2,4,6-tris(dimethylaminomethyl)phenol

Endpoint summary

Description of key information

A short term 14 day toxicity study was used as dose range finding study for the OECD 422 study. The dose levels were 0, 150, 250, 500 and 1000 mg/kg bw/d. The maximum tolerated dose was 150 mg/kg bw/d. Therefore, this dose was used as highest dose for the OECD 422 (SafePharm 2006). An OECD 422 study with Sprague-Dawley rats (SafePharm, 2006) is used as dose range finding study for the sub-chronic OECD 408 study (LPT, 2017).

In this dose range finding study, the oral administration of test item to rats by gavage at a maximum dose level of 150 mg/kg/day resulted in toxicologically significant histopathological findings in the liver, spleen and brain. Similar brain histopathology was observed for a few animals at 50 mg/kg/day. The No Observed Effect Level (NOEL) for systemic toxicity was therefore considered to be 15 mg/kg/day (SafePharm, 2006).

The 90 day oral exposure to 15, 50, and 150 mg test item/kg bw (male and female Sprague-Dawley rats; OECD 408; LPT, 2017) led to adverse test item-related effects at 50 mg test item/kg b.w./day in form of a decreased drinking water consumption and of histopathological changes, at 150 mg test item/kg b.w./day in form of a reduced body weight, a decreased food and drinking water consumption, changes in biochemical parameters, organ weights and at histopathological examination compared to the control group. By morphology and distribution, the findings are indicative for phospholipidosis, which were confirmed in additional examinations.

Selected animals from the control, intermediate and high dose groups were re-evaluated (Weber, 2018): The previously reported findings were confirmed. The alterations were not associated with further inflammatory and/or degenerative lesions, and showed partial or complete recovery. In addition, there were reduced reproductive organ weights (epididymides, prostate glands, seminal vesicles, and ovaries and uterus) without related with specific changes in oocytes or sperm cells but with vacuolation of arteries and smooth muscle cells. Therefore, a primary effect on reproductive organs cannot be considered. The same is true for the effects endocrine organs, i.e., the pituitary gland and adrenal glands. The findings are consistent with phospholipidosis. A primary affection of the endocrine system cannot be concluded. In conclusion, the test item caused no findings at 15 mg/kg b.w. /day. Furthermore, at higher doses, there was a lack of concurrent adverse toxicity or dysfunction in the affected organs.

The electron microscopy evaluation revealed the presence of membrane bound vacuoles indicative for lysosome containing myelin figures. They were noted in the brain plexus epithelia, in smooth muscle fibers of the heart, in liver macrophages, in renal tubular cells and in the smooth muscle fibers of lungs. The finding of lysosomes containing myelin figures is proving the hypothesis of phospholipidosis (Chatman et al., 2009; Nolte et al., 2016), which is regarded to be not relevant for human health.

Under the present test conditions of this study, the NOAEL (No-Observed-Adverse-Effect-Level) is 15 mg test item/kg b.w./day by oral administration for 90 days. Furthermore, at higher doses, there was a lack of concurrent adverse toxicity or dysfunction in the affected organs.

No valid repeated-dose toxicity studies are available for the dermal and inhalation routes of exposure. Data waiver are claimed.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2005-05-06 to 2005-12-09

Reliability:

1 (reliable without restriction)

Justification for type of information:

This OECD 422 study is used as dose range finding study for the OECD 408 study (LPT, 2017).

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:Charles River (UK) Limited, Margate, Kent
- Strain:Sprague-Dawley Cr1:CD (SD) IGS BR strain
- Sex. male and female
- Age: Young adult rats, approx 8 weeks old at the start of the treatment
- Weight at study initiation: Males:193 - 251 g, females:150 - 195 g
- Housing:group-housed, with up to 5 animals of the same sex and dose group/cage, with the exception of the mating and gestation/delivery period
- Diet (e.g. ad libitum): Rodent PMI 5002 (Certified) diet, BCM IPS Limited, London, UK
- Water (e.g. ad libitum): tap water
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 2 °C
- Humidity (%): 55 +/- 15 %
- Air changes (per hr): was at least fifteen air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours daily

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

PEG400

Details on oral exposure:

Vehicle: Polyethylene glycol 400
The test material was administered daily by gavage. Control animals were treated in an identical manner with 4 ml/kg/day of polyethylene glycol 400.
The volume of test and control material administered to each animal was based on the most recent bodyweight and was adjusted at regular intervals.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability and homogeneity of the test material formulations were determined by Safepharm Analytical Laboratory. The concentration of test item in the test material formulations
was determined spectrophotometrically.
The analytical method has been satisfactorily validated in terms of linearity, specificity and accuracy for the purposes of the study.

Duration of treatment / exposure:

Up to 54 consecutive days

Frequency of treatment:

Daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control Group

Dose / conc.:

15 mg/kg bw/day (nominal)

Remarks:

Low Group

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

Intermediate Group

Dose / conc.:

150 mg/kg bw/day (nominal)

Remarks:

High Group

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.
The dose levels were chosen based on the results of a preliminary range-finder presented in Part 2 of this report. The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test material, and the results of the study are believed to be of value in predicting the likely toxicity of the test material to man and to screen for potential adverse effects on reproduction.

Positive control:

not required

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS:
All animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before and after dosing, and one and five hours after dosing, during the working week. Animals were observed immediately before and after dosing, and one hour after dosing at weekends and bank holidays (except for females during parturition where applicable).

DETAILED CLINICAL OBSERVATIONS:
All animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before and after dosing, and one and five hours after dosing, during the working week.
Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functionalhehavioural toxicity. Functional performance tests were also performed on
five selected males and females per dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.

Detailed individual clinical observations were performed for each animal using a purpose-built arena. The following parameters were observed:
Gait
Tremors
Twitches
Convulsions
Bizarre/Abnormal/Stereotypie behaviour
Salivation
Pilo-erection
Exophthalmia
Lachrymation
Hyper/Hypothermia
Skin colour
Respiration
Palpebral closure
Urination
Defecation
Transfer arousal
Tail elevation

Functional Performance Tests
Motor Activify. Purpose-built 44 infra-red beam automated activity monitors were used to assess
motor activity. Animals were randomly allocated to the activity monitors. The tests were
performed at approximately the same time each day, under similar laboratory conditions. The
evaluation period was thirty minutes for each animal. The percentage of time each animal was
active and mobile was recorded for the overall thirty minute period and also during the final 20%
of the period (considered to be the asymptotic period).

Forelimb/Hindlimb Grips Strength.
An automated grip strength meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. The animal was pulled by the base of the tail until its grip was broken. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal.

Sensory Reactivity
Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. The following parameters were observed:
Grasp response
Vocalisation
Toe pinch
Tail pinch
Finger approach
Touch escape
Pupil reflex
Startle reflex
Blink reflex


BODY WEIGHT:
Individual bodyweights were recorded on Day 0 (the day before the start of treatment) and then weekly for males until termination. Females were weighed weekly until mating was evident.
Bodyweights were then recorded on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum.

FOOD CONSUMPTION:
During the maturation period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating phase. For females showing evidence of mating,
food consumption was recorded for the periods covering Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded on Days 1 and 4 postpartum.

WATER CONSUMPTION:
Water intake was observed daily by visual inspection of water bottles for any overt change.

Sacrifice and pathology:

Adult males were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 43. Adult females were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 5 post partum.
All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Organ Weights
The following organs, removed from adult animals that were killed at the end of the study, were dissected free from fat and weighed before fixation:
Adrenals
Brain
Epididymides
Heart
Kidneys
Liver
Ovaries
Spleen
Testes
Thymus

Histopathology
Samples of the following tissues were preserved from five males and five females from each dose group, in buffered 10% formalin. The tissues shown in bold were also removed from the remaining animals:
Adrenals
Aorta (thoracic)
Bone & bone marrow (femur including stifle joint)
Bone & bone marrow (sternum)
Brain (including cerebrum, cerebellum and pons)
Caecum
Coagulating gland *
Colon
Duodenum
Epididymides *
Eyes
Gross lesions
Heart
Ileum
Jejunum
Kidneys
Liver
Lungs (with bronchi)
Lymph nodes (cervical and mesenteric)
Mammary gland
Muscle (skeletal)
Ovaries *
Pancreas
Pituitary *
Prostate *
Oesophagus
Rectum
Salivary glands (submaxillary)
Sciatic nerve
Seminal vesicles *
Skin (hind limb)
Spinal cord (cervical)
Spleen
Stomach
Thyroidlparathyroid
Trachea
Testes *
Thymus
Urinary bladder
UterusICewix
Vagina

All tissues were despatched to Propath (Principal Investigator: T Hilling). The tissues from five selected control and 150 mg/kg/day dose group animals were prepared as paraffin blocks, sectioned at nominal thickness of 5 pm and stained with haematoxylin and eosin for subsequent microscopic examination. The tissues markes with * from the remaining control and 150 mg/kg/day were also processed.
Since there were indications of treatment-related changes in the brain, liver and kidneys, examination was subsequently extended to include similarly prepared sections of these tissues from five selected males and females from the 50 and 15 mg/kg/day dose groups.
Microscopic examination was conducted by the Study Pathologist. All findings were entered into the ROELEE Pathology computerisation system for tabulation and report production.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Two males treated with 150 or 50 mg/kg/day displayed an isolated incident of noisy respiration on Day 38 and Day 42 respectively, additionally incidents of increased salivation were detected immediately after dosing in animals of either sex from the 150 mg/kg/day treatment group (from Day 33 in males and from Day 26 in females). Observations of this nature are commonly reported following oral administration of an unpalatable or slightly irritant test material formulation and, in the absence of correlating histopathological evidence and, in isolation, are considered not to be toxicologically significant. Incidents of generalised redbrown staining of the fur and mouth/snout was observed in control animals of either sex and animals of either sex from all treatment groups from Day 15 onwards. As the staining is not confined to test group animals it was considered incidental and therefore of no toxicological significance.

Generalised fur loss was detected in a single female treated with 150 mgkglday from Day 3 onwards. One other female in this treatment group developed fix loss on Day 21 but then recovered by Day 23. This observation is sometimes observed when females are approaching their littering period so in isolation is considered unrelated to toxicity.

One female treated with 50 mgikglday developed a damaged tail tip on Day 9 of the study, this is a physical injury and is unrelated to treatment.

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths during the study.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There were no adverse effects on bodyweights for males throughout the study.
Bodyweight gains for treated females throughout the maturation, gestation and lactation phases of the study were considered to have been unaffected by treatment. Lower bodyweight gain was evident at both 50 and 150 mgkglday, compared with the controls, during the last week of gestation, but this was considered to reflect the lower litter size at these dosages rather than any effect on the parental females.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

For females receiving 150 mg/kg/day, food consumption and food utilisation were unaffected by treatment during the maturation phase of the study. Subsequent food intake during the first two weeks of gestation was lower than controls, with differences attaining statistical significance (p<0.05), although food conversion efficiency for this period was unaffected. Differences in food intake for the last week of gestation and during early lactation were considered to reflect the lower litter size at this dosage.
There was no adverse effect of treatment on food consumption of females during the maturation, gestation or lactation phases of the study at 15 or 50 mg/kg/day. Food conversion efficiency during maturation and the first two weeks of gestation at these dosages were also unaffected by treatment.

Food efficiency:

no effects observed

Description (incidence and severity):

see "Food consumption"

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

no effects observed

Description (incidence and severity):

Daily visual inspection of water bottles revealed no intergroup differences.

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

No treatment-related changes were detected in the haematological parameters examined.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No treatment-related changes were detected in the blood chemical parameters examined.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Behavioural Assessment: There were no treatment-related behavioural changes. All inter and intra group differences in urination, defecation and transfer arousal scores were considered to be a result of normal variation for rats of the strain and age used and were of no toxicological importance.
Functional Performance Tests: There were no treatment-related changes in the functional performance parameters measured. Statistical analysis of the data revealed no significant intergroup differences.
Sensory Reactivity Assessments: There were no treatment-related changes in sensory reactivity. All inter and intra group differences in sensory reactivity scores were considered to be a result of normal variation for rats of the strain and age used and were of no toxicological importance.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No toxicologically significant effects of treatment were detected.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related macroscopic abnormalities were detected for adult animals at terminal kill.
One female treated with 150 mg/kg/day had a fluid filled sack approximatly 1 mm2 situated on lower right horn of uterus. This is an isolated finding and is considered to be of no toxicological
significance. Additionally the brain of one female treated with 15 mg/kg/day and the right adrenal of a control female were both damaged during the necropsy procedure and are not treatment related.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

There were no treatment-related changes observed in the reproductive or accessory reproductive organs for rats of either sex.
Liver: Periportal lipid vacuolation of hepatocytes was observed in animals of either sex treated with 150 mg/kg/day, but not at any other dose level. The nature of the vacuolation was not
established but it was considered not to result from lipid accumulation.
Spleen: Vacuolated cells were observed scattered throughout the red pulp of three male and of one female rat treated with 150 mg/kg/day. Animals from the remaining treatment levels were not similarly affected.
Brain: Vacuolation of the ventricular choroid plexus was seen for animals of either sex treated with 150 mg/kg/day, and for three males treated with 50 mg/kg/day

Other effects:

no effects observed

Details on results:

There were no unscheduled deaths during the study and no clinical signs of toxicity were observed. A functional observational battery did not detect any treatment-related behavioral effects, changes in sensory reactivity, or changes in the functional performance parameters measured. Bodyweight gains were considered to be unaffected by treatment. No toxicologically significant effects on dietary intake or food efficiency were detected and no overt intergroup differences in water consumption were detected. No significant hematological or serum chemistry effects were detected prior to mating. Organ weight analysis and macroscopic necropsy did not indicate any adverse effects of treatment, however microscopic examination revealed histopathological changes, involving minimal to moderate vacuolation of cells, for the liver, spleen and brain (ventricular choroids plexus) in 5/10 males and 6/10 females at 150 mg/kg/day. No similar histopathological changes in the liver and spleen were apparent at 50 mg/kg/day, however, 3/5 males did show minimal vacuolation of the ventricular choroid plexus of the brain.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Histopathology

Critical effects observed:

not specified

The oral administration of Phenol, 2,4,6-tris [(dimethylamino)methyl] to rats by gavage at a maximum dose level of 150 mg/kg/day resulted in toxicologically significant histopathological findings in the liver, spleen and brain. Similar brain histopathology was observed for a few animals at 50 mg/kg/day. The No Observed Effect Level (NOEL) for systemic toxicity was therefore considered to be 15 mg/kg/day.

Mortality - There were no unscheduled deaths during this study.

Clinical Observations - No clinical observable signs of toxicity were observed.

Behavioral Assessment - No treatment-related effects were detected.

Function Performance Tests - There were no treatment-related changes in the functional performance parameters measured.

Sensory Reactivity Assessments - There were no treatment-related changes in sensory reactivity.

Bodyweight - Bodyweight gains for males throughout the study and for females during the maturation, gestation or lactation phases of the study were considered to be unaffected by treatment.

Food Consumption - No toxicologically significant effects on dietary intake or food efficiency were detected for males throughout the treatment period or for females during maturation, gestation or lactation phases of the study.

Water Consumption - No overt intergroup differences were detected.

Hematology - No significant hematological effects were detected prior to mating.

Blood Chemistry - No significant biochemical effects were detected prior to mating.

Necropsy - No treatment-related effects were detected at terminal kill.

Organ Weights - No adverse effects of treatment were detected.

Histopathology:

Liver - Periportal lipid vacuolation of hepatocytes was observed in animals of either sex treated with 150 mg/kg/day, but not at any other dose level.

Spleen - Vacuolated cells were observed scattered throughout the red pulp of three male and of one female rat treated with 150 mg/kg/day. Animals from the remaining treatment levels were not similarly affected.

Brain - Vacuolation of the ventricular choroid plexus was seen for animals of either sex treated with 150 mg/kg/day, and for three males treated with 50 mg/kg/day.

Conclusions:

The oral administration of test item to rats by gavage at a maximum dose level of 150 mg/kg/day resulted in toxicologically significant histopathological findings in the liver, spleen and brain. Similar brain histopathology was observed for a few animals at 50 mg/kg/day. The No Observed Effect Level (NOEL) for systemic toxicity was therefore considered to be 15 mg/kg/day.

Executive summary:

Introduction.The study was designed to investigate the systemic toxicity and potential adverse effects on reproduction (including offspring development) of the test material and complies with the recommendations of the OECD Guidelines for Testing of Chemicals No. 422 "Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test" (adopted 22 March 1996).

Methods.The test material was administered by gavage to three groups each of ten male and ten female Sprague-Dawley Crl.:CD (SD) IGS BR strain rats, Rats were exposed via oral gavage to test item at dose levels of 0, 15, 50, and 150 mg/kg/day for 43 days.

A control group of ten males and ten females was dosed with vehicle alone (polyethylene glycol 400).

Clinical signs, behavioural assessments, bodyweight development, food and water consumption were monitored during the study. Haematology and blood chemistry were evaluated prior to mating on five selected males and females from each dose group.

Pairing of animals within each dose group was undertaken on a one male to one female basis on Day 15 of the study, to produce litters.

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of developmental landmarks.

Functional observations were performed on five selected parental males from each dose group after the completion of the mating phase, and for five selected parental females from each dose group on Day 4 postpartum.

Males were terminated on Day 43, followed by the termination of all surviving females and offspring on Day 5 postpartum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

 

Results.

Mortality. There were no unscheduled deaths during the study.

Clinical Observations. No clinical observable signs of toxicity were observed.

Behavioural Assessment. No treatment-related effects were detected.

Functional Performance Tests. There were no treatment-related changes in the functional performance parameters measured.

Sensory Reactivity Assessments. There were no treatment-related changes in sensory reactivity.

Bodyweight, Bodyweight gains for males throughout the study and for females during the maturation, gestation or lactation were considered to be unaffected by treatment.

Food Consumption. No toxicologically significant effects on dietary intake or food efficiency were detected for males throughout the treatment period or for females during maturation, gestation or lactation phases of the study.

Water Consumption. No overt intergroup differences were detected.

Haematology. No significant haematological effects were detected prior to mating.

Blood Chemistry. No significant biochemical effects were detected prior to mating.

Reproductive Screening:

 

Pathology:

Necropsy.

No treatment-related effects were detected at terminal kill.

Organ Weights. No adverse effects of treatment were detected.

Histopathology.

Liver: Periportal lipid vacuolation of hepatocytes was observed in animals of either sex treated with 150 mg/kg/day, but not at any other dose level.

Spleen: Vacuolated cells were observed scattered throughout the red pulp of three male and of one female rat treated with 150 mg/kg/day. Animals from the remaining treatment levels were not similarly affected.

Brain: Vacuolation of the ventricular choroid plexus was seen for animals of either sex treated with 150 mg/kg/day, and for three males treated with 50 mg/kg/day.

Conclusion:

Rats were exposed via oral gavage to test item at dose levels of 0, 15, 50, and 150 mg/kg/day for 43 days. There were no unscheduled deaths during the study and no clinical signs of toxicity were observed. A functional observational battery did not detect any treatment-related behavioral effects, changes in sensory reactivity, or changes in the functional performance parameters measured. Bodyweight gains were considered to be unaffected by treatment. No toxicologically significant effects on dietary intake or food efficiency were detected and no overt intergroup differences in water consumption were detected. No significant hematological or serum chemistry effects were detected prior to mating. Organ weight analysis and macroscopic necropsy did not indicate any adverse effects of treatment, however microscopic examination revealed histopathological changes, involving minimal to moderate vacuolation of cells, for the liver, spleen and brain (ventricular choroids plexus) in 5/10 males and 6/10 females at 150 mg/kg/day. No similar histopathological changes in the liver and spleen were apparent at 50 mg/kg/day, however, 3/5 males did show minimal vacuolation of the ventricular choroid plexus of the brain. The no observed effect level (NOEL) for systemic toxicity was 15 mg/kg/day.

 

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-06-22 to 2018-02-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

adopted September 21, 1998

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

May 30, 2008

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

The test item was diluted in the vehicle to the appropriate concentrations.
Test item formulations with concentrations of 3.75, 12.5 or 37.5 mg/mL were prepared.
The administration formulations were freshly prepared daily.

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ORGANISMS:
- Species: Rat
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Strain: Rat / CD®-1 / Crl:CD(SD)
- Age: Males 62 days, females: 63 days
- body weight: Males: 293.4 g - 327.5 g, females: 216.3 g - 277.5.0 g
- Diet: ad libitum, Commercial ssniff R/M-H V1534 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany)
- Water: ad libitum
- Acclimatisation period: 8 (males) or 9 (females) days
-Housing: kept singly in MAKROLON cages

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 °C +/- 3° C
- Humidity (%): 55% +/- 10 %
- Illumination: 12 hours artifical fluorescent light and 12 hours dark

ENVIRONMENTAL ENRICHMENT
- Animals received one piece of wood (certified for animal use) to gnaw on once weekly at change of the cages.
- Octagon-shaped red-tinted huts (polycarbonate) were placed in the cages to offer the animals a resting and hiding place

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

PEG400

Details on oral exposure:

ADMINISTRATION: 
- Frequency: once daily for 90 days
- Dose volume: 4 mL/kg b.w./day
- Dose: 0, 100, 300 or 1000 mg/kg/bw
- Vehicle: PEG400
- Animals:
Main study animals: 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 for groups 1 and 4.

- DOSAGE PREPARATION:
- The administration formulations were freshly prepared every day.
The test item will be diluted in the vehicle (PEG400) to the appropriate concentrations and will be administered orally at a constant volume/kg b.w. once daily for 90 days.
The dose of the test item is adapted to the animal's body weight daily up to and including test week 6, thereafter weekly.
The control animals receive the vehicle at a constant volume orally once daily in the same way.
In addition, the stability, homogeneity, and concentration of the application formulations will be monitored.

Analytical verification of doses or concentrations:

yes

Remarks:

The analysis of the test item-vehicle mixtures from test days 1 and 90 was performed by using an HPLC-UV detection method. The following parameters were determined: - Linearity - Accuracy - Precision - Sensitivity - Specificity - Stability.

Details on analytical verification of doses or concentrations:

For the analysis of the administration formulations, samples of approximately 4 mL were taken at the following times and stored at -20°C or colder until analysis:
For each test item formulation, the stability and concentration of the test item in the formulations were analysed.
On the first administration day:
Analysis of stability and concentration
Immediately after preparation of the formulations as well as after 8 and 24 hours storage of formulations at room temperature.
(3 samples/test item group).
Number of samples: 3 x 3 = 18

On the last administration day:
Analysis of concentration
During treatment always before administration to the last animal of the group (1 sample/test item group).
Number of samples: 1 x 3 = 3

Total number of all samples: 12

The results indicate that all test item formulations were correctly prepared by LPT, and were stable for at least 24 hours.

Duration of treatment / exposure:

90 test days
14 recovery days for selected animals

Frequency of treatment:

Once daily for 90 consecutive days

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Vehicle control

Dose / conc.:

15 mg/kg bw/day (nominal)

Remarks:

Low dose

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

Intermediate dose

Dose / conc.:

150 mg/kg bw/day (nominal)

Remarks:

High dose

No. of animals per sex per dose:

Main study: 10 animals/sex/group
Recovery animals: 5 animals/sex/group for groups 1 and 4

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels for this study had been selected by the Sponsor based on available toxiciological data of an OECD 422 study already conducted.

- Selection of species:
The rat was selected because of its proven suitability in toxicology studies and to comply with regulatory requirements for testing in a rodent animal species.

- Identification of animals:
Each rat received a continuous number. According to a differentiated number scheme, points were set on paws and/or tail by tattoo. Additionally, the animal cages were numbered with study number, animal number, sex, and treatment group.

Positive control:

not required

Observations and examinations performed and frequency:

Dated and signed records of all activities relating to the day by day running and maintenance of the study within the animal unit as well as to the group observations and examinations outlined in this procedure will be recorded in the appropriate documentation. In addition, observations related to individual animals will be made throughout the study and will be recorded.
The following observations will be made during the course of the study:

Clinical signs
Individual animals will be observed before and after dosing for any signs of behavioural changes, reaction to treatment, or illness.
In addition, animals will be checked regularly throughout the working day from 7.30 a.m. to 4.30 p.m. by cage-side observations.
On Saturdays and Sundays animals will be checked regularly from 8.00 a.m. to 12.00 noon with a final check performed at approximately 4.00 p.m.
Cage-side observations will include skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behavior patterns. The onset, intensity and duration of any signs observed will be recorded.
Dated and signed records of appearance, change, and disappearance of clinical signs of individual animals will be maintained on clinical history sheets.
Special attention will be paid to ascertain if there are any signs of irritation after oral dosing, such as increased salivation, redness of the oral cavity etc.
Additionally, once before the first exposure (to allow for within-subject comparisons) and once a week thereafter, detailed clinical observations will be made in all animals; in test week 13 these observations will be performed prior to any laboratory investigations. These observations will be made outside the home cage in a standard arena and at the same time, each time. Signs noted will include changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, pilo-erection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling) or bizarre behaviour (e.g. self-mutilation, walking backwards) will also be recorded.

Mortality
Checks were made early in the morning and again in the afternoon of each working day to look for dead or moribund animals.

Body weight
The weight of each rat was recorded at group allocation (test day -8/-9), on test day 1 (before first administration), and once a week thereafter always on the same day of the week throughout the experimental period (test days 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, 85 and 90 for all animals, and test days 97 and 104 for the animals scheduled for the recovery). Furthermore, the weight of each rat was recorded daily from the day of commencement of treatment up to and including test week 6 for dose adjustment only.

Food consumption
The quantity of food left by individual animals was recorded on a weekly basis throughout the experimental period. The food intake per animal (g/animal/week) was calculated using the total amount of food given to and left by each rat in each group on completion of a treatment week. The relative food consumption (in g/kg b.w./day) was determined

Water consumption
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 residue was discarded.
No possible water loss due to spilling was noted based on visual appraisal.

Neurological screening
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 Gad ), as well as the assessment of grip strength (Meyer ) and motor activity assessment were conducted for all animals as described below. The observations were made 1 to 2 hours after dosing.
Observational screening:
Righting reflex
Body temperature
Salivation
Startle response
Respiration
Mouth breathing
Urination
Convulsions
Pilo-erection
Diarrhoea
Pupil size
Pupil response
Lacrimation
Impaired gait
Stereotypy
Toe pinch
Tail pinch
Wire manoeuvre
Hind-leg splay
Positional passivity
Tremors
Positive geotropism
Limb rotation
Auditory function

Functional tests:
Grip strength
Locomotor activity

Laboratory examinations
Blood samples were taken from the retrobulbar venous plexus under light 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).
The blood samples obtained were divided into tubes as follows:
EDTA anticoagulant (whole blood) for haematological investigations
Citrate anticoagulant (plasma) for coagulation tests
Li-Heparin anticoagulant (plasma) for biochemical tests

Haematology: ADVIATM 120 Siemens Diagnostics GmbH, 35463 Fernwald, Germany
Haemoglobin content (HGB), mmol/L blood
Erythrocytes (RBC), 10E6/µL blood
Leucocytes (WBC), 10E3/µL blood
Reticulocytes (Reti), % of the erythrocytes
Platelets (PLT), 10E3/µL blood
Haematocrit value (HCT), %
Differential blood count (relative) #, %
Differential blood count (absolute) #, 10E3/µL blood
Mean corpuscular volume (MCV), fL
Mean corpuscular haemoglobin(MCH), fmol
Mean corpuscular haemoglobin concentration (MCHC), mmol/L blood
#: Neutrophilic, eosinophilic and basophilic granulocytes, lymphocytes and monocytes. Large unstained cells were simultaneously quantified during measurement of the differential blood count.

Coagulation: Amax Destiny Plus™ Tcoag Deutschland GmbH, 32657 Lemgo, Germany
Thromboplastin time (TPT) sec
Activated partial thromboplastin time (aPTT), sec

Clinical biochemistry: KONELAB 30i Thermo Fisher Scientific, 63303 Dreieich, Germany
Albumin g/L plasma
Globulin g/L plasma (by substraction)
Albumin/globulin ratio (non-dimensional) (by calculation)
Bile acids µmol/L plasma
Total bilirubin µmol/L plasma
Total cholesterol mmol/L plasma
Creatinine µmol/L plasma
Glucose mmol/L plasma
Protein (total) g/L plasma
Triglycerides mmol/L plasma
Urea (in blood) mmol/L plasma
Calcium mmol/L plasma
Chloride mmol/L plasma
Potassium mmol/L plasma
Sodium mmol/L plasma
Alanine amino-transferase (ALAT) U/L plasma
Alkaline phosphatase (aP) U/L plasma
Aspartate aminotransferase (ASAT) U/L plasma
Lactate dehydrogenase (LDH) U/L plasma


Ophthalmological and auditory examinations
Examinations were performed predose, in test week 13 (end of the treatment period) and in test week 15 (end of recovery
The eyes were examined with a HEINE ophthalmoscope. After examination of the pupillary reflex, mydriasis was produced after instillation of STULLN® eye drops into the cornea. The following ocular structures were then examined:
• Adnexa oculi (i.e. lids, lacrimal apparatus), conjunctiva
• Cornea, anterior chamber
• Lens, vitreous body, fundus (retina, optic disc)
The auditory acuity was checked with a simple noise test.

Sacrifice and pathology:

Necropsy
- On test day 91 (approx. 24 hours after the last administration), the main study animals were dissected following a randomisation scheme.
- Necropsy of all animals scheduled for the recovery period was performed on test day 105
- The animals were euthanized under CO2 atmosphere, 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 examined under suitable illumination.
- The liver and the kidneys were examined.
- Any abnormalities in the appearance and size of the gonads, adrenal glands, uterus, intra-abdominal lymph nodes and accessory reproductive organs were recorded.
The weights of the following organs of all animals were determined before fixation:
Adrenal gland (2)
Brain
Epididymis (2)
Testicle (2)
Ovary (2)
Heart
Kidney (2)
Liver
Spleen
Prostate and seminal vesicles (with coagulating glands, as a whole)
Thymus
Uterus (incl. cervix)
Paired organs were weighed individually and identified as left or right


Histopathology
The organs or parts of organs listed below of all animals were fixed in 7% buffered formalin. The eyes were fixed in Davidson's solution, and the testes in modified Davidson's solution for optimum fixation.
Adrenal gland (2)
Aorta abdominalis
Bone (os femoris with joint)
Bone marrow (os femoris)
Brain (3 levels: cerebrum, cerebellum, medulla/pons)
Epididymis (2)
Eye with optic nerve (2)
Gross lesions observed
Heart (3 levels: right and left ventricle, septum)
Intestine, large (colon, rectum)
Intestine, small (duodenum, jejunum, ileum, incl. Peyer´s patches), Swiss roll method
Kidney and ureter (2)
Liver (2 lobes)
Lungs (with mainstem bronchi and bronchioles (preserved by inflation with fixative and then immersion))
Lymph node (one cervical)
Lmph node (one mesenteric)
Mammary gland
Muscle (skeletal, leg)
Nerve (sciatic)
Oesophagus
Ovary and oviducts (2)
Pancreas
Pituitary
Prostate and seminal vesicles with coagulating glands
Salivary glands (mandibular, sublingual and parotid gland)
Skin (left flank)
Spinal cord (3 sections: cervical, mid-thoracic, lumbar)
Spleen
Stomach
Testicle (2)
Thymus
Thyroid (2) (incl. parathyroids)
Tissue masses or tumours (including regional lymph nodes)
Trachea (incl. larynx)
Urinary bladder
Uterus (incl. cervix)
Vagina
- The afore-listed organs of all main study and recovery animals of groups 1 and 4 were examined histologically after preparation of paraffin sections and haematoxylin-eosin staining.
- Due to test item related changes, targets organs of the animals of the low and intermediate dose level (group 2 and 3) were examined histopatholocically, too.
- In addition, frozen sections of the heart, liver and one kidney were made, stained with Oil Red O and examined microscopically.
- Parathyroids were examined microscopically only if identified macroscopically and if in the plane of section, or in case they were noted as grossly enlarged.

Other examinations:

Assessment on Histomorphological Findings
A number of lesions were noted during pathology evaluation in organs from animals treated for 90 days once daily by oral application of 2,4,6-tris[(dimethylamino)methyl]phenol at doses of 50 and 150 mg/kg b.w./day.
The aim of this assessment was to discuss a possible mechanism for those findings noted during the study.
To prepare the present assessment, the report of the LPT study 34963 was provided. Furthermore, the sections from all organs and all animals were available.
Selected animals from the control, intermediate and high dose groups were re-evaluated. Furthermore an electron microscopy was performed to confirm the suspiction of phospholipidosis.

Statistics:

Toxicology and pathology data were captured, whenever possible, using the departmental computerized systems (Provantis®). Raw data not fully compatible with the computerized systems were maintained on paper according to appropriate SOPs.
The test item-treated groups 2 to 4 were compared with the control group 1.
The following statistical methods were used for the data captured with the Provantis system:
Multiple t-test based on DUNNETT, C. W. New tables for multiple comparisons with a control Biometrics, 482 – 491 (September 1964):
Body weight / Food consumption / / Haematology / Coagulation / Clinical chemistry / Relative and absolute organ weights (p
Exact test of R. A. FISHER : Histology (p
The following settings were used for the statistical evaluation of the parametrical values captured by Provantis:

Homogeneity of variances and normality of distribution were tested using the BARTLETT’s and SHAPIRO-WILKS test. In case of heterogeneity and/or non-normality 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), inter-group comparisons with the control group were made by the DUNNETT’s test (p ≤ 0.01 and p ≤ 0.05).

The following statistical methods were used for the data not captured with the Provantis system:
STUDENT's t-test:
Numerical functional tests: Body temperature / Hind leg splay / Grip strength / Spontaneous motility (p ≤ 0.05 and p ≤ 0.01)
The following limits were used:
- p = 0.05 / 0.01 ^ t = 2.0484 / 2.7633 (for 28 degrees of freedom)
- p = 0.05 / 0.01 ^ t = 2.0687 / 2.8073 (for 23 degrees of freedom)
- p = 0.05 / 0.01 ^ t = 2.3060 / 3.3554 (for 8 degrees of freedom)
These statistical procedures were used for all data. Significantly different data are indicated in the text tables of the results section and in the result tables of the tables section of this report.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Treatment period
None of the male and female rats treated with 15 or 50 mg test item/kg b.w./day and none of the male animals treated with 150 mg test item/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 noted for all female animals treated with 150 mg test item/kg b.w./day from test day 40 onwards.
Recovery period (restricted to groups 1 and 4)
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.

Detailed clinical observations
Detailed clinical observations in form of an assessment of external appearance, body posture, movement and coordination capabilities, and behaviour were performed for all animals pre- and post-dose on test day 1, and once weekly thereafter throughout the treatment period (test weeks 1 to 13). The observations were made within 2 hours after dosing.
All parameters of the detailed clinical observations of all animals scheduled for the control or treatment groups were in the normal range at pre-dose examination on test day 1.
All male and female control animals revealed normal values for each parameter set examined throughout the course of the study.
None of the male and female animals treated with 15 or 50 mg test item/kg b.w./day and none of the male animals treated with 150 mg test item/kg b.w./day by oral administration for 90 days revealed any test item-related changes in external appearance, body posture, movement and coordination capabilities in test weeks 1 to 13.
The female animals treated with the high dose of 150 mg test item/kg b.w./day revealed pilo-erection in test weeks 7 (corresponds to test day 42) to 13.

Mortality:

no mortality observed

Description (incidence):

Treatment period
None of the animals died or had to be sacrificed prematurely. All male and female animals treated with 15, 50 or 150 mg test item/ kg b.w./day survived until their scheduled sacrifice on test day 91.
Recovery period (restricted to groups 1 and 4)
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):

Treatment period
No test item-related influence was observed on the body weight and the body weight gain of the male and female animals treated with 15 or 50 mg test item/kg b.w./day by oral administration for 90 days compared to the control animals throughout the treatment period. No test item-related differences were noted for the body weight at autopsy between the low and intermediate dosed animals and the control animals.
The body weight of the male and female animals treated with 150 mg test item/kg b.w./day was reduced by up to 13% for the males and by up to 12% for the females compared to the control animals as of test day 8 (statistically significant at p ≤ 0.05 or p ≤ 0.01 on test days 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, 85 and 90 for the male and female animals). Accordingly, the body weight gain from test day 1 to test day 90 was reduced by approx. 20% for the males and by approx. 15% for the females compared to the control animals. The body weight at autopsy of the high dosed animals was reduced by 11% for the male rats and by 14% for the female rats.
Recovery period (restricted to groups 1 and 4)
The body weight of the male animals previously treated with 150 mg test item/kg b.w./day was still reduced by 11% (statistically significant at p ≤ 0.01 on test days 97 and 104) at the end of the recovery period. The body weight at autopsy was also reduced by 11% for the males. In contrast, the body weight and the body weight at autopsy of the previously high dosed female animals had normalized during the 14-day treatment-free recovery period.

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 15 or 50 mg test item/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 150 mg test item/kg b.w./day by oral administration was reduced by up to 10% for the males and by up to 19% for the females compared to the control animals starting in test week 1 (statistically significant at p ≤ 0.05 or p ≤ 0.01 in test weeks 1 to 4 and 10 to 13 for the males and in test weeks 1 to 13 for the females).
Recovery period (restricted to groups 1 and 4)
The food consumption of the male and female animals previously treated with 150 mg test item/kg b.w./day had normalized and the values were within the range of the control group at the end of the 14-day treatment-free recovery period.

Food efficiency:

not specified

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 15 mg test item/kg b.w./day and for the male animals treated with 50 mg test item/kg b.w./day by oral administration compared to the control animals throughout the 90-day treatment period.
The drinking water consumption of the female animals treated with 50 mg test item/kg b.w./day by oral administration was slightly decreased by up to 14% compared to the control animals starting in test week 5 (statistically significant at p ≤ 0.05 or at p ≤ 0.01 in test weeks 5 to 8) evaluated by weekly quantitative assessment.
The drinking water consumption of the male and female animals treated with 150 mg test item/kg b.w./day by oral administration was decreased by up to 11% for the males and by up to 28% for the females compared to the control animals starting in test week 1 (statistically significant at p ≤ 0.05 or at p ≤ 0.01 in test weeks 1 to 4, 6 to 8 and 12 for the males and in test weeks 1 to 2 and 4 to 13 for the females) evaluated by weekly quantitative assessment.
Recovery period (restricted to groups 1 and 4)
The drinking water consumption of the female animals previously treated with 150 mg test item/kg b.w./day was still decreased by 30% or 19% in test weeks 14 and 15, respectively (statistically significant at p ≤ 0.05 in test week 14). In contrast, the drinking water consumption of the previously high dosed male animals had normalized during the 14-day treatment-free recovery period.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Treatment and recovery period (recovery restricted to groups 1 and 4)
The ophthalmological examination did not reveal any test item-related changes of the eyes and the optic region in any animal after repeated oral treatment with 15, 50 or 150 mg test item/kg b.w./day in test week 13 and test week 15.
No test item-related pathological changes were noted on the adnexa oculi (i.e. lids, lacrimal apparatus), conjunctiva, cornea, anterior chamber, lens, vitreous body, and fundus (retina, optic disc).
There was no indication of any impairment to auditory acuity.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Haematology and coagulation
Treatment period and recovery period (restricted to groups 1 and 4)
No test item-related influence was observed on the haematological parameters for the male and female animals treated with 15, 50 or 150 mg test item/kg b.w./day by oral administration for 90 days compared to the control animals at the end of the treatment period (test day 91) and at the end of the recovery period (test day 105).
No test item-related effects were observed for the haemoglobin content (HGB), the numbers of erythrocytes (RBC), leucocytes (WBC) and platelets (PLT), the relative reticulocyte count (Reti), the haematocrit value (HCT), the relative and absolute differential blood count, the thromboplastin time (TPT), the activated partial thromboplastin time (aPTT), the mean corpuscular volume (MCV), the mean corpuscular haemoglobin (MCH), and the mean corpuscular haemoglobin concentration (MCHC) on test day 91 and on test day 105.
The following statistically significant differences in haematological parameters compared to the control animals noted on test day 91 or on test day 105 are not considered to be test item-related but to be coincidental: see table "any other information on results".

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Treatment period
No test item-related influence was observed on the biochemical parameters for the male and female animals treated with 15 or 50 mg test item/kg b.w./day by oral administration for 90 days compared to the control animals at the end of the treatment period (test day 91).
The following test item-related changes were noted for the male and female animals treated with 150 mg test item/kg b.w./day on test day 91:
Test item-related changes in biochemical parameters compared to the control group 1 [%].
Parameter Group 4, 150 mg/kg
males females
Test day 91
Albumin -7** -18**
Globulin -7 -16**
Protein -7** -17**
Urea +42** +62**
*/**: statistically significant at p ≤ 0.05 / p ≤ 0.01

No test item-related effects were noted on the albumin/globulin ratio, on the plasma levels of bile acids, total bilirubin, total cholesterol, creatinine, glucose, triglycerides, calcium, chloride, potassium, and sodium on test day 91. No test item-related influence was noted on the plasma enzyme activities of alanine amino-transferase (ALAT), alkaline phosphatase (aP), aspartate aminotransferase (ASAT), and lactate dehydrogenase (LDH). All data are within the limits of normal biological variability.

The following statistically significant differences in biochemical parameters compared to the control animals noted on test day 91 are not considered to be test item-related but to be coincidental:
see table "any other information on results"

Recovery period (restricted to groups 1 and 4)
No recovery was noted for the findings previously noted for the parameters of the clinical chemistry: The following test item-related changes were still noted for the male and female animals previously treated with 150 mg test item/kg/b.w./day at the end of the 14-day treatment-free recovery period on test day 105:
Test item-related changes in biochemical parameters compared to the control group 1 [%]
Parameter Group 4, 150 mg/kg
males females
Test day 105
Albumin -6* -9**
Globulin -8** -5
Protein -7** -7*
Urea +38** +51**
*/**: statistically significant at p ≤ 0.05 / p ≤ 0.01

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Neurological screening
The neurological screening was performed on all main study and recovery animals (groups 1 and 4: n = 15 per group and sex, groups 2 and 3: n = 10 per group and sex) at the end of treatment (in test week 13) 1 to 2 hours after dosing, and on all recovery animals (groups 1 and 4: n = 5 per group and sex) at the end of the recovery period (in test week 15).
Treatment period and recovery period (restricted to groups 1 and 4)
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 15, 50 or 150 mg test item/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 150 mg test item/kg b.w./day in test week 15.
The statistically significant increase (at p

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Treatment period
No test item-related changes were noted for the relative and absolute organ weights of the male and female animals treated with 15 or 50 mg test item/kg b.w./day by oral administration for 90 days compared to the control animals at the end of the treatment period (test day 91).
The following test item-related changes were noted for the male and female animals treated with 150 mg test item/kg b.w./day on test day 91:
Test item-related changes in organ weights compared to the control group 1 [%]
Organ Group 4, 150 mg/kg
males females
Test day 91
Epididymis (left, abs.) -13** N/A
Epididymis (right, abs.) -11* N/A
Prostate and Seminal Ves. (rel.) -13** N/A
Prostate and Seminal Ves. (abs.) -22** N/A
Ovary (left, abs.) N/A -26
Ovary (right, abs.) N/A -27*
Liver (rel.) none +42**
Liver (abs.) none +23**
Spleen (rel.) none +51**
Spleen (abs.) none +30**
Uterus (incl. cervix, abs.) N/A -28*
*/**: statistically significant at p ≤ 0.05 / p ≤ 0.01
N/A: not applicable

The following statistically significant differences in organ weights compared to the control animals on test days 91 are not considered to be test item-related but mostly caused by the reduced body weight in the high dose group: see table "any other information on results".

Recovery period (restricted to groups 1 and 4)
No recovery was noted for the findings previously noted for the relative and absolute organ weights: The following test item-related changes were noted for the male and female animals previously treated with 150 mg test item/kg b.w./day at the end of the 14-day treatment-free recovery period on test day 105:
Test item-related changes in organ weights compared to the control group 1 [%]
Organ Group 4, 150 mg/kg
males females
Test day 105
Epididymis (left, abs.) -21 N/A
Epididymis (right, abs.) -26* N/A
Prostate and Seminal Ves. (rel.) -11 N/A
Prostate and Seminal Ves. (abs.) -22** N/A
Liver (rel.) none +35**
Liver (abs.) none +29**
Spleen (rel.) none +41*
Spleen (abs.) none +35
*/**: statistically significant at p ≤ 0.05 / p ≤ 0.01
N/A: not applicable

Statistically significant differences in organ weights compared to the control animals on test days 91 and 105 that are not considered to be test item-related but mostly caused by the reduced body weight in the high dose group are listed in the text table: see table "any other information on results".

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Macroscopic post mortem findings
Treatment and recovery period (recovery restricted to groups 1 and 4)
The macroscopic inspection at necropsy did not reveal any test item-related changes in the organs and tissues of the animals treated with 15, 50 or 150 mg test item/kg b.w./day by repeated oral administration after terminal sacrifice at the end of the treatment period (test day 91) and at the end of the recovery period (test day 105).
However, macroscopic changes were noted in the thyroid (reduced in size), heart (enlarged), kidneys (stained pale discoloured), uterus (dilated, filled with clear liquid), spleen (enlarged) and liver (enlarged) 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:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment period
No test item-related changes were noted in the selected target organs of the male and female animals treated with 15 mg test item/kg b.w./day by oral administration for 90 days.
The histomorphological examination of the selected target organs of the male and female animals treated with 50 mg test item/kg b.w./day by oral administration for 90 days revealed the following test item-related changes in numerous organs:
Vacuolations / vacuolated (swollen) cells were found in the media of arteries in many organs such as adrenal gland, aorta, bone, heart, kidneys, lungs, skeletal muscle, pancreas, salivary glands, stomach, and thymus.
Furthermore, vacuolation was noted in the smooth muscles/muscularis of the coagulating glands, lungs (bronchus), seminal vesicles, stomach and ureters.
Vacuolization (swellings) of epithelial / glandular epithelial cells was observed in salivary glands (parotis), lungs (bronchus, alveoles) and liver (centrilobular).
In addition, vacuolation of interstitial / reticular cells and macrophages were noted in thyroids, heart and spleen.
No vacuolization was noted in the brain.

The histomorphological examination of the organs of the male and female animals treated with 150 mg test item/kg b.w./day by oral administration for 90 days revealed the following test item-related changes in numerous organs:
Vacuolations / vacuolated (swollen) cells were found in the media of arteries in many organs such as adrenal gland, aorta, bone, cervix, epididymis, oesophagus, eye, heart, duodenum, ileum, jejunum, kidneys, larynx, liver, lungs, lymph nodes, mammary glands, skeletal muscle, ovaries, oviducts, pancreas, prostate, salivary glands, skin, stomach, testes, thymus, uterus and vagina.
Furthermore, vacuolation was noted in the smooth muscles/muscularis of the cervix, coagulating glands, colon, duodenum, jejunum, ileum, rectum, lungs (bronchus), oviducts, prostate, seminal vesicles, stomach, trachea, ureters, uterus and vagina.
Vacuolization (swellings) of epithelial / glandular epithelial cells was observed in the brain (choroid plexus), eyes (iris), parathyroids, salivary glands (parotis), larynx, trachea, lungs (bronchus, alveoles), liver (centrilobular, with minimal centrilobular infiltration of inflammatory cells), stomach (pars glandularis) and kidneys (proximal tubuli).
In addition, vacuolation of interstitial / reticular cells and macrophages were noted in thyroids, heart, spleen and lymph nodes.
And finally, vacuolation was observed in the pituitary (pars nervosa) of the female animals.
All other changes noted are regarded as spontaneous and to be within the normal background pathology commonly seen in rats of this strain and age.

Recovery period (restricted to groups 1 and 4)
Vacuolation was still noted for the male and female animals previously treated with 150 mg test item/kg b.w./day at the end of the 14-day treatment-free recovery period, however, the severity of the vacuolation had slightly subsided in some organs.

The additional histopathological examination of the organs of the low and intermediate dosed animals revealed vacuolization in various organs of the animals treated with 50 mg test item/kg b.w./day by oral administration for 90 days. However, less organs compared to the high dose group (150 mg/kg) were affected at 50 mg/kg and the organs were affected to a lesser degree.

No test item-related findings were noted for the animals treated with 15 mg test item/kg b.w./day by oral administration for 90 days.

Histopathological findings: neoplastic:

not specified

Description (incidence and severity):

see above

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Assessment on Histomorphological Findings
Selected animals from the control, intermediate and high dose groups were re-evaluated. The previously reported findings were confirmed. Representative pictures from selected organs were taken by an Olympus UC-30 camera.

As seen in the report attached to this study record endpoint following findings are described: (figures 1-17 see below and assessment attached to this study endpoint record)
The vascular lesions in arteries, i.e., vacuoles of the media smooth muscle cells appeared to be as sharply demarcated optically empty spaces (except of the margins, where there appeared narrow, irregular rims of a pinkish substance (Figures 1-6)
Similar alterations were noted in smooth muscle fibers of some organs. i.e., seminal vesicles (Figure 7).
In heart myofibers, a minor vacuolation caused a vacuolar degeneration (/Figure 8).
Epithelial vacuolation was morphological similar to cell edema, i.e., it resembled swelling with displacement of cytoplasm and cytoplasmic structures. In epithelial cells, the vacuoles were not as sharply demarcated as in arterial smooth muscle cells (Figures 9-11).
In the liver, vacuoles resembled lipid storage (Figure 12).
The choroid plexus cells were enlarged and, within the cytoplasm, very tiny vacuoles were present that caused a pale appearing cytoplasm (Figures 13-15).
Vacuoles in the spleen are considered to represent Histiocytosis. The cells contained occasionally remnants of cells in the vacuolar cytoplasm (Figures 16, 17).

A number of lesions were noted during pathology evaluation in organs from animals treated for 90 days once daily by oral application of 2,4,6-tris[(dimethylamino)methyl]phenol at doses of 50 and 150 mg/kg b.w./day.

The noted findings may be grouped in:
 -            Vacuolation of smooth muscle fibers in the arterial media in multiple organs
-            Vacuolation of smooth muscle fibers in different organs
-            Vacuolar degeneration of cardiac myofibers
-            Epithelial vacuolation in several organs, e.g., parathyroid glands, renal glomeruli
-            Vacuolation in liver
-            Vacuolation of choroid plexus cells
-            Vacuolation and increased histiocytes in the spleen.
 
The alterations were not associated with further inflammatory and/or degenerative lesions, and showed partial or complete recovery.
 
By morphology and distribution, the findings are indicative for phospholipidosis, which is not an adverse effect and not relevant for human health.

The overall picture of the lesions, i.e., the vacuolation and the distribution of the findings is deemed to be indicative for phospholipidosis. Phospholipidosis can affect multiple and different organs (van Meer, 2006). This is especially true for the medial affection of arteries in multiple organs (Ishikawa et al., 1988; Shayman and Abe, 2013). Renal affections are described occasionally. i.e., namely a podocyte affection in the renal glomeruli is described (Costa et al., 2013; Scheurle et al., 2014). The latter is deemed to be the cuase of the glomerular vacuolation. Macrophages are involved in the facilitationo of the removal/clearance of the of the lamellar body/xenobiotic complex (vanMeer, 2006), and therefore, Kuppfer’s cells and histiocytes are involved that are represented in the present study by vacuolated cells in the liver and spleen. The involvement of neuronal structures, and especially the plexus is less common, but is also a known phenomenon (Benitz and Kramer, 1968; Koizumi et al., 1986). Increased organ weights are considered to be related to the phospholipidosis.
 
In addition, there were reduced reproductive organ weights (epididymides, prostate glands, seminal vesicles, and ovaries and uterus) without related with specific changes in oocytes or sperm cells but with vacuolation of arteries and smooth muscle cells. Therefore a primary effect on reproductive organs cannot be considered.
The same is true for the effects endocrine organs, i.e., the pituitary gland and adrenal glands. The findings are consistent with phospholipidosis. A primary affection of the endocrine system cannot be conluded.

Conclusion
The test item caused no findings at 15 mg/kg b.w. /day. Furthermore, at higher doses, there was a lack of concurrent adverse toxicity or dysfunction in the affected organs.  
 
The diagnosis of Phospholipidosis was confirmed by Transmission electron microscopy, which is considered to be the most sensitive method for confirming the presence of PL…’ (Chatman et al., 2009). Therefore, organs were selected for evaluation by transmission electron microscopy.
The electron microscopy evaluation revealed the presence of membrane bound vacuoles indicative for lysosome containing myelin figures. They were noted in the brain plexus epithelia, in smooth muscle fibers of the heart, in liver macrophages, in renal tubular cells and in the smooth muscle fibers of lungs. The finding of lysosomes containing myelin figures is proving the hypothesis of phospholipidosis (Chatman et al., 2009; Nolte et al., 2016).
Further pictures of the electron microscopy can be found in the attached report.


 

Details on results:

In conclusion, adverse test item-related effects were noted at 50 mg test item/kg b.w./day in form of a decreased drinking water consumption and histopathological examination, at 150 mg test item/kg b.w./day in form of a reduced body weight, a decreased food and drinking water consumption, changes in biochemical parameters, organ weights and at histopathological examination compared to the control group.

Key result

Dose descriptor:

NOAEL

Effect level:

15 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

other: Reduced body weight, decreased food / water consumption, changes in biochem. parameters / organ weights and at histopath. examination

Key result

Critical effects observed:

not specified

Lowest effective dose / conc.:

50 mg/kg bw/day (nominal)

System:

other: a reduced body weight, a decreased food and drinking water consumption, changes in biochemical parameters and organ weights and at histopathological examination

 Haematology and coagulation

Statistically significant changes in haematological parameters considered not test item-related

Parameter	Reference table no.	Increase­ Decrease¯	Group / Sex	Test day	Statistical significance	Reason
HGB	9-1	¯ ¯ ¯	2 f 4 f 4 f	91 91 105	p ≤ 0.01 p ≤ 0.01 p ≤ 0.05	A, B A A
RBC		¯ ¯ ¯ ¯	4 m 4 m 4 f 4 f	91 105 91 105	p ≤ 0.01 p ≤ 0.01 p ≤ 0.01 p ≤ 0.01	A A A A
WBC		+ +	4 f 4 f	91 105	p ≤ 0.01 p ≤ 0.05	A, C A, C
Neut (abs.)		+	4 f	91	p ≤ 0.01	A, C
Lym (abs.)		+ +	4 f 4 f	91 105	p ≤ 0.01 p ≤ 0.05	A, C A, C
Mono (abs.)		+ +	4 f 4 f	91 105	p ≤ 0.01 p ≤ 0.01	A, C A, C
LUC (abs.)		+ +	4 f 4 f	91 105	p ≤ 0.01 p ≤ 0.01	A, C A, C
Baso (abs.)		+ +	2 f 4 f	91 91	p ≤ 0.05 p ≤ 0.01	A, B, C A, C

Parameter	Reference table no.	Increase­ Decrease¯	Group / Sex	Test day	Statistical significance	Reason
Reti	9-1	+ +	4 m 4 f	105 105	p ≤ 0.01 p ≤ 0.05	A A
HCT		¯ ¯	2 f 4 f	91 91	p ≤ 0.01 p ≤ 0.01	A, B A
MCV		+ +	4 m 4 f	91 105	p ≤ 0.01 p ≤ 0.01	A A
MCH		+ +	4 m 4 f	91 105	p ≤ 0.01 p ≤ 0.01	A A
m:         male f:           female A:          the difference to the control group is considered to have no toxicological significance B:          lacking dose dependence C:          effect is due to the relative low or high value observed for the control group

 Clinical chemistry

Statistically significant changes in biochemical parameters considered not test item-related

Parameter	Reference table no.	Increase­ Decrease¯	Group / Sex	Test day	Statistical significance	Reason
Creatinine		+	4 m	91	p ≤ 0.01	A
Calcium		¯ ¯ ¯	3 m 4 m 4 f	91 91 91	p ≤ 0.01 p ≤ 0.01 p ≤ 0.01	A A A
Chloride		¯ ¯	3 m 4 m	91 91	p ≤ 0.05 p ≤ 0.05	A A
Potassium		+	4 f	91	p ≤ 0.05	A
ALAT		¯ ¯	4 m 4 f	91 91	p ≤ 0.01 p ≤ 0.05	A A
LDH		+	4 f	91	p ≤ 0.01	A
m:         male f:           female A:          the difference to the control group is considered to have no toxicological significance

Organ weights

Statistically significant organ weight changes unrelated to the test item

Statistically significant changes in organ weights in comparison to the control group considered not test-item-related
Organ	Reference table nos.	Increase­ Decrease¯	Group / Sex	Test day	Statistical significance	Reason
Brain (relative)	14-1	+ +	4 m 4 f	91 91	p ≤ 0.01 p ≤ 0.01	B B
Testis, left (relative)		+	4 m	91	p ≤ 0.05	B
Testis, right (relative)		+	4 m	91	p ≤ 0.01	B
Heart (relative)		+ +	4 m 4 f	91 91	p ≤ 0.05 p ≤ 0.01	B B
Kidney, left (relative)		+	4 f	91	p ≤ 0.01	B
Kidney, right (relative)		+	4 f	91	p ≤ 0.01	B
Liver (relative)		+	4 m	91	p ≤ 0.01	B
Spleen (relative)		+	4 m	91	p ≤ 0.05	B
Kidney, left (absolute)	15-1	¯	4 m	91	p ≤ 0.05	A
m:         male f:           female A:          the slight change compared to the control group is considered to be without toxicological relevance B:          change is due to the reduced body weight

Statistically significant organ weight changes unrelated to the test item

Statistically significant changes in organ weights in comparison to the control group considered not test-item-related
Organ	Reference table nos.	Increase­ Decrease¯	Group / Sex		Test day	Statistical significance	Reason
Brain (relative)	14-1	+	4 m	105		p ≤ 0.05	B
Heart (relative)		+ +	4 m 4 f	105 105		p ≤ 0.05 p ≤ 0.01	B A
Kidney, left (relative)		+	4 m	105		p ≤ 0.05	B
Liver (relative)		+	4 m	105		p ≤ 0.01	B
Brain (absolute)	15-1	¯	4 f	105		p ≤ 0.05	A
Heart (absolute)		+	4 f	105		p ≤ 0.01	A
m:         male f:           female A:          the slight change compared to the control group is considered to be without toxicological relevance B:          change is due to the reduced body weight

Test item formulation analysis

The analysis of the test item-vehicle mixtures from test days 1 and 90 was performed by LPT using an HPLC-UV detection method that was re-validated at LPT.

Parameter	Sampling / Handling	Range of test item concentration in percent of nominal concentration
Concentration	Immediately after preparation and at study termination	91.9% - 101.1%
Stability	Left at room temperature for 8 h or 24 h	91.3% - 101.6%

The results indicate that all test item formulations were correctly prepared by LPT, and were stable for at least 24 hours.

Conclusions:

In conclusion, adverse test item-related effects were noted starting at 50 mg test item/kg b.w./day in form of a decreased drinking water consumption and histopathological changes. At 150 mg test item/kg b.w./day changes were noted such as reduced body weight, a decreased food and drinking water consumption, changes in biochemical parameters and organ weights and at histopathological examination in comparison to the control group.

Under the present test conditions of this study, the NOAEL (No-Observed-Adverse-Effect-Level) is 15 mg test item/ 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 of test item 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 with 15, 50 or 150 mg test item/kg b.w./day. The control animals received the vehicle (PEG400).

No deaths occurred.

Pilo-erection was noted for all female animals treated with 150 mg test item/kg b.w./day from test day 40 onwards. The male animals were not affected.

The body weight, the body weight gain, the body weight at autopsy and the relative food consumption of the male and female animals treated with 150 mg test item/kg b.w./day were reduced.

The drinking water consumption (by weekly quantitative assessment) of the female animals treated with 50 mg test item/kg b.w./day and of the male and female animals treated with 150 mg test item/kg b.w./day by oral administration was decreased compared to the control animals.

The plasma levels of albumin, globulin and protein were decreased and the plasma level of urea was increased for the male and female animals treated with 150 mg test item/kg b.w./day compared to the control animals.

The relative and/or absolute weights of the epididymides, the prostate and the seminal vesicles of the male animals and of the ovaries and uterus of the female animals were reduced and the relative and absolute liver and spleen weights of the female animals treated with the high dose of 150 mg test item/kg b.w./day were increased compared to the control animals. No effects were observed at the mid and low dose.

Vacuolations / vacuolated (swollen) cells were found in the media of arteries, in the smooth muscles / muscularis, of epithelial / glandular epithelial cells, of interstitial / reticular cells and macrophages in many organs and in the pituitary of the male and/or female animals treated with 50 or 150 mg test item/kg b.w./day. However, less organs of the animals treated with 50 mg test item/kg b.w./day were affected compared to the high dose group and the findings were of a less severity. Vacuolizations in the brain (choroid plexus) were only observed at the high dose of 150 mg test item/kg b.w./day. As these effects in the brain are not clinical detectable (No test item-related effects were observed in the neurological screening; see 1.2), they are not expected to be related to neurotoxic effects of the test item.

Selected animals from the control, intermediate and high dose groups were re-evaluated (Weber, 2018; see attachment above): The previously reported findings were confirmed. The alterations were not associated with further inflammatory and/or degenerative lesions, and showed partial or complete recovery. By morphology and distribution, the findings are indicative for phospholipidosis. In addition, there were reduced reproductive organ weights (epididymides, prostate glands, seminal vesicles, and ovaries and uterus) without related with specific changes in oocytes or sperm cells but with vacuolation of arteries and smooth muscle cells. Therefore a primary effect on reproductive organs cannot be considered. The same is true for the effects endocrine organs, i.e., the pituitary gland and adrenal glands. The findings are consistent with phospholipidosis. A primary affection of the endocrine system cannot be concluded. In conlusion, the test item caused no findings at 15 mg/kg b.w. /day. Furthermore, at higher doses, there was a lack of concurrent adverse toxicity or dysfunction in the affected organs.

The electron microscopy evaluation revealed the presence of membrane bound vacuoles indicative for lysosome containing myelin figures. They were noted in the brain plexus epithelia, in smooth muscle fibers of the heart, in liver macrophages, in renal tubular cells and in the smooth muscle fibers of lungs. The finding of lysosomes containing myelin figures is proving the hypothesis of phospholipidosis (Chatman et al., 2009; Nolte et al., 2016), which is regarded to be not relevant for human health.

No test item-related changes were observed for any of the parameter of the neurological screening, haematological and coagulation parameters, the eyes or optic region, the auditory acuity, and at macroscopic inspection at necropsy at any dose level.

At the end of the recovery period, the body weight and the body weight at autopsy of the male animals and the drinking water consumption of the female animals previously treated with 150 mg test item/ kg b.w./day were still decreased. Further, the plasma levels of albumin, globulin and protein were still decreased and the plasma level of urea was still increased for the male and female animals previously treated with 150 mg test item/kg b.w./day at the end of the recovery period. In addition, the relative and/or absolute weights of the epididymides, the prostate and the seminal vesicles of the previously high dosed males were still reduced and the relative and/or absolute weights of several organs were still influenced. At histopathological examination, vacuolation was still noted for the male and female animals previously treated with 150 mg test item/ kg b.w./day, however, the severity of the vacuolation had slightly subsided in some organs.

All other changes had subsided at the end of the 14-day treatment-free recovery period.

In conclusion, adverse test item-related effects were noted starting at 50 mg test item/kg b.w./day in form of a decreased drinking water consumption of histopathological changes. At 150 mg test item/kg b.w./day changes were noted in form of a reduced body weight, a decreased food and drinking water consumption, changes in biochemical parameters and organ weights and at histopathological examination compared to the control group.

Under the present test conditions of this study, the NOAEL (No-Observed-Adverse-Effect-Level) is 15 mg test item/ kg b.w./day by oral administration for 90 days.

 

Reference 3

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

Dose Range finding study for the OECD 422 study in rats

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2006

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Short term study with reduced scope of examination

Qualifier:

no guideline followed

Principles of method if other than guideline:

Short term toxicity study used as dose range finding study for OECD 422 study (SafePaharm 2006) with only limited scope of examination (clinical observations, body weight, necropsy)

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

other: Sprague-Dawley Crl: CD (SD) IGS BR

Details on species / strain selection:

Sprague-Dawley Crl: CD (SD) IGS BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

ANIMALS
15 male and 15 female Sprague-Dawley CRL: CD (SD) IGS BR strain rats were obtained from Charles River (UK) Limited, Margate, Kent.
After an acclimatisation period of at least seven days, animals were selected at random and given a unique number within the rangefinder
by ear punching.
At the start of treatment the males weighed 248 to 366 g and the females weighed 177 to 234 g.

HOUSING
The animals were housed in groups of three by sex in polypropylene grid-floor cages suspended over trays containing absorbent paper.
Free access to mains drinking water and food (Rodent PM1 5002 (Certified) Diet, BCM IPS Limited, London, UK) was allowed throughout the
acclimatisation and treatment period.
The animals were housed in a single air-conditioned room within the Safephm Barrier Maintained Rodent Facility.
The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours
continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerised system,
and printouts of the mean temperature and humidities were included in the records. The temperature and relative humidity were controlled
to remain within target ranges of 21 +/- 2°C and 55 +/- 15% respectively.

Route of administration:

oral: gavage

Vehicle:

arachis oil

Remarks:

Arachis oil BP

Details on oral exposure:

The test material was administered daily, for up to fourteen consecutive days, by gavage using a suitable dosing cannula attached to a disposable plastic syringe. Control animals were treated
in an identical manner with 4 ml/kg/day of Arachis oil BP. The volume of test and control material administered to each animal was based on the most recent
bodyweight and was adjusted at Days 4,8 and 11.

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

The concentration and stability of the test material formulations were not determined analytically.
For the purpose of the rangefmder, the test material was prepared as a solution in Arachis oil BP. A fresh formulation was made each day and the animals were dosed
within three hours of preparation.

Duration of treatment / exposure:

14 days

Frequency of treatment:

daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

control group

Dose / conc.:

150 mg/kg bw/day (nominal)

Remarks:

37.5 mg/ml;

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

62.5 mg/ml

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

125 mg/ml

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

250 mg/ml

No. of animals per sex per dose:

15 males and females per dose group

Control animals:

yes, concurrent vehicle

Details on study design:

Short term toxicity study used as dose range finding study for OECD 422 study (SafePaharm 2006) with only limited scope of examination (clinical observations, body weight, necropsy)

Positive control:

no

Observations and examinations performed and frequency:

Clinical Observations
All animals were examined for overt signs of toxicity, ill health or behavioral change immediately before dosing and one hour after dosing.
All observations were recorded.

Bodyweight
Individual body weights were recorded on Days 1,4, 8, 1 1 and 14 of the treatment period.

Sacrifice and pathology:

Necropsy
On completion of the treatment period, all animals were killed by cervical dislocation and immediately subjected to an internal and external macroscopic examination.
No tissues were retained.

Other examinations:

not performed

Statistics:

no data

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Animals treated with 1000 mg/kg/day on Day 3 showed instances of hunched posture, tip-toe gait and noisy respiration.
Instants of red/brown staining were also observed around the snout, anogenital region and the fur with one female also showed pitosis.
Two males were found dead on Day 4; subsequently the remaining animals in the treatment group were killed in extremis. Similar
clinical observations along with pilo-erection were observed in animals of either sex treated with 500 or 250 mg/kg/day starting on
Day 5 and Day 8 respectively, leading to the termination of the 500 mg/kg/day treatment group on Day 8 due to the severity of the
clinical observations displayed. Animals treated with 250 mg/kg/day continued to display these clinical observations for the remainder
of the study. Animals of either sex treated with 150 mg/kg/day showed instants of hunched posture, tip-toe gait, noisy respiration,
pilo-erection and redbrown staining around snout/mouth from Day 11 for the remainder of the study. Increased salivation was detected
immediately after dosing for animals of either sex in all treatment groups from Day 3 onwards.

Mortality:

mortality observed, treatment-related

Description (incidence):

Two males treated with 1000 mg/kg/day were found dead on Day 4, subsequently all remaining animals of either sex were terminated due to the severity of the clinical observations. Animals of
either sex treated with 500 mg/kg/day were terminated on Day 8 due to the severity of the clinical observations.
No unscheduled deaths were observed in animals of either sex treated with 150 or 250 mg/kg/day.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Animals of either sex from all treatment groups showed reduction of bodyweight gains throughout the treatment period with severe weight loss detected in animals treated
with 1000 or 500 mg/kg/day.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

All animals treated with 1000 mg/kg/day showed haemorrhage of the glandular region of the stomach and epithelial sloughing of the non-glandular region
of the stomach. The two males of this treatment group that were found dead also showed haemorrhage of the lungs and patchy pallor of the liver.
The remaining male in the treatment group showed pallor of the liver and kidneys. One female of this treatment group had pallor of the kidneys.
Epithelial sloughing of the non-glandular region of the stomach was seen in the majority of animals treated with 500 mg/kg/day with one female also showing
thickening of the non-glandular region of the stomach. One animal of either sex treated with 250 mg/kg/day had a mottled appearance to all
lobes of the lungs, with one animal of either sex in the same treatment group showing pallor of the lungs. One male and two females treated with 150 mg/kg/day
showed pallor of the lungs the male also showed a mottled appearance of all lobes of the lungs. One animal of either sex showed red patches on all lobes of the lungs
and one female had increased renal pelvic cavitations of both kidneys.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Dose descriptor:

LOAEL

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

gross pathology

Conclusions:

The dose levels for the main study were chosen, following consultation with the Sponsor, as:
Low dose: 15 mg/kg/day
Intermediate dose: 50 mg/kg/day
High dose: 150 mg/kg/day
plus a control group treated with vehicle alone (Arachis oil BP).
Air Products

Executive summary:

The substance was tested in a 14 day short term toxicity study in rats with dose levels of 0, 150, 250, 500 and 1000 mg/kg bw/d. The aim was to determine suitable dose groups for the OECD 422 study. At 1000 and 500 mg/kg mortality occured and the animals had to be terminated due to the severity of the clinical observations. The clinical sings such as staining arround the snout, pilo-erection, hunched posture, tip-toe gait and noisy respiration was also obsereved in lower dose groups. Salivation was present in all dosed groups. Body weight was severe decreased in 500 mg/kg bw/d and 1000 mg/kg bw/d groups. Necropsy findings in the stomach can all be related to the corrosive properties of the test compounds. effects were haemorrhage of the glandular regions of the stomach and epithelial sloughing of the non-glandular region of the stomach. Some effects such as mottlled appearance or paloor occured in the kidney and the lungs.

The dose levels for the OECD 422 study were dtermined to be 0, 15, 50 and 150 mg/kg bw/d.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

15 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Klimisch 1

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Data waiving:

other justification

Justification for data waiving:

other:

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

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: dermal

Data waiving:

other justification

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Because of the results of the oral repeated dose toxicity studies (according to OECD 422 and OECD 408) the substance is not classified according to CLP regulation (1272/2008).