyroid including parathyroid
5.11.3. Histotechnology
All organ and tissue samples, as defined under Histopathology (following), were processed,
embedded in paraffin wax (Klinipath, Duiven, The Netherlands), cut at a thickness of 2-4
micrometers and stained with haematoxylin and eosin (Klinipath, Duiven, The Netherlands).
5.11.4. Histopathology
The following slides were examined by a pathologist:
• all tissues collected at the scheduled sacrifice from all Main Group 1 and 4 animals,
• all tissues from all animals of all dose groups which were terminated in extremis,
• duodenum, jejunum, ileum, mesenteric lymph node, liver and brain of all males and
females of Groups 2 and 3 and recovery groups and kidneys of all males of Group 2
and 3 and recovery groups, based on (possible) treatment-related changes in these
organs in Group 4,
• all gross lesions.
All abnormalities were described and included in the report. An attempt was made to
correlate gross observations with microscopic findings.

Other examinations:

Clinical Laboratory Investigations
Clinical laboratory investigations were conducted at the end of treatment (Main and Recovery animals) and at the end of recovery (Recovery animals). Blood samples were collected under anaesthesia using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) between 7.00 and 10.30 a.m.
Animals were deprived of food overnight (for a maximum of 24 hours), but water was available. Blood samples were drawn from the retro-orbital sinus and collected into tubes (Greiner Bio-One GmbH, Kremsmünster, Austria) prepared with K3-EDTA for haematological parameters (0.5 mL),
with citrate for clotting tests (0.45 mL) and Li-heparin treated tubes for clinical biochemistry parameters (0.5 mL). An additional blood sample (0.25 mL) was collected into serum tubes for determination of bile acids.
Urine samples were collected overnight (approximately 15-20 hrs) from the animals. During the sampling period animals were deprived of food but water was available. Urine was collected into a specimen vial, using a metabolism cage.

Haematology
The following parameters were determined in blood prepared with K3-EDTA as an anticoagulant, using the ADVIA® 2120i Hematology System (Siemens Healthcare Diagnostics B.V., Den Haag, The Netherlands):
Parameter (Abbreviation) Unit
White blood cells (WBC) 109/L
Differential leucocyte count:
Neutrophils %WBC
Lymphocytes %WBC
Monocytes %WBC
Eosinophils %WBC
Basophils %WBC
Red blood cells 1012/L
Reticulocytes %RBC
Red blood cell distribution width (RDW) %
Haemoglobin mmol/L
Haematocrit L/L
Mean corpuscular volume (MCV) fL
Mean corpuscular haemoglobin (MCH) fmol
Mean corpuscular haemoglobin concentration
(MCHC) mmol/L
Platelets 109/L
The following clotting parameters were determined in plasma prepared with citrate as anticoagulant, using the STA Compact® (Diagnostica Stago S.A.S., Asnières, France):
Parameter (Abbreviation) Unit
Prothrombin Time (PT) s
Activated Partial Thromboplastin Time (APTT) s

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Breathing related ailments (rales, labored respiration, or gasping) were frequently observed amongst the 250 mg/kg animals sacrificed moribund and occasionally amongst the surviving animals, including a single 50 mg/kg male, two 250 mg/kg males, and two 250 mg/kg females. Female no. 45 (10 mg/kg) displayed rales for a good part of a week prior to scheduled sacrifice.
Piloerection and hunched posture were briefly observed in a couple of surviving 250 mg/kg animals, and gas distention of the abdomen and lean appearance was also observed in a small number of surviving 250 mg/kg animals. No findings were noted during the arena observations in this study. In addition, there were no toxicologically relevant clinical signs present during the recovery phase.
The chromodacryorrhoea and watery discharge from the right eye of animal no. 53 (250 mg/kg, female) occurred as a result of the blood sampling procedure.
Salivation seen after dosing amongst the treatment groups was not considered toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response related to taste of the test item rather than a sign of systemic toxicity. Furthermore, salivation subsided across all treatment groups during the recovery phase.
Any other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related

Mortality:

mortality observed, treatment-related

Description (incidence):

At 250 mg/kg, there were three males and three females (one female at 450 mg/kg) sacrificed in moribund condition, all considered to be test item-related.
Female no. 61 (450 mg/kg) was sacrificed on the second day of treatment, and displayed clinical signs that included labored respiration, gasping and vocalization (squeaking sounds).
Histopathology revealed acute erosion/ulceration of the stomach (correlating with macroscopic finding irregular surface) and duodenum, which was considered to have contributed to the moribundity, as well as the distension with gas of the large intestines (gross finding without microscopic correlate).
After reducing the dose levels, there were another 5 premature sacrifices in the 250 mg/kg (previously 450 mg/kg) animals. These occurred after 5 (male no 26), 6 (male no. 27), 7 (female no. 57), 16 (male no. 24) and 28 treatment days (female no. 59, assigned to the
recovery group).
Male nos. 24 and 26 displayed breathing related ailments such as rales, gasping, and/or
labored respirations on the days leading up to sacrifice. Reduced prostate and epididymis size
(both sides) was noted in male no. 24, while male no. 26 displayed gas distention of the
gastro-intestinal tract, swollen lungs, and foci in the preputial glands.
Clinical signs for male no. 27 included labored respiration, piloerection, and hunched posture
on the day of sacrifice, while macroscopic examination also revealed gas distention of the
gastro-intestinal tract and swollen lungs.
Female no. 57 displayed hunched posture, piloerection and lean appearance on the days
leading up to sacrifice, and had gelatinous contents in the intestines, which correlated with
mucus depletion of the rectum.
On many occasions during the treatment period, including the day of sacrifice (recovery day
1), female no. 59 displayed rales, with gasping and swollen abdomen being observed on the
day of sacrifice. Macroscopic examination revealed gas distention of the gastro-intestinal
tract, swollen lungs, irregular stomach surface and liver foci.
The principal microscopic findings of these 5 premature sacrifices include:
• Intestines: mild alterations in one or more parts, such as: erosions, inflammation,
hemorrhage, hypertrophy, mucus depletion and/or foamy vacuolated hypertrophic
neurons of the enteric cytoplasm (3 males, 3 females: up to moderate).
• Thymus: atrophy (up to slight) and/or increased lymphocytolysis (3 males, 1 female: up
to marked).
• Mesenteric and mandibular lymph node: atrophy (1 female: slight).
• Spleen: atrophy (2 males: slight).
• Bone marrow: an increase in (mature) granulocytes (3 males, 2 females: up to marked).
Furthermore, there were test item-related findings in brain, enteric nervous system (ENS) of
the (small) intestines, duodenum, liver and mesenteric lymph node, similar to what was
observed in the scheduled sacrifice animals. These are listed under Microscopic Examination
(Section 7.2.11). Additional findings of note were observed in the epididymis (reduced sperm
and cellular debris), testes (missing stages: spermiation) of male nos. 26 and 27, which were
most likely in concordance with the young age of these premature decedents.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight gain and relative body weights were slightly lower in 250 mg/kg males and females across the treatment period, when compared to controls. This effect was not always
statistically significant. The lower body weight gain continues to be present during the recovery period in 250 mg/kg animals.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption before or after correction for body weight was slightly less in 250 mg/kg male and female groups, when compared to controls. These effects was present across the
treatment period but stronger in the first two weeks of the study and resolved during recovery.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

A statistically significant increase in prothrombin time (PT) was seen in 250 mg/kg males when compared to controls at the end of treatment, while platelet levels were increased in 250 mg/kg females in a statistically significant manner.
Relative lymphocyte levels were statistically significantly lower and relative neutrophil levels were higher (trend) in 250 mg/kg females when compared to controls at the end of treatment.
None of the effects described were present at the end of the recovery period. Any other statistically significant changes in haematology parameters were considered to be unrelated to
treatment as these occurred in the absence of a dose-related trend or during the recovery phase.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Levels of the liver enzymes alanine aminotransferase (ALAT) and aspartate transaminase
(AST) were statistically significantly increased in 250 mg/kg males and females when
compared to controls at the end of treatment, with the largest increases seen in males. Urea
and creatinine levels were statistically significantly reduced in 250 mg/kg male and females
when compared to controls at the end of treatment. Reductions in total protein and albumin
levels were also observed in 250 mg/kg animals at the end of treatment, with statistical
significance seen in males only. Ion levels were also affected by treatment, with statistically
significant increases in sodium levels in 10, 50 and 250 mg/kg males and statistically
significant reductions in calcium levels in 250 mg/kg males.
All the effects described were absent at the end of the recovery phase. Any other statistically
significant changes in clinical biochemistry parameters were considered to be unrelated to
treatment as these occurred in the absence of a dose-related trend.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Urinalysis did not reveal any toxicologically relevant treatment-related effects.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

There were no test item-related alterations in organ weights in Main and Recovery groups. Some organ weight differences were statistically significant when compared to the control
group but were considered to be the result of a test item-related effect on body weight.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item-related gross observations in the scheduled sacrificed Main and Recovery rats. Test item related findings of the unscheduled sacrificed rats, are described
under the mortality paragraph.
The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Vacuolation choroid plexus of the brain was observed at 250 mg/kg in:
• Scheduled Main Sacrifices in both sexes, at minimal degree.
• 1 Unscheduled Sacrificed male, after 16 treatment days, at minimal degree.
• Scheduled Recovery Sacrifices in both sexes, at minimal degree and more or less
similar incidences.
There was no recovery for the vacuolation of the choroid plexus of the brain in both sexes.
intestines, was observed at 250 mg/kg in:
• All Scheduled Main Sacrifices in both sexes, up to slight degree.
• 1 Unscheduled Sacrificed male, after 16 treatment days at minimal degree.
• 1 Unscheduled Sacrificed Recovery female after 28 treatment days/first Day of
Recovery, at minimal degree.
• All Scheduled Recovery Sacrifices in both sexes, up to slight degree.
There was no Recovery for this finding in both sexes.
Mucosal hypertrophy of the duodenum, was observed at 250 mg/kg in:
• Most Scheduled Main Sacrifices in both sexes, at minimal degree.
• 1 Unscheduled Sacrificed male, after 16 treatment days, at minimal degree.
• 1 Unscheduled Sacrificed female after 28 treatment days/first Day of Recovery, at
minimal degree.
There was a complete recovery of this finding for both sexes after a 14-day treatment-free
period.
was observed at 250 mg/kg in:
• All Scheduled Main Sacrifices in both sexes, up to moderate degree.
• 1 Unscheduled Sacrificed male, after 16 treatment days at minimal degree.
• 1 Unscheduled Sacrificed female after 28 treatment days/first Day of Recovery, at
minimal degree.
• All Scheduled Recovery Sacrifices in both sexes, up to moderate degree.
There was no recovery of this finding in both sexes after a 14-day treatment-free period.
Increased incidence of single cell necrosis of the liver was observed at 250 mg/kg in:
• Scheduled Main Sacrifices at minimal degree in all rats, compared to a single incidence
in males of the Control group.
• Scheduled Recovery Sacrifices in all males, at minimal degree.
There was no Recovery in males and complete recovery in females.
Microvesicular vacuolation (centrilobular) of the hepatocytes of the liver was observed at 250
mg/kg in:
• Scheduled Main Sacrifices up to slight degree in half of the males and at minimal
degree in a female.
• Scheduled Recovery Sacrifices in a female, at minimal degree.
• 3 Unscheduled Sacrificed males, starting after 5 treatment days and 1 Unscheduled
Sacrificed female after 7 treatment days, at minimal degree.
There was no Recovery in females and complete recovery in males.

Other effects:

not examined

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: neoplastic

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

urinalysis

Critical effects observed:

no

Wistar rats were treated with PACM BADGE ADDUCT for 28 consecutive days by daily oral gavage, at dose levels of 10, 50, 250 mg/kg followed by a 14-day treatment-free recovery period.

Test formulations prepared were considered homogeneous at the concentrations tested and analysis of the accuracy revealed acceptable levels.

Adverse test item-related morphologic alterations were present in males and females treated at the high dose, and consisted of premature decedents displaying alterations in the gastrointestinal

tract. The gastro-intestinal tract was targeted at an early stage in the study, with one premature sacrifice displaying acute stomach and duodenal erosion/ulcerations. Despite lowering the doses from Day 3 onwards, another five rats of the high dose group had to be sacrificed prematurely with signs of gastro-intestinal alterations related to their moribundity, such as distension with gas, gelatinous contents and/or mild inflammation and/or hemorrhage of parts of the intestines.

Gastro-intestinal changes in the scheduled sacrificed rats were noted in the duodenum, consisting of mucosal hypertrophy, mesenteric lymph node, consisting of macrophage aggregates and enteric nervous system (ENS) of the (small) intestines, consisting of foamy vacuolated hypertrophic neurons. These changes were also observed in one unscheduled sacrificed high dose male after 16 treatment days and one unscheduled sacrificed high dose female at the first day of the recovery period. There was complete recovery for the findings of the duodenal mucosa and no recovery for the findings in the mesenteric lymph node and enteric nervous system. It is described that morphologic changes of the neurons of the ENS can alter intestinal functionality (Ref. 6, Ref. 7) and intestinal morphology. Therefore, the

intestinal findings of this study were evaluated as associated findings. Since the gastrointestinal findings were related with moribundity, they were considered as adverse. Non-adverse test item-related morphologic alterations were observed in the brain and liver of

both males and females treated at 250 mg/kg. Liver alterations consisted of microvesicular vacuolation and/or increased incidence of single cell necrosis, at low degrees. Recovery of the single cell necrosis was complete for females but not for males, while recovery of the microvesicular vacuolation was complete for males but not for females after a 14-day treatment free period. A subjective correlation was made with significantly increased serum levels of the liver enzymes ALAT and ASAT at the end of treatment. However after a 14-day treatment-free recovery period, these values were normalised. As a result of the recorded low severities, these histologic liver alterations were considered to be non-adverse.

The brain also displayed test item-related changes in the form of vacuolation of epithelial cells of the choroid plexus. The vacuolation was intracytoplasmic (apical), from microvesicular to larger vacuoles with sometimes nuclear displacement in the epithelial layer.This was especially present at the more lateral parts of the choroid plexus at the level of the cerebellum. It is described that there are regional differences in structure and function of the choroid plexus. Based on the low degree (minimal) and absence of other test item-related changes in the adjacent periventricular region of the brain, this

vacuolation of the epithelial cells of the choroid plexus was considered to be non- adverse.

Secondary changes resulting from the adverse gastrointestinal tract alterations include atrophy (or increased lymphocytolysis) of the thymus and/or mesenteric and/or mandibular lymph node and/or an increased amount of (mature) granulocytes of the bone marrow.

Regarding clinical signs, the most frequent were breathing related ailments (rales, gasping, labored respiration), predominantly in 250 mg/kg moribund animals. These findings may be related to the irritation caused by the aspiration of the test item from the digestive system, which could also be reflected in the swollen lungs (with no microscopic outcomes) seen in 250 mg/kg animals. Furthermore, the less frequent clinical signs such as piloerection, hunched posture, gas distention of the abdomen, and lean appearance are also likely related to gastrointestinal irritation. Secondary test-item related effects were also reflected in other parameters, such as reduced relative and absolute body weight, and food intake in 250 mg/kg animals during the treatment period.

Test-item related changes in the remaining haematological and clinical biochemistry parameters, mainly observed in 250 mg/kg animals at the end of treatment, were all normalized by the end of the recovery period. These included changes in clotting parameters (PT and platelet levels), relative lymphocyte, total protein, and albumin levels in 250 mg/kg males and/or females. Furthermore, changes observed in kidney parameters such as urea, creatinine and ion levels (sodium, calcium, chloride) in the 250 mg/kg groups did not have any morphological correlates, or findings from urinalysis. As a result, these haematological and clinical biochemical changes were not considered to be adverse.

Based on the findings described in this report, in particular the high levels of mortality and the adverse changes in the gastro-intestinal tracts of animals treated for 2 days at 450 mg/kg and 26 days at 250 mg/kg, a No Observed Adverse Effect Level (NOAEL) of 50 mg/kg was established for PACM BADGE ADDUCT.

Conclusions:

Based on the findings of 28 day repeat dose toxicity study with PACM BADGE ADDUCT No Observed Adverse Effect Level (NOAEL) of 50 mg/kg was established.

Executive summary:

Wistar rats were treated with PACM BADGE ADDUCT for 28 consecutive days by daily oral gavage, at dose levels of 10, 50, 250 mg/kg followed by a 14-day treatment-free recovery period.

Test formulations prepared were considered homogeneous at the concentrations tested and analysis of the accuracy revealed acceptable levels.

Adverse test item-related morphologic alterations were present in males and females treated at the high dose, and consisted of premature decedents displaying alterations in the gastrointestinal

tract. The gastro-intestinal tract was targeted at an early stage in the study, with one premature sacrifice displaying acute stomach and duodenal erosion/ulcerations. Despite lowering the doses from Day 3 onwards, another five rats of the high dose group had to be sacrificed prematurely with signs of gastro-intestinal alterations related to their moribundity, such as distension with gas, gelatinous contents and/or mild inflammation and/or hemorrhage of parts of the intestines.

Gastro-intestinal changes in the scheduled sacrificed rats were noted in the duodenum, consisting of mucosal hypertrophy, mesenteric lymph node, consisting of macrophage aggregates and enteric nervous system (ENS) of the (small) intestines, consisting of foamy vacuolated hypertrophic neurons. These changes were also observed in one unscheduled sacrificed high dose male after 16 treatment days and one unscheduled sacrificed high dose female at the first day of the recovery period. There was complete recovery for the findings of the duodenal mucosa and no recovery for the findings in the mesenteric lymph node and enteric nervous system. It is described that morphologic changes of the neurons of the ENS can alter intestinal functionality (Ref. 6, Ref. 7) and intestinal morphology. Therefore, the

intestinal findings of this study were evaluated as associated findings. Since the gastrointestinal findings were related with moribundity, they were considered as adverse. Non-adverse test item-related morphologic alterations were observed in the brain and liver of

both males and females treated at 250 mg/kg. Liver alterations consisted of microvesicular vacuolation and/or increased incidence of single cell necrosis, at low degrees. Recovery of the single cell necrosis was complete for females but not for males, while recovery of the microvesicular vacuolation was complete for males but not for females after a 14-day treatment free period. A subjective correlation was made with significantly increased serum levels of the liver enzymes ALAT and ASAT at the end of treatment. However after a 14-day treatment-free recovery period, these values were normalised. As a result of the recorded low severities, these histologic liver alterations were considered to be non-adverse.

The brain also displayed test item-related changes in the form of vacuolation of epithelial cells of the choroid plexus. The vacuolation was intracytoplasmic (apical), from microvesicular to larger vacuoles with sometimes nuclear displacement in the epithelial layer.This was especially present at the more lateral parts of the choroid plexus at the level of the cerebellum. It is described that there are regional differences in structure and function of the choroid plexus. Based on the low degree (minimal) and absence of other test item-related changes in the adjacent periventricular region of the brain, this

vacuolation of the epithelial cells of the choroid plexus was considered to be non- adverse.

Secondary changes resulting from the adverse gastrointestinal tract alterations include atrophy (or increased lymphocytolysis) of the thymus and/or mesenteric and/or mandibular lymph node and/or an increased amount of (mature) granulocytes of the bone marrow.

Regarding clinical signs, the most frequent were breathing related ailments (rales, gasping, labored respiration), predominantly in 250 mg/kg moribund animals. These findings may be related to the irritation caused by the aspiration of the test item from the digestive system, which could also be reflected in the swollen lungs (with no microscopic outcomes) seen in 250 mg/kg animals. Furthermore, the less frequent clinical signs such as piloerection, hunched posture, gas distention of the abdomen, and lean appearance are also likely related to gastrointestinal irritation. Secondary test-item related effects were also reflected in other parameters, such as reduced relative and absolute body weight, and food intake in 250 mg/kg animals during the treatment period.

Test-item related changes in the remaining haematological and clinical biochemistry parameters, mainly observed in 250 mg/kg animals at the end of treatment, were all normalized by the end of the recovery period. These included changes in clotting parameters (PT and platelet levels), relative lymphocyte, total protein, and albumin levels in 250 mg/kg males and/or females. Furthermore, changes observed in kidney parameters such as urea, creatinine and ion levels (sodium, calcium, chloride) in the 250 mg/kg groups did not have any morphological correlates, or findings from urinalysis. As a result, these haematological and clinical biochemical changes were not considered to be adverse.

Based on the findings described in this report, in particular the high levels of mortality and the adverse changes in the gastro-intestinal tracts of animals treated for 2 days at 450 mg/kg and 26 days at 250 mg/kg, a No Observed Adverse Effect Level (NOAEL) of 50 mg/kg was established for PACM BADGE ADDUCT.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

50 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available