N-[3-(dimethylamino)propyl]-N,N',N'-trimethylpropane-1,3-diamine

Endpoint summary

Description of key information

In a repeat dose study was according to OECD Guidelines (No. 422/GLP), oral administration of the test substance to Wistar rats by gavage (50, 100 and 200 mg/kg/day) for 42 day resulted in treatment-related clinical signs, changes in the body weights, feed consumption and microscopic effects in male and female animals dosed at 100 and 200 mg/kg/day. No toxicologically significant effects were detected in both sex treated with 50 mg/kg/day.

The vacuolation detected in the various organs of high dose and intermediate dose group were considered to be treatment related and no recovery of microscopic changes were noted in the satellite group. Hence, the 'No Observed Effect Level' (NOEL) for systemic toxicity is considered to be 50 mg/kg/day.  

Treatment-related reproduction / developmental toxicity effect of influencing the  sex ratio (decrease in the % male offspring) were observed in the high dose group (200 mg/kg/day).

Test item related microscopic changes were observed in the testes and epididymides of male and ovaries of female animals in the high dose group (200 mg/kg/day).

No treatment-related effects were observed on reproduction/ development indices of animals in the 50 and 100  mg/kg/day dose groups. Mating index, fertility index, gestation length, pre-implantation loss, post-implantation loss, sex ratio and offspring growth and development were unaffected.

Some of the observed effects appear consistent with the animals being administered a corrosive/irritating substance. However, a No Observed Effect Level (NOEL) for systemic toxicity was derived at 50 mg/kg/day, whilst the No Observed Effect Level (NOEL) for reproductive effects was determined as 100 mg/kg/day.

A 90 -day repeat-dose oral study according to OECD TG 408 was performed. The rats were treated with 10,30 or 100/65 mg PU‑2018-788 (Polycat 77)/kg b.w./day by daily oral administration. Due to a reduction of body weight of the high dosed animals greater than recommended, the initial dose of 100 mg/kg b.w. was reduced to 65 mg/kg b.w. as of test day 15. The control animals received the vehicle (distilled water).
None of the animals died or had to be sacrificed prematurely.
In conclusion, adverse test item-related effects were noted a t30 mg PU‑2018-788 (Polycat 77)/kg b.w./day (in form of histopathological changes) and at 100/65 mg PU‑2018-788 (Polycat 77)/kg b.w./day (in form of a reduced body weight, a decreased food consumption, an increased drinking water consumption, and at histopathological examination) compared to the control group.Based on the above results, the no-observed-effect level (NOEL) was10 mg PU‑2018-788 (Polycat 77)/kg b.w./day by oral administration for 90 days.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January-October 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was performed according to OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test” (adopted 22 March 1996). A GLP certificate is provided. The study was conducted in compliance with OECD Principles of Good Laboratory Practice, as revised in 1997 and adopted November 26th, 1997 by decision of the OECD Council [C(97)186/Final]. A dose range finding study (DRF) was conducted to confirm the doses for the main study and solubility test was performed before the study initiation. Hence, these are excluded from the statement. Study procedures were periodically inspected with the exception of solubility test and dose range finding study. The study plan and report were audited by the Quality Assurance. Overall, the study is considered a high-quality, was performed to guidelines, and produced interpretable results that are relevant for deltermining repeat-dose toxicity.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Name of the Test Item: POLYCAT® 77 Catalyst
Chemical Name: N-[3-dimethylamino)propyl]-N,N,N'-trimethylpropane-1,3 diamine
CAS No: 3855-32-1
Description: Clear Colourless liquid
Purity (By GC): 96.9%
Batch No.: 1226246
Manufacture Date :October 27, 2011
Expiry Date: October 27, 2014
Stability of test item: Stable

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Total number of animals used: 120 (60 male, 60 Female)
Age on first treatment: 9-10 weeks
Body weight when treated: For Allocation A and B Male : 210.7 to 239.6 g Female : 182.1 to 197.1 g (Nulliparous and Non-pregnant (Weight variation was within the 20% of mean body weight of each sex))
Identification: By unique cage number and individual animal numbers marked with indelible marker pen on the base of the tail. The animals were marked before the start of test item administration and weekly thereafter. The animals were marked with the temporary animal numbers on the tip of the tail at start of acclimatization.
Acclimatization: 7 and 8 Days for step I and II animals of Allocation A and B under laboratory conditions, after veterinary examination. Only animals without any visible signs of illness were used for the study


Animals were selected and/or grouped based on stratified randomization by using body weights taken before treatment. Computerized statistical programme was used for randomization.
Daily dose levels:
Group 1: 0 mg/kg body weight
Group 2: 50 mg/kg body weight
Group 3: 100 mg/kg body weight
Group 4: 200 mg/kg body weight
Group 1S: 0mg/kg body weight
Group 4S: 200 mg/kg body weight

The animal room was air-conditioned with adequate air changes per hour (at least 10 air changes). The experimental room was continuously monitored for temperature and relative humidity. The ranges for room temperature and relative humidity were 20.7°C to 22.8°C and 52 to 65%, respectively. The animals were provided with a light cycle of 12 hours light and 12 hours dark. Initially (acclimatization and randomization period), all animals were housed in groups of two/ three in polycarbonate cages (approximate internal dimensions of 365 mm x 202 mm x 180 mm height) with corn cob bedding. After randomization, males and females were housed individually. During the mating phase, animals were housed on one male: one female basis within each dose group. After successful mating, the females were returned to their original cages and housed individually during gestation and lactation. Results of analyses for contaminants of corn cob will be archived at RCC Laboratories India Pvt. Ltd.

Route of administration:

oral: gavage

Vehicle:

water

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of the dose formulations were taken immediately after dilution of the concentrates with the diluent (vehicle) on treatment start date (Day 1 – Premating i.e., 19/02/2013) and towards treatment end date (After first dam sacrifice i.e., 03/04/2013) for homogeneity (mean of homogeneity were given as dose concentration) analysis. On week 5, samples of all dose formulations was analysed for dose concentrations by analysing triplicate samples.

Duration of treatment / exposure:

Allocation A (Males were administered with test item daily up to 42 Days; Females were administered with test item daily during premating, mating, gestation periods and up to Day 4 post partum)


Group1-Control : 10 males, 10 females
Group 2-Low dose : 10 males, 10 females
Group 3-Intermediate dose : 10 males, 10 females
Group 4 - High dose : 10 males, 10 females

Frequency of treatment:

Daily

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

Doses / Concentrations:
50 mg.kg.day
Basis:
nominal in water

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Doses / Concentrations:
100 mg.kg.day
Basis:
nominal in water

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

Doses / Concentrations:
200 mg.kg.day
Basis:
nominal in water

No. of animals per sex per dose:

Group1-Control: 10 males, 10 females
Group 2-Low dose: 10 males, 10 females
Group 3-Intermediate dose: 10 males, 10 females
Group 4 - High dose: 10 males, 10 females

Control animals:

yes

Positive control:

No

Observations and examinations performed and frequency:

All the animals were regularly weighed and subjected to gross examination and observation of clinical parmaters (Haematology, biochemistry),

Sacrifice and pathology:

The male animals were sacrificed on Day 43 and female animals were sacrificed Day 5 post partum.

The following tissues were subjected to histopatholical examination:
Adrenal glands
Aorta
Bone marrow (Sternum)
Brain (3 levels)
Cecum
Colon
Duodenum
Epididymides
Heart
Ileum, with Peyer’s patches
Jejunum
Kidneys
Liver
Lungs (inflated with NBF at necropsy)
Lymph nodes (mesenteric, axillary)
Oesophagus
Ovaries Pancreas
Rectum
Skeletal muscle
Spinal cord (cervical, mid thoracic, lumbar)
Spleen
Stomach
Testes
Thymus
Thyroid
Trachea
Urinary bladder (inflated with NBF at necropsy)
Uterus
All Gross lesions

Other examinations:

Feed consumption.

Statistics:

Statistical methods were used to analyze the Body weight, feed consumption, hematological, biochemical parameters and organ weight data, Pre-coital interval, gestation length, litter size and litter weights, sex ratio, corpora lutea and implantation sites, Implantation losses, viability indices, offspring body weight and body weight change.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No clinical signs were observed in the control group (0 mg/kg bodyweight) and low dose group (50 mg/kg body weight) male and female animals.

In males, piloerection was observed in the intermediate dose group (100 mg/kg body weight) from treatment Day 38 to Day 42. Piloerection and dullness was observed in the high dose group (200 mg/kg body weight) on treatment Day 14 and continued to exhibit the signs till the last observation period (Day 42).
In females, piloerection was observed in the intermediate dose group (100 mg/kg body weight) towards end of gestation and lactation period. In addition, cannibalism (eating its own pups on Day I observation) and no lactation was noted in one animal.
In high dose group (200 mg/kg bodyweight), piloerection was observed in all the animals at the end of premating period and throughout the gestation and lactation periods, in addition dullness was observed during gestation and lactation periods in animal number 74 & 79. In lactation phase one animal from high dose showed cannibalism (eating its own pups).
The high dose satellite group also showed piloerection in all the animals. In addition dullness was observed in two animals (animal number 111 and 118) during the treatment and recovery period.

Mortality:

no mortality observed

Description (incidence):

There were no mortalities observed in the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No significance in the body weight and body weight gain (%) were observed in low dose group animals of both sexes when compared with control group.

In males, intermediate dose group showed decreased body weight and body weight gain (%) when compared with control group on few occasions. However, no significant changes in the body weight and body weight gain (%) were observed in the female animals of intermediate dose group when compared with control group.

In males, the body weights and body weight gain (%) were significantly decreased in the high dose group when compared with control group animals. The same was observed in the high dose satellite group (recovery group) when compared with respective satellite control group.

In females, the body weights and body weight gain (%) were significantly decreased in the high dose group when compared with control group animals at premating, gestation and lactation periods. The high dose satellite group (recovery group) females also showed decreased body weight and body weight gain (%) when compared with respective satellite control group.

The body weight and body weight gain (%) was significantly decreased in high dose animals of both sexes and intermediate dose group male animals when compared with control group and this effect was not reversed in the satellite group animals and hence these are considered to be treatment related,

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

No significance was observed in the feed consumption in low dose group animals of both sexes when compared with control group.

Intermediate dose group male and female animals showed decreased feed consumption when compared with control group on few occasions. No significant changes in the feed consumption were observed in the female animals of the intermediate dose group when compared with control group during the gestation and lactation periods.
In males, the feed consumption was significantly decreased in the high dose group when compared with control group animals. Similarly, decreased feed consumption was observed in the high dose satellite group (recovery group) when compared with respective satellite control group during the treatment and recovery period.
In females, the feed consumption was significantly decreased in the high dose group when compared with control group animals during premating and gestation periods. No significant variation in the feed consumption was observed during the lactation period. The high dose satellite group (recovery group) also showed decreased feed consumption when compared with respective satellite control group during the treatment and recovery period.

This change of feed consumption in high dose group male and female animals can be considered as treatment effect. Since, decrease of body weight and body weight gain (%) is correlated with decreased feed consumption in high dose group animals.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

No treatment related significance was observed in any of the haematology parameters.

However, activated partial thromboplastin time was significantly decreased in low and high dose males when compared with control group. Since the change was not dose dependent and was not observed in female animals of any group, it is considered to be not attributable to the test item.
Significant increase in the platelet count, decrease in the RBC and eosinophil count of male high dose satellite group was observed when compared to respective control group and was not considered treatment related since the values lies in the normal biological range.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No treatment related significance was observed in any of the biochemistry parameters.

Creatinine levels decreased in all treated males when compared with control group animals.
In females, sodium levels were increased in high dose group when compared with intermediate and decreased in intermediate when compared with low dose group.
Alkaline phosphatase, alanine transaminase, triglycerides and sodium levels were decreased in the high dose satellite group females when compared with respective control.
The significance of these differences can't be attributed to treatment due to marginal increase or decrease in individual control values.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Treatment with test item had no effect on urinary parameters in both the sexes with an exception of decreased epithelial cells in low dose group when compared with control and increased epithelial cells in intermediate dose group when compared with low dose group in males. In the high dose satellite group, urinary volume increased in males and decreased in females when compared with respective control. In addition, decreased leukocyte count in female animals were observed. However, this significance is solely of individual variation not to represent any biological significance.

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

In Allocation A male animals, there was significant decrease in absolute weight of thymus in high dose (G4) and intermediate dose (G3) groups when compared with control group (Gl) and also in high dose (G4) when compared with low dose (G2) group while in case of female animals there was significant decrease in absolute weight of thymus in high dose (G4) and intermediate dose (G3) when compared with low dose (G2) and significant increase absolute weight of thymus in low dose (G2) when compared with control (G1) group.

There was significant increase in relative weight of liver and testes in high dose (G4) and intermediate dose (G3) groups when compared with control (GI) and low dose (G2) groups and significant increase in relative weight of brain in high dose (G4) and intermediate dose (G3) groups when compared with control (G1) group and also in high dose (G4) when compared with low dose (G2) group in male animals.

In the case of female animals, significant decrease in relative weight of thymus in intermediate dose (G3) groups when compared with low dose (G2) group while significant increase in relative weight of thymus in high dose (G4) group when compared with intermediate dose (G3) group and also in low dose (G2) group when compared with control (G1) group was observed. In addition, significant increase in relative weight of kidneys of female animals in high dose (G4) and intermediate dose (G3) groups when compared with control (Gl) and low dose (G2) groups.

In Allocation B male animals, there was significant decrease in absolute weight of epididymides in male animals while significant increase in absolute weight of heart, thymus and spleen in case of female animals of high dose satellite (G4S) group when compared with respective control satellite (GIS) group. Whereas, significant increase in relative weight of adrenals, kidneys, liver, heart, spleen, testes and brain in male animals and kidneys, liver, heart, thymus and spleen in female animals of high dose satellite (G4S) group when compared with respective control satellite (G1S) group was observed.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Necropsy performed at the end of the treatment period revealed no abnormality in any of the male and female control animals (G1), intermediate (G3) and high dose (G4) groups.
Fragile liver, enlarged kidney, reddened stomach and small sized testes, epididymides, seminal vesicles and prostate in one male animal and distended uterus with watery content in one female animal of low dose (G2) group was observed.
Following the recovery period, no abnormality was observed in male animals whereas, distended uterus with watery content was observed in two female animals of high dose satellite (G4S) group.
These findings were considered to be gender, congenital and/or physiology related and considered as biological variations.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Moderate centrilobular vacuolation, vacuolation inkidney, bladder, arteries, parietal layer of stomach, testes. Apoptotic necrosis in thymus.

No abnormality was observed attributable to the test item at low dose group (G2).

Microscopic examination of high dose (G4) and intermediate dose (G3) group animals revealed test item related minimal to moderate centrilobular and/or arterial vacuolation in liver; minimal to moderate arterial vacuolation and/or minimal to mild vacuolation of glomerular tuft in kidneys, minimal to mild arterial vacuolation in urinary bladder; minimal to marked vacuolation of arteries, trabeculae, red and pulp in spleen; minimal to mild arterial vacuolation in thymus; minimal to moderate arterial and/or cortical vacuolation in axillary and/or mesenteric lymph nodes; minimal to moderate vacuolation of arteries, bronchiolar epithelium and/or muscularis in lungs; minimal to moderate epithelial vacuolation in trachea; minimal to moderate vacuolation of vasavasorum, aorta and/or pulmonary artery in heart; minimal to moderate arterial and/or exocrine vacuolation in pancreas, mild to moderate vacuolation of tunica muscularis, arterial and/or parietal layer of stomach; minimal to mild arterial vacuolation in skeletal muscle; minimal to marked vacuolation and necrosis of choroid plexus in brain in both sexes, minimal to mild arterial vacuolation in ovaries of female animals.

Microscopic examination of high dose satellite (G4S) group animals revealed test item related minimal to moderate centrilobular vacuolation, necrosis and/or fibrosis in liver; minimal to mild arterial vacuolation and/or minimal to mild vacuolation of glomerular tuft in kidneys; minimal to moderate arterial vacuolation and/or epithelial vacuolation in urinary bladder; minimal to marked vacuolation of arteries, trabeculae, red and/or white pulp in spleen; minimal to mild arterial vacuolation and/or increased incidence of apoptotic necrosis in thymus; minimal to moderate arterial and/or cortical vacuolation in axillary and/or mesenteric lymph nodes; mild to marked vacuolation of arteries, bronchiolar epithelium and/or muscularis in lungs; minimal to moderate epithelial vacuolation in trachea; minimal to mild vacuolation of vasavasorum, aorta and/or pulmonary artery in heart; minimal to marked arterial and/or exocrine vacuolation in pancreas; minimal to marked vacuolation of tunica muscularis, arterial and/or parietal layer of stomach; minimal to mild arterial vacuolation in skeletal muscle; mild to marked vacuolation of choroid plexus in brain in both sexes, mild arterial vacuolation in testes of male animals and minimal to mild arterial vacuolation in ovaries of female animals.
The lesions observed in high dose satellite (G4S) group did not reverse after recovery period and seems to be more severe.

Additionally, male and female animals from control and high dose groups and target organs from low dose, intermediate dose and recovery dose group animals showed lesions such as minimal to mild individual cell necrosis, lymphocytic infiltration and/or sinusoidal dilatation in liver; minimal to mild necrosis, basophilic tubules, vacuolation of tubular epithelium, tubular dilatation and/or proteinaceous material in kidneys; minimal to mild lymphocytic infiltration, alveolar histiocytosis, haemorrhages and/or presence of keratinized cyst in lungs; minimal to mild vacuolation, dilatation and/or presence of accessory adrenocortical tissue in adrenals; minimal to mild lymphoid necrosis in axillary lymph node; minimal lymphoid necrosis in spleen; minimal to mild necrosis or apoptotic necrosis in pancreas, minimal to mild lymphoid or apoptotic necrosis in thymus and presence of ectopic thymus or ultimobranchial cyst in thyroid in both sexes and mild oligospermia in epididymis of male animals.

The histopathological examination of organs showing macroscopic findings revealed mild sinusoidal dilatation in liver and mild congestion of stomach, minimal tubular hypertrophy of kidneys, moderate atrophy of testes, azoospermia and/or necrotic debris in epididymides and mild atrophy of seminal vesicles and prostate in one male animal of low dose (G2) group, whereas mild polymorphonuclear cells infiltration and luminal dilatation of uterus in one female animal of low dose (G2) group and two female animals of high dose satellite (G4S) group.

The changes observed other than the test item related lesions in various tissues during evaluation of control, low, intermediate, high dose and satellite dose group animals were comparable and hence considered incidental. These observed changes can usually be considered to be species, age, gender, congenital, physiological or mode of death related and are covered in background historical data of pathology

Histopathological findings: neoplastic:

no effects observed

Key result

Dose descriptor:

NOAEL

Remarks:

Systemic effects

Effect level:

>= 50 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Dose descriptor:

NOAEL

Remarks:

Reproductive effects

Effect level:

>= 100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

not specified

No toxicologically significant effects were detected in both sex treated with 50 mg/kg/day. The vacuolation detected in the various organs of high dose and intermediate dose group were considered to be treatment related and no recovery of microscopic changes were noted in the satellite group. Hence, the 'No Observed Effect Level' (NOEL) for systemic toxicity is considered to be 50 mg/kg/day. 

No treatment-related effects were observed on reproduction/ development such as mating index, fertility index, gestation length, pre-implantation loss, post-implantation loss, sex ratio and offspring growth and development in the animals treated at 50 and 100 mg/kg/day therefore, a ‘No Observed Effect Level’ (NOEL) for reproductive toxicity is considered to be 100 mg/kg/day.

Conclusions:

This study was performed according to OECD Guidelines (No. 422/GLP).

Oral administration of the test substance to Wistar rats by gavage (50, 100 and 200 mg/kg/day) resulted in treatment-related clinical signs, changes in the body weights, feed consumption and microscopic effects in male and female animals dosed at 100 and 200 mg/kg/day. No toxicologically significant effects were detected in both sex treated with 50 mg/kg/day.

The vacuolation detected in the various organs of high dose and intermediate dose group were considered to be treatment related and no recovery of microscopic changes were noted in the satellite group. Hence, the 'No Observed Effect Level' (NOEL) for systemic toxicity is considered to be 50 mg/kg/day.

Treatment-related reproduction / developmental toxicity effect of influencing the sex ratio (decrease in the % male offspring) were observed in the high dose group (200 mg/kg/day).

Test item related microscopic changes were observed in the testes and epididymides of male and ovaries of female animals in the high dose group (200 mg/kg/day).

No treatment-related effects were observed on reproduction/ development indices of animals in the 50 and 100 mg/kg/day dose groups. Mating index, fertility index, gestation length, pre-implantation loss, post-implantation loss, sex ratio and offspring growth and development were unaffected.

Some of the observed effects appear consistent with the animals being administered a corrosive/irritating substance. However, a No Observed Effect Level (NOEL) for systemic toxicity was derived at 50 mg/kg/day, whilst the No Observed Effect Level (NOEL) for reproductive effects was determined as 100 mg/kg/day.

Executive summary:

The test item, POLYCAT®77 (formulated in distilled water) was administered by gavage to three treatment groups, each of ten male and ten female Wistar rats, for up to 42 Days for males, up to two weeks premating phase, two weeks mating, three weeks gestation and early lactation for females, at dose levels of 50, 100 and 200 mg/kg/Day. A control group of ten males and ten females was dosed with vehicle alone (Distilled water). In addition, ten males and ten females were allocated to satellite group Group-1s, Group-4S, to represent recovery groups for control (0 mg/kg bw/Day), high dose (test item at 200 mg/kg bw/Day) respectively. Satellite group animals were treated along with main group (Allocation A) animals and dosing was stopped on the Day of first dam sacrifice. Satellite group animals were observed for a further 14-day recovery period.

The treatment with test item resulted in no mortalities. All the animals survived to the scheduled sacrifice. There were test item related clinical signs such as piloerection and dullness observed in the intermediate and high dose groups, significant decrease of food consumption and body weights in the intermediate and high dose group were correlated and considered to be treatment related. The clinical signs, body weight and feed consumption between low dose group and control group were comparable.

No test item effects were observed in any of the hematological, clinical biochemistry and urine parameters in the treated groups including the satellite group which represents the recovery.

The body weight and body weight gain (%) was significantly decreased in high dose animals of both sexes and intermediate dose group male animals when compared with control group and this effect was not reversed in the satellite group animals and hence these are considered to be treatment related.

In females, the body weights and body weight gain (%) were significantly decreased in the high dose group when compared with control group animals at premating, gestation and lactation periods. The high dose satellite group (recovery group) females also showed decreased body weight and body weight gain (%) when compared with respective satellite control group.

Post-mortem examinations revealed no test item-related macroscopic findings.

No abnormality was observed attributable to the test item at low dose group. Histopathological findings of high dose and intermediate dose includes minimal to moderate centrilobular and/or arterial vacuolation in liver; minimal to moderate arterial vacuolation and/or minimal to mild vacuolation of glomerular tuft in kidneys; minimal to mild arterial vacuolation in urinary bladder; minimal to marked vacuolation of arteries, trabeculae, red and/or white pulp in spleen; minimal to mild arterial vacuolation in thymus; minimal to moderate arterial and/or cortical vacuolation in axillary and/or mesenteric lymph nodes; minimal to moderate vacuolation of arteries, bronchiolar epithelium and/or muscularis in lungs; minimal to moderate epithelial vacuolation in trachea; minimal to moderate vacuolation of vasavasorum, aorta and/or pulmonary artery in heart; minimal to moderate arterial and/or exocrine vacuolation in pancreas; minimal to moderate vacuolation of tunica muscularis, arterial and/or parietal layer of stomach; minimal to mild arterial vacuolation in skeletal muscle; minimal to marked vacuolation and necrosis of choroid plexus in brain in both sexes, whereas, mild arterial vacuolation in testes and/or epididymides of male animals and minimal to mild arterial vacuolation in ovaries of female animals. These lesions observed in high dose satellite (G4S) group were not reversed after recovery period and were likely to be increased in severity and needs a longer recovery period. No other distinguishable microscopic changes were seen with relation to the test item.

The oral administration of POLYCAT® 77 Catalyst to Wistar rats by oral gavage, at dose levels of 50, 100 and 200 mg/kg/day, resulted in treatment-related clinical signs, changes in the body weights, feed consumption and microscopic effects in male and female animals treated at 100 mg/kg/day and 200 mg/kg/day. No toxicologically significant effects were detected in both sex treated with 50 mg/kg/day. The vacuolation detected in the various organs of high dose and intermediate dose group were considered to be treatment related and no recovery of microscopic changes were noted in the satellite group. Hence, the 'No Observed Effect Level' (NOEL) for systemic toxicity is considered to be 50 mg/kg/day.

No treatment-related effects were observed for reproduction/ development parameters such as precoital interval, mating index, fertility index, gestation length, pre-implantation loss, post-implantation loss, sex ratio and offspring growth and development at any dosage in the animals treated with 50 and 100 mg/kg/Day. However significant change in sex ratio on day I did not show any dose response and also no related reproductive findings were noted and it is therefore considered to have arisen by chance or a biological variation.  The reproductive and developmental NOEL (no observed effect level) is estimated as 200 mg/kg bw/day.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Nov 2018-July 2019

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)

Deviations:

yes

Remarks:

Minor deviations that were not reflected by Study Plan amendments did not affect the validity and integrity of the scientific results obtained during the study.

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

minor deviations that were not reflected by Study Plan amendments did not affect the validity and integrity of the scientific results obtained during the study.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Designation: PU-2018-788 (Polycat 77)
Chemical name: N-[3-(Dimethylamino)propyl]-N,N’,N’-trimethylpropan-1,3-diamin
CAS no.: 3855-32-1
Batch no.:2302776
Receipt no. 66072
Date of receipt: 25 June 2018
Characteristics: Colourless liquid
Storage conditions: At +10°C to +25°C, in a tightly closed container, in a dry, cool and well-ventilated place.
Production date: 13 April 2017
Stability / expiry date: 13 April 2020
Purity: 96.3%

Species:

rat

Strain:

CD-1

Remarks:

Rat / CD® / Crl:CD(SD)

Details on species / strain selection:

Rat / CD® / Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

100 animals (50 males and 50 females)
Main study:
80 animals (40 male and 40 female rats);
10 animals/sex/group.
Recovery animals:
20 animals (10 male and 10 female rats);
5 animals/sex/group for groups 1 and 4.

The animals were kept singly in MAKROLON cages (type III plus) with a basal surface of approx. 39 cm × 23 cm and a height of approx. 18 cm at a room temperature of 22°C ± 3°C (maximum range) and a relative humidity of 55% ± 10% (maximum range). Deviations from the maximum range caused for example during cleaning procedures were dealt with in SOPs.
The rooms were lit (about 150 lux at approx. 1.5 metres room height) and darkened for periods of 12 hours each.
Granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt, Germany) was used as bedding material for the cages. The cages were changed and cleaned once a week.
Periodic analysis of the bedding material for contaminants based on EPA/USA is conducted at least once a year by LUFA-ITL.

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Distilled Water

Details on oral exposure:

Dose levels: Group 1: 0 mg/kg b.w./day (vehicle control)
Group 2: 10 mg/kg b.w./day
Group 3: 30 mg/kg b.w./day
Group 4: 100/65 mg/kg b.w./day
Due to a reduction of body weight of the group 4 animals greater than recommended, the initial dose of 100 mg/kg b.w. was reduced to 65 mg/kg b.w. as of test day 15.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

GC-FID method for the quantification of PU-2018-788 in tap water formulations.
The results indicate that all test item formulations were correctly prepared by LPT, and were stable for at least 24 hours. The measured concentrations ranged from 97.4% to 107.4% in the test item formulation samples.

Duration of treatment / exposure:

90 Days

Frequency of treatment:

Once daily for 90 consecutive days

Dose / conc.:

10 mg/kg bw/day (nominal)

Remarks:

Nominal in water

Dose / conc.:

30 mg/kg bw/day (nominal)

Remarks:

Nominal in water

Dose / conc.:

65 mg/kg bw/day (nominal)

Remarks:

Due to a reduction of body weight of the group 4 animals greater than recommended,
the initial dose of 100 mg/kg b.w. was reduced to 65 mg/kg b.w. as of test day 15.

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control group

No. of animals per sex per dose:

10 animals/sex/group.

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

Checks were made early in the morning and again in the afternoon of each working day to look for dead or moribund animals. On Saturdays and Sundays a similar procedure was followed with a final check at approximately 03:30 p.m.

The weight of each rat was recorded at group allocation (test day -8), daily from the day of commencement of treatment up to and including test week 6 for dose adjustment, thereafter weekly throughout the experimental period.
The quantity of food left by individual animals was recorded on a weekly basis throughout the experimental period (treatment and recovery).
Drinking water consumption was recorded weekly by weighing the water bottles when filled and the residues upon removal at the end of the test week.

The animals were observed individually at least once daily, preferably at the same time each day for any signs of behavioural changes, reaction to treatment or illness. Any signs of illness or reaction to treatment were recorded individually for
each animal.
In addition, the animals were checked regularly throughout the working day from 07:00 a.m. to 03:45 p.m. by cage side observations. On Saturdays and Sundays, the animals were checked regularly from 07:00 a.m. to 11:00 a.m. with a final
check performed at approximately 03:30 p.m.

Blood samples were taken from the retrobulbar venous plexus under isoflurane anaesthesia from animals fasted overnight. The blood samples were collected from all main study animals on test day 91 (end of the treatment period), and from all
recovery animals on test day 105 (end of the recovery period).

At the end of the treatment period (in test week 13, before blood sampling for laboratory examinations), and at the end of the recovery period (in test week 15)
screening of sensory reactivity to stimuli of different types (e.g. auditory, visual and proprioceptive stimuli; based on Gad5), as well as the assessment of grip
strength (Meyer6) and motor activity assessment were conducted for all animals.

Sacrifice and pathology:

Starting on test day 91 (approx. 24 hours after the last administration), the main study animals were dissected following a randomisation scheme. Main study animals not dissected on test day 91 were dosed until one day before sacrifice.
Necropsy of all animals scheduled for the recovery period was performed on test day 105.

The animals were euthanized by carbon dioxide (CO2), exsanguinated by cutting the aorta abdominalis, weighed, dissected, and inspected macroscopically under the direction of a pathologist.

All superficial tissues were examined visually and by palpation and the cranial roof was removed to allow observation of the brain, pituitary gland and cranial nerves.
After ventral midline incision and skin reflection all subcutaneous tissues were examined. The condition of the thoracic viscera was noted with due attention to
the thymus, lymph nodes and heart.
The abdominal viscera were examined before and after removal, the urinary bladder was examined externally and by palpation. The gastro-intestinal tract was
examined as a whole and the stomach and caecum were incised and examined.
The lungs were removed and all pleural surfaces were examined under suitable illumination. The liver and the kidneys were examined. Any abnormalities in the
appearance and size of the gonads, adrenal glands, uterus, intraabdominal lymph nodes and accessory reproductive organs was recorded.

Other examinations:

Ophthalmological and auditory examinations were performed predose, in test week 13 (end of the treatment period) and in test week 15 (end of recovery)

Histopathology: Due to test item-related changes in the animals of the high dose group 4, sections of target organs of the animals of the low and intermediate dose level groups and Oil Red O-stained sections of selected organs of the animals of groups 1 and 4 were prepared (for the histological visualization of fat cells and neutral fat) and evaluated.

Statistics:

Homogeneity of variances and normality of distribution were tested using the BARTLETT’s and SHAPIRO-WILK's test. In case of heterogeneity and/or nonnormality of distribution, stepwise transformation of the values into logarithmic or
rank values was performed prior to ANOVA. If the ANOVA yielded a significant effect (p ≤ 0.05), intergroup comparisons with the control group was made by the DUNNETT’s test (p ≤ 0.01 and p ≤ 0.05).

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

None of the male and female rats treated with 10 or 30 mg PU 2018-788 (Polycat 77)/kg b.w./day by oral administration for 90 days revealed any test item-related changes in behaviour, external appearance, or consistency of faeces.
Pilo-erection was observed for 2 of 15 male and 10 of 15 female animals treated with 100 mg PU 2018-788 (Polycat 77)/kg b.w./day by oral administration on up to 6 days starting on test day 9. Furthermore, one of 15 males (no. 81) treated with 100 mg PU 2018-788 (Polycat 77)/kg b.w./day by oral administration revealed slightly reduced motility consistently during the day on test days 13 and 14.
After the dose reduction to 65 mg/kg b.w. on test day 15, pilo-erection was still observed for 2 of 15 male and 12 of 15 female animals treated with 100/65 mg PU 2018-788 (Polycat 77)/kg b.w./day by oral administration on up to 16 days starting on test day 15 and again for 1 of 15 female animals (no. 91) on test days 49 to 71. Furthermore, one of 15 males (no. 81) treated with 100/65 mg PU 2018-788 (Polycat 77)/kg b.w./day by oral administration still revealed slightly reduced motility consistently during the day on test day 15.
These changes are regarded to be test item-related.

In addition, a haemorrhagic canthus of the right eye was noted for 1 of 10 female animals (no. 66) treated with the intermediate dose of 30 mg PU 2018-788 (Polycat 77)/kg b.w./day by oral administration for 90 days on test days 57 to 59. This finding is considered to be coincidental and not related to the administration of the test item.

No changes in behaviour, external appearance, or consistency of faeces were noted for the previously high-dosed male and female animals during the 14-day treatment-free recovery period.

Mortality:

no mortality observed

Description (incidence):

None of the animals died or had to be sacrificed prematurely. All male and female animals treated with 10, 30 or 100/65 mg PU 2018-788 (Polycat 77)/kg b.w./day survived until their scheduled sacrifice on test day 91/92.
None of the previously high-dosed animals died or had to be sacrificed prematurely during the recovery period.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No test item-related influence was observed on the body weight, the body weight gain and the body weight at autopsy of the male and female animals treated with 10 or 30 mg PU 2018-788 (Polycat 77)/kg b.w./day by oral administration for 90 days compared to the control animals throughout the treatment period.
The body weight of the male animals treated with 100 mg PU 2018-788 (Polycat 77)/kg b.w./day was decreased by 8% on test day 15 compared to the control animals. After the dose reduction to 65 mg/kg b.w. on test day 15, the decrease in body weight was less pronounced (decrease in body weight by 3% to 6% compared to the control up to test day 71). From test day 78 to 90 (end of treatment period), the decrease in body weight was again more pronounced with differences compared to the control group by up to 8% (statistically significant at p ≤ 0.05 on test days 85 and 90). The body weight gain and the body weight at autopsy were reduced accordingly.
The body weight of the female animals treated with 100 mg PU 2018-788 (Polycat 77)/kg b.w./day was decreased by up to 17% on test days 8 and 15 compared to the control animals (statistically significant at p ≤ 0.01 on test days 8 and 15). After the dose reduction to 65 mg/kg b.w. on test day 15, the decrease in body weight was less pronounced (decrease in body weight by mainly 8% to 9% compared to the control up to test day 90, statistically significant at p ≤ 0.01 on test days 22 and 36 to 90). The body weight gain and the body weight at autopsy were reduced accordingly.

Recovery period (restricted to groups 1 and 4)
The body weight of the animals previously treated with 100/65 mg PU 2018-788 (Polycat 77)/kg b.w./day was still reduced by 13% or 12% (statistically significant at p ≤ 0.05 on test day 97) for the males and by 7% or 8% for the females (statistically significant at p ≤ 0.05 on test days 97 and 104) during the recovery period. The body weight at autopsy was reduced accordingly.
However, the high dosed animals revealed a body weight gain of 5% for the males and of 4% for the females compared to a body weight gain of 3% or 2% for the male and female control animals, respectively, from test day 90 to 104, indicated a slight trend to recovery.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Treatment period
No test item-related influence was observed on the relative food consumption of the male and female animals treated with 10 or 30 mg PU 2018-788 (Polycat 77)/kg b.w./day by oral administration compared to the control animals throughout the 90 day treatment period.
The relative food consumption of the male and female animals treated with 100 mg PU 2018-788 (Polycat 77)/kg b.w./day by oral administration was reduced by up to 19% for the males and by up to 45% for the females compared to the control animals in test weeks 1 and 2 (statistically significant at p ≤ 0.01 in test weeks 1 and 2 for the females). After the dose reduction to 65 mg/kg b.w. on test day 15, the food consumption of the high dosed animals was within the range of the control group.

Recovery period (restricted to groups 1 and 4)
The food consumption of the male and female animals previously treated with 100/65 mg PU 2018-788 (Polycat 77)/kg b.w./day exceeded the food consumption of the control group by 14% to 25% (males) or by 10% to 11% (females) during the 14-day treatment-free recovery period (statistically significant at p ≤ 0.01 in test week 14 and at p ≤ 0.05 in test week 15 for the males and at p ≤ 0.05 in test 14 for the females).

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

Treatment period
No test item-related influence was observed on the drinking water consumption of the male and female animals treated with 10 or 30 mg PU 2018-788 (Polycat 77)/kg b.w./day by oral administration compared to the control animals throughout the 90-day treatment period.
The drinking water consumption of the male and female animals treated with 100/65 mg PU 2018-788 (Polycat 77)/kg b.w./day by oral administration was slightly increased by up to 16% for the males in test weeks 3 to 5 and 13 and by up to 17% for the females in test weeks 3, 4, 7 and 8 (statistically significant at
p ≤ 0.05 in test week 3 for the females) compared to the control animals, evaluated by weekly quantitative assessment. This finding is considered to be test item-related and might be due to the irritating properties of the test item.
Furthermore, a reduced drinking water consumption was noted for 1 of 15 male (no. 81) and 8 of 15 female animals (nos. 87, 88, 89, 91, 93, 94, 96 and 97) treated with 100/65 mg PU 2018-788 (Polycat 77)/kg b.w./day by oral administration for 90 days on up to 9 days starting on test day 11, assessed by visual appraisal during the daily inspections. This observation was also reflected by the individual quantitatively assessed values. However, this finding was only noted for individual animals is not considered to be toxicologically relevant as a general trend to an increased water consumption was observed.

Recovery period (restricted to groups 1 and 4)
The drinking water consumption of the male animals previously treated with 100/65 mg PU 2018-788 (Polycat 77)/kg b.w./day was still increased by up to 17% in test weeks 14 and 15 (statistically significant at p ≤ 0.05 in test week 14). The drinking water consumption of the female animals previously treated with 100/65 mg PU 2018-788 (Polycat 77)/kg b.w./day was within the range of the control group during the 14-day treatment-free recovery period.

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

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):

The macroscopic inspection at necropsy did not reveal any test item-related changes in the organs and tissues of the animals treated with 10, 30 or 100/65 mg PU 2018-788 (Polycat 77)/kg b.w./day by repeated oral administration after terminal sacrifice at the end of the treatment period (test day 91/92) and at the end of the recovery period (test day 105).
However, macroscopic changes were noted in the adrenal glands (pale), testes (reduced in size, soft consistency), epididymides (reduced in size), thymus (reduced in size), liver (enlarged, increased lobular pattern), spleen (enlarged), hepatic lymph nodes (enlarged), prostate (reduced in size), seminal vesicles (reduced in size), pituitary (enlarged), uterus (dilated) and vulva area (tissue enlargement) of single animals of all groups. All changes were considered either coincidental, or to lie within the normal background alterations which may be seen in untreated rats of this age and breed.

Neuropathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related influence was noted on any of the parameters examined during the functional observation tests, on the fore- and hind limb grip strength, or on the spontaneous motility for any of the male and female animals after repeated
oral treatment with 10, 30 or 100/65 mg PU-2018-788 (Polycat 77)/kg b.w./day in test week 13.
No test item-related influence was noted on any of the above listed parameters for any of the male and female animals previously treated with 100/65 mg PU-2018-
788 (Polycat 77)/kg b.w./day in test week 15.
Sstatistically significant differences in neurological screening parameters compared to the control animals noted in test week 13 are not considered to be test item-related but to be coincidental

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment period and recovery period (restricted to groups 1 and 4)
The histopathological examination was performed by Test Site 1 (Global Pathology Support B.V., The Netherlands) and full histopathology was restricted to a variety of organs and tissues from the control and the high dose animals (groups 1 and 4), target organs (heart, kidneys, liver, lungs, mesenteric lymph node, pancreas, Peyer‘s patches, spleen, stomach and trachea) were examined of group 2 (low dose) and group 3 (intermediate dose).
Histopathology assessment of the target organs revealed test item-related changes in the animals treated with 30 mg PU 2018-788 (Polycat 77)/kg b.w./day by oral administration for 90 days in both sexes. Test item-related changes were observed in:
• Lungs (degeneration, bronchi, terminal bronchioles);
• Peyer’s patches (lymphoid depletion, vacuolation);
• Spleen (vacuolation, medial or adventitial, artery) and
• Trachea (degeneration, epithelium).
Histopathology evaluation of the organs of the animals treated with 100/65 mg PU 2018-788 (Polycat 77)/kg b.w./day by oral administration for 90 days revealed test item-related changes in a number of organs mostly in both sexes. Test item-related changes were observed in:
• Heart (mononuclear cell infiltrate/fibrosis, myocardium, multifocal (males only); vacuolation, myocardium, medial or adventitial, artery);
• Kidneys (degeneration, tubule, proximal & distal tubules);
• Liver (centrolobular vacuolation);
• Lungs (degeneration, bronchi, terminal bronchioles);
• Lymph node, mesenteric (vacuolation);
• Pancreas, exocrine (lobular atrophy, vacuolation);
• Peyer’s patches (lymphoid depletion (males), vacuolation, increased absence);
• Spleen (lymphoid depletion, vacuolation);
• Stomach, muscular tunics (vacuolation) and
• Trachea (epithelial degeneration).
 

In the previously high dosed recovery animals, similar changes were observed also often attaining statistical significance, with the exception of changes in the heart, in the kidney of female animals and of lobular atrophy of the pancreas.
After detailed histopathological examination performed on one testicle and one epididymis (group 1 and 4 animals), no test item-related effects were observed.
Oil Red O-staining did not reveal any toxicological relevant differences for the majority of selected target organs in all treated groups when compared to controls.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Key result

Dose descriptor:

NOEL

Effect level:

10 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

30 mg/kg bw/day (nominal)

System:

other: Multiple

Organ:

heart

kidney

liver

lungs

lymph node

pancreas

spleen

stomach

trachea

other: Peyer's patches

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Table 1: Dose Groups (Male/Female)

 

	Control	N-[3-(Dimethylamino)propyl]-N,N’,N’-trimethylpropan-1,3-diamin
Dose Group	1	2	3	4
Dose (mg/kg/day)	0	10	30	100/65*

 *Due to a reduction of body weight of the group 4 animals greater than recommended, the initial dose of 100 mg/kg b.w. was reduced to 65 mg/kg b.w. as of test day 15.

Tables of findings see attached background material, pdf-file.

Conclusions:

In conclusion, adverse test item-related effects were noted at 30 mg PU 2018-788 (Polycat 77)/kg b.w./day (in form of histopathological changes) and at 100/65 mg PU 2018-788 (Polycat 77)/kg b.w./day (in form of a reduced body weight, a decreased food consumption, an increased drinking water consumption, and at histopathological examination) compared to the control group.
Based on the above results, the no-observed-effect level (NOEL) was 10 mg PU 2018-788 (Polycat 77)/kg b.w./day by oral administration for 90 days.

Executive summary:

The aim of this repeated dose toxicity study was to obtain information on the toxicity ofPU‑2018-788 (Polycat 77)when given to rats by daily oral administration via gavage for 90 days and to assess the reversibility of any effects after a treatment-free recovery period. The rats were treated with10,30or100/65 mgPU‑2018-788 (Polycat 77)/kg b.w./day. Due to a reduction of body weight of the high dosed animals greater than recommended, the initial dose of 100 mg/kg b.w. was reduced to 65 mg/kg b.w. as of test day 15. The control animals received the vehicle (distilled water).

None of the animals died or had to be sacrificed prematurely.

Pilo-erection was observed for 2 of 15 male and 10 of 15 female animals treated with100 mgPU‑2018-788 (Polycat 77)/kg b.w./day by oral administration on up to 6 days starting on test day 9. Furthermore, one of 15 males (no. 81) treated with100 mgPU‑2018-788 (Polycat 77)/kg b.w./day by oral administration revealed slightly reduced motility consistently during the day on test days 13 and 14. After the dose reduction to 65 mg/kg b.w. on test day 15, pilo-erection was still observed for 2 of 15 male and 12 of 15 female animals treated with100/65 mgPU‑2018-788 (Polycat 77)/kg b.w./day by oral administration on up to 16 days starting on test day 15 and again for 1 of 15 female animals (no. 91) on test days 49 to 71. Furthermore, one of 15 males (no. 81) treated with100/65 mgPU‑2018-788 (Polycat 77)/kg b.w./day by oral administration still revealed slightly reduced motility consistently during the day on test day 15. These changes are regarded to be test item-related.

The body weight, the body weight gain, the body weight at autopsy and the relative food consumption of the male and female animals treated with100 mgPU‑2018-788 (Polycat 77)/kg b.w./day were reduced compared to the control animals. After the dose reduction to 65 mg/kg b.w. on test day 15, the decrease in body weight was less pronounced, and the food consumption was within the range of the control group.

The drinking water consumption (by weekly quantitative assessment) of the male and female animals treated with100/65 mgPU‑2018-788 (Polycat 77)/kg b.w./day by oral administration was increased compared to the control animals.

Histopathological evaluation of the organs of the animals treated with10,30or100/65 mgPU‑2018-788 (Polycat 77)/kg b.w./day by oral administration for 90 days revealed test item-related changes in a number of organs mostly in both sexes of the intermediate dose (30 mg/kg) and high dose (100/65 mg/kg).

 

Test item-related changes were observed in theheart(mononuclear cell infiltrate/fibrosis, myocardium, multifocal (males only); vacuolation, myocardium, medial or adventitial, artery),kidneys(degeneration, tubule, proximal & distal tubules),liver(centrolobular vacuolation),lungs(degeneration, bronchi, terminal bronchioles),lymph node, mesenteric(vacuolation),pancreas, exocrine (lobular atrophy, vacuolation),Peyer’s patches(lymphoid depletion, vacuolation, increased absence),spleen(lymphoid depletion, vacuolation),stomach, muscular tunics (vacuolation) andtrachea(epithelial degeneration).

No test item-related changes were observed during the detailed clinical observations, for any of the parameters of the neurological screening, for any of the haematological, coagulation, and clinical chemistry parameters, the thyroid hormone levels, the eyes or optic region, the auditory acuity, at macroscopic inspection at necropsy, and the organ weights at any dose level.

At the end of the recovery period, the body weight and body weight gain of the male and female animals were still reduced, however, body weight gain indicated a slight trend to recovery. The drinking water consumption of themaleanimals was still increased during the recovery period. The drinking water consumption of thefemaleanimals had normalized during the 14-day treatment-free recovery period. At histopathological examination, similar changes were observed also often attaining statistical significance, with the exception of changes in the heart, kidney (females) and lobular atrophy absence on the pancreas. All other changes had subsided during or at the end of the 14-day treatment-free recovery period.

In conclusion, adverse test item-related effects were noted at30 mgPU‑2018-788 (Polycat 77)/kg b.w./day (in form of histopathological changes) and at100/65 mgPU‑2018-788 (Polycat 77)/kg b.w./day (in form of a reduced body weight, a decreased food consumption, an increased drinking water consumption, and at histopathological examination) compared to the control group.

Based on the above results, the no-observed-effect level (NOEL) was10 mgPU‑2018-788 (Polycat 77)/kg b.w./day by oral administration for 90 days.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

10 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Study was performed in 2019 according to OECD Guidelines (No. 408/GLP) and is considered of good quality.

Organ:

heart

kidney

liver

lungs

lymph node

mesenteric lymph node

pancreas

spleen

stomach

trachea

other: Peyer's patches

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

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In a repeated dose study was according to OECD Guidelines (No. 422/GLP), oral administration of the test substance to Wistar rats by gavage (50, 100 and 200 mg/kg/day) for 42 day resulted in treatment-related clinical signs, changes in the body weights, feed consumption and microscopic effects in male and female animals dosed at 100 and 200 mg/kg/day. No toxicologically significant effects were detected in both sex treated with 50 mg/kg/day.

The vacuolation detected in the various organs of high dose and intermediate dose group were considered to be treatment related and no recovery of microscopic changes were noted in the satellite group. Hence, the 'No Observed Effect Level' (NOEL) for systemic toxicity is considered to be 50 mg/kg/day.  

Treatment-related reproduction / developmental toxicity effect of influencing the  sex ratio (decrease in the % male offspring) were observed in the high dose group (200 mg/kg/day).

Test item related microscopic changes were observed in the testes and epididymides of male and ovaries of female animals in the high dose group (200 mg/kg/day).

No treatment-related effects were observed on reproduction/ development indices of animals in the 50 and 100  mg/kg/day dose groups. Mating index, fertility index, gestation length, pre-implantation loss, post-implantation loss, sex ratio and offspring growth and development were unaffected.

Some of the observed effects appear consistent with the animals being administered a corrosive/irritating substance. However, a No Observed Effect Level (NOEL) for systemic toxicity was derived at 50 mg/kg/day, whilst the No Observed Effect Level (NOEL) for reproductive effects was determined as 100 mg/kg/day.

A 90 -day repeat-dose oral study according to OECD TG 408, oral administration of the test substance to CD rats by gavage (10, 30 and 100/65 mg/kg/day)

resulted in treatment-related clinical signs, changes in the body weights, food consumption, drinking water consumption and microscopic effects in male and female animals dosed at 30 and 100/65 mg/kg/day. No toxicologically significant effects were detected in both sex treated with 10 mg/kg/day.

The vacuolation detected in the various organs of high dose and intermediate dose group were considered to be treatment related and no recovery of microscopic changes were noted in the satellite group. Hence, the 'No Observed Effect Level' (NOEL) for systemic toxicity is considered to be 10 mg/kg/day.  

Animal studies conducted with this substance and other tertiary amines (e.g. CAS 33329-35-0) generally show severe irritation to irreversible skin corrosion, destruction of membranes and eye damage. On this basis, tertiary amines are typically classified as harmful to the skin, causing irritation or corrosion, and with the potential to cause serious eye damage. Observed corrosive properties are a general feature of the tertiary amines and are the dominant effects for human health endpoints.

The substance under evaluation is corrosive, and classified for Skin Corr 1b (H314) Acute Tox. 3 (H312). The substance can cause severe burns and irreversible skin damage on contact, as confirmed in Rabbits. Contact with the liquid is likely to cause severe eye damage. Animals dosed by gavage generally exhibit forestomach corrosion, and exhibit behavioural signs of distresss (piloerection, vocalisation).

Industrial exposure to these substances is generally highly restricted. The substance is used as part of an isocyanate system which contains other hazardous substances (sensitizers). In general the hazards and risk of using such sytems are well understood from many years of industrial and professional experience.

Low-level occupational exposure already manifests as irritation (eyes/skin/mucous membranes) and is a potent indicator of exposure (i.e. exposed individuals would be aware and self-remove). Vapour exposure in occupational settings is known to cause a temporal visual impairment. (Glaucopsia, ‘blue haze’, halovision). Sustained exposures are unlikely due to the severe irritative effects.

Subsequent to intended use as a catalyst, the substance is bound in a matrix and no longer a risk.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
A 90 -day Repeated Dose Toxicity Study was performed according to OECD Guidelines (No. 408/GLP) and is considered of good quality. Recorded results show a consistent treatment-related effect.

Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: heart, kidneys, liver, lungs, lymph node, mensenteric, pancreas, Peyer's patches, spleen, stomach, trachea