Cyclohexane, 5-isocyanato-1-(isocyanatomethyl)-1,3,3-trimethyl-, homopolymer, acetone oxime-blocked

Endpoint summary

Description of key information

Oral route:

There is a 90 day sub-chronic oral toxicity study available.

Overall Conclusion:

All described observations, although statistically significant, were not considered of any biological or any toxicological significance since no correlating effects were found at the histopathological evaluation in particular in the liver. Moreover, most changes noted had subsided at the end of the 4 -week recovery period.

Under the present test conditions of this study, the no observed adverse effect level (NOAEL) was above 1000 mg test item/kg b.w./day.

Study was conducted in accordance with ECHA Decision TPE-D-2114306081-68-01/F Helsinki, 30 July 2015

Inhalation route:

A subacute 28 day or subchronic 90 day inhalation toxicity study is not needed, because a subchronic oral  90 day toxicity study is available for the substance.  According to ECHA decision TPE-D-2114306081-68-01/F Helsinki, 30 July 2015, ECHA considers that testing by oral route is most appropriate. Cited from the decision:  "In light of the physicochemical properties of the substance, a solid with a high molecular weight, very low vapour pressure and low water solubility and the information provided on the uses and human exposure (i.e. no uses with spray application), ECHA considers that testing by the oral route is most appropriate."

Dermal route:

A 14-day range finding study of cyclohexane, 5-isocyanato-1-(isocyanatomethyl)-1,3,3-trimethyl-, homopolymer, acetone oxime-blocked in rats by dermal administration (0, 30, 100 and 1000 mg/kg bw/day; 6 hours/day, 5 days/week; based on OECD 411) did not cause any signs of local or systemic intolerance. None of the rats died prematurely. No test item-related influence was noted on the body weight, food and drinking water consumption. Macroscopic examination at necropsy revealed no test item-related changes at any of the tested groups. Therefore, under the conditions of this study the highest concentration (1000 mg/kg bw /day) is considered to be the No Observed Adverse Effect Level (NOAEL).

In addition to No Observed Adverse Effect Level (NOAEL > 1000 mg/kg bw /day) via oral route and the physicochemical properties (described in chapter 4) it can be concluded that further testing by dermal route is not needed.

Cited from the decision:  "In light of the physicochemical properties of the substance, a solid with a high molecular weight, very low vapour pressure and low water solubility and the information provided on the uses and human exposure (i.e. no uses with spray application), ECHA considers that testing by the oral route is most appropriate."

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

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2015-11-04 to 2015-12-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Study was conducted as dose range finding study (DRF) according ECHA Decision TPE-D-2114306081-68-01/F Helsinki, 30 July 2015

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:

2001/59/EC; Official Journal of European Communities, L 255 2001.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

CD-1

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: 56 days, females: 57 days
- body weight: Males: 292.1 g - 321.0 g, females: 198.4 g - 232.1 g
- Diet: ad libitum, Commercial diet ssniff® R/Z V1324 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany)
- Water: ad libitum
- Acclimatisation period: 8 days
-Housing: kept singly in MAKROLON cages
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 °C +/- 3° C
- Humidity (%): 55% +/- 15 %
- Illumination: 12 hours artifical fluorescent light and 12 hours dark
- Ventilation rate: between fifteen to twenty air changes per hour.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

ADMINISTRATION: 
- Frequency: once daily for 14 days
- Dose volume: 3 mL/kg b.w./day
- Dose: 0, 100, 300 or 1000 mg/kg/bw
- Animals: 5 male and 5 female rats/dose
- DOSAGE PREPARATION:
- The test item formulations were freshly prepared on every administration day. The test item was suspended in the vehicle to the appropriate
concentrations. The following administration formulations were prepared:
Group 2: 33.3 mg/mL: 400 mg test item were dissolved and diluted ad 12 mL vehicle
Group 3: 100 mg/mL: 1200 mg test item were dissolved and diluted ad 12 mL vehicle
Group 4: 333 mg/mL: 4000 mg test item were dissolved and diluted ad 12 mL vehicle
The amount of the test item was adjusted to the animal's current body weight on each administration day.
- The test item formulations were administered orally at a constant volume once daily.
- The control animals received the vehicle at the same administration volume daily in the same way.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For each test item mixed with a vehicle, tests by appropriate analytical methods will be carried out for the main study (LPT Study No. 32702) to
determine the concentration, homogeneity and, if needed, stability of the test item in the formulations.

Duration of treatment / exposure:

14 days

Frequency of treatment:

daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Vehicle control

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Low dose

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Intermediate dose

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

High dose

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale:
The dose levels were selected in agreement with the Sponsor/Monitor and were based on available toxicological data
(LD50 cut-off for the rat, oral: > 2000 mg/kg b.w.).

Selection of route of administration: According to international guidelines (OECD 408 and ECHA request).

Positive control:

not required

Observations and examinations performed and frequency:

Dated and signed records of all activities related 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 the Study Plan were recorded in appropriate documentation. In addition, observations related to
individual animals were made throughout the study and recorded.

- Clinical signs:
The animals were observed individually before and after dosing at each time of dosing for any signs of behavioural changes, reaction to
treatment or illness.
- Mortality: Further checks were made early in the morning and again in the afternoon of each working day to look for dead or moribund animals.
This would have allowed post mortem examination to be carried out during the working period of that day.
On Saturdays and Sundays, a similar procedure was followed with a final check at approximately 4.00 p.m.
- Body weight: The body weight of each rat was recorded at the time of group allocation (on test day -8), on the day of commencement of
treatment (test day 1) and weekly thereafter.
- Food and drinking water 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 animal in
each group on completion of a treatment week.
The drinking water consumption was monitored daily by visual appraisal throughout the study.

Sacrifice and pathology:

PATHOLOGY
- On test day 15 (approximately 24 hours after the last administration) all animals were dissected. For this purpose the selection of animals followed
a randomization scheme.
- The animals were euthanized by carbon dioxide (CO2) inhalation, 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. 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 conditions of the thoracic viscera
were noted with due attention to the thymus, lymph nodes, and heart.
- The abdominal viscera were examined before and after removal. The urinary bladder was examined externally and by palpation. The
gastro-intestinal tract was examined as a whole, and the stomach and caecum were incised and examined. The lungs were removed and all pleural
surfaces were examined under suitable illumination. The liver and the kidneys were examined. Any abnormalities in the appearance and size of the
gonads, adrenal glands, uterus, intra-abdominal lymph nodes, and accessory reproductive organs were recorded.
- The weights of the following organs of all animals were determined:
Epididymis (2)
Liver
Ovary (2)
Prostate
Seminal vesicle
Testicle (2)
Uterus (incl. cervix and oviducts)
Vagina
Paired organs were weighed individually and identified as left or right.

Statistics:

Toxicology data were captured, as far as possible, using the departmental computerized systems (Provantis® Integrated preclinical software, Instem LSS Ltd., version 8.2.0). Raw data not fully compatible with the computerized systems were maintained on paper according to appropriate SOPs.
The test item groups 2 to 4 were compared to the control group 1 using the following statistical method:
Test: Multiple t-test based on DUNNETT, C. W. New tables for multiple comparisons Biometrics, 482-491 (September 1964)
Parameter: Body weight / Food consumption / Absolute and relative organ weights (p  0.01 and p  0.05)

Clinical signs:

no effects observed

Description (incidence and severity):

No abnormalities were observed in behaviour, external appearance, or condition of faeces at any oral dose level of test item

Mortality:

no mortality observed

Description (incidence):

None of the male and female rats treated orally with 100, 300 or 1000 mg test item/kg b.w. daily for 14 days died during the course of the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No influence was noted.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No influence was noted.

Food efficiency:

not specified

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

no effects observed

Description (incidence and severity):

No influence was noted.

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No influence was noted.

Gross pathological findings:

no effects observed

Description (incidence and severity):

At necropsy on test day 15, no macroscopic changes were noted.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Details on results:

Mortality:
None of the male and female rats treated orally with 100, 300 or 1000 mg test item/kg b.w. daily for 14 days died during the course of the study.

Behaviour, external appearance and faeces:
None of the male and female rats treated orally with 100, 300 or 1000 mg test item/kg b.w. daily for 14 days revealed any changes in behaviour or
external appearance during the course of the study.
The faeces of all animals were of a normal consistency.

Body weight:
No influence was noted on the body weight of the male and female rats treated orally with 100, 300 or 1000 mg test item/kg b.w. daily for 14 days
during the course of the study

Food and drinking water consumption:
No test item-related influence was noted on the food consumption of the male and female rats treated orally with 100, 300 or
1000 mg test item/kg b.w. daily for 14 days during the course of the study.
The visual appraisal of the drinking water consumption did not reveal any test item-related influence.

Macroscopic post mortem findings:
At necropsy on test day 15 no macroscopic changes were observed in the male and female rats treated orally with 100, 300 or
1000 mg test item/kg b.w. daily for 14 days.

Organ weights:
The relative and absolute organ weights were not influenced in the male and female rats treated orally with 100, 300 or
1000 mg test item/kg b.w. daily for 14 days

Critical effects observed:

not specified

no other information

Conclusions:

After consideration of these data, the following dose levels were selected for the 90-day toxicity main study of test item administered to CD® rats
by oral administration:
Group 1:Control (vehicle), Group 2: 100 mg test itemkg b.w./day, p.o, Group 3: 300 mg test item/kg b.w./day, p.o, Group 4: 1000 mg/kg b.w./day, p.o.

Executive summary:

The aim of this 14-day dose-range-finding study was to select the dose levels for a study with repeated oral administration of test item in rats. The following main study will be based on OECD guideline 408.

The animals were treated orally with 100, 300 or 1000 mg test item/kg b.w.daily for 14 days. The control group was treated with the vehicle in the same way.

None of the animals died during the course of the study.

No signs of systemic intolerance reactions were observed at any of the tested dose levels. The body weight, body weight gain, food and drinking water consumption were not influenced.

At necropsy on test day 15, neither macroscopic changes nor changes in the body weight at autopsy, and no changes of the relative and absolute organ weights were observed.

After consideration of these data, the following dose levels ere selected for the 90-day toxicity main study of test item administered to CD® rats by oral administration:

Group 1:	Control (vehicle)
Group 2:	100 mg test item/kg b.w./day, p.o
Group 3:	300 mg test item/kg b.w./day, p.o
Group 4:	1000 mg test item/kg b.w./day, p.o.

 

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-02-19 to 2016-06-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Study was conducted according ECHA Decision TPE-D-2114306081-68-01/F Helsinki, 30 July 2015

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:

2001/59/EC; Official Journal of European Communities, L 255 2001.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

CD-1

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 and females: 64 days
- body weight: Males: 278.0 g - 325.5 g, females: 198.6 g - 234.2 g
- Diet: ad libitum, Commercial ssniff-R/M-H V1534 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany)
- Water: ad libitum
- Acclimatisation period: 9 days
-Housing: kept singly in MAKROLON cages
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 °C +/- 3° C
- Humidity (%): 55% +/- 15 %
- Illumination: 12 hours artifical fluorescent light and 12 hours dark
- Ventilation rate: between fifteen to twenty air changes per hour.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

ADMINISTRATION: 
- Frequency: once daily for 90 days
- Dose volume: 3 mL/kg b.w./day
- Dose: 0, 100, 300 or 1000 mg/kg/bw
- Vehicle: Corn oil, Batch no. 15050501, Caesar & Loretz GmbH, 40721 Hilden, Germany
- 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 was suspended in the vehicle to the appropriate concentrations using a magnetic stirrer. Stirring of the formulations was continued until the last animal of the dose group had been dosed.
- The dose of the test item was adapted to the animal's current body weight daily up to and including test week 6, and weekly thereafter.
- The test item formulations were administered orally at a constant volume once daily.
- The control animals received the vehicle at a constant volume of 3 mL/kg b.w. orally once daily in the same way.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For the analysis of the administration formulations, samples of approximately 4 mL were taken (divided into 2 aliquots of 2 mL) at the following times and stored at
-20°C or colder until analysis:
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: 2 x 3 x 3 = 18
Homogeneity
At the start of administration, during (middle) administration and before administration to the last animal of the test item group.
(3 samples/test item group).
Number of samples: 2 x 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: 2 x 1 x 3 = 6

Sum of all samples: 2 x 21 = 42

Duration of treatment / exposure:

90 days

Frequency of treatment:

daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Vehicle control

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Low dose

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Intermediate dose

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

High dose

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels for this study have been selected in agreement with the Monitor based on available toxicological data generated during a preliminary dose-range- finding study.
In this 14-day dose-range-finding study, the animals were treated orally with 100, 300 or 1000 mg test item/kg b.w. daily for 14 days. The control group was treated with the vehicle in the same way.
None of the animals died during the course of the study.
No signs of systemic intolerance reactions were observed at any of the tested dose levels. The body weight, body weight gain, food and drinking water consumption were not influenced.
At necropsy on test day 15, neither macroscopic changes nor changes in the body weight at autopsy, and no changes of the relative and absolute organ weights were observed.

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

- Identification of animals: Each rat received a continuous number: According to a number scheme, points were set on paws and/or tail by tattoo. Additionally, the animal cages were labelled 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 the Study Plan were recorded in the appropriate documentation. In addition, observations related to individual animals made throughout the study were recorded.
The following observations were made during the course of the study:
-Cage side observations:
- Clinical signs: Cageside observations included skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behaviour patterns. The onset, intensity and duration of any signs observed were recorded.
The animals were observed individually before and after dosing at each time of dosing for any signs of behavioural changes, reaction to
treatment or illness.
- Mortality: Further checks were made early in the morning and again in the afternoon of each work-ing day to look for dead or moribund animals. On Saturdays and Sundays a similar procedure was followed except that the final check was carried out at approximately 4.00 p.m.
- Body weight: The weight of each rat was recorded at the time of group allocation (test day -9), daily from the day of commencement of treatment up to and including test week 6 for dose adjustment and weekly thereafter throughout the experimental period. .
- Food and drinking water consumption: The quantity of food left by individual animals was recorded on a weekly basis through-out the experimental period. Food intake per rat (g/rat/week) was calculated using the total amount of food given to and left by each rat in each group upon completion of a treatment week.
The drinking water consumption was monitored daily by visual appraisal throughout the study.

- Neurological screening: In test week 13 (before any blood sampling for laboratory examinations) and at the end of the recovery period in test week 17, screening of sensory reactivity to stimuli of differ-ent types (e.g. auditory, visual and proprioceptive stimuli, based on GAD ), as well as the assessment of grip strength (according to MEYER ), and motor activity assessment were conducted in all animals outside the home cage as described below.
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 collected 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
No anticoagulant (serum) for bile acid determination
Blood samples were collected at the following times:
- At the end of test week 13 (before necropsy): All main study animals
- At the end of the recovery period (on the day of dissection): All recovery animals

Haematology: ADVIATM 120 Siemens Diagnostics GmbH, 35463 Fernwald, Germany
Haemoglobin content (HGB), mmol/L blood
Erythrocytes (RBC), 106/µL blood
Leucocytes (WBC), 103/µL blood
Reticulocytes (Reti), %
Platelets (PLT), 103/µL blood
Haematocrit value (HCT), %
Differential blood count (relative)#, %
Differential blood count (absolute)#, 103/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 substraction)
Bile acids µmol/L serum
Bilirubin (total) µmol/L plasma
Cholesterol (total) 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

Urinalysis:
Urine samples were collected from animals fasted overnight at the following times and the parameters listed below were determined:
- At the end of test week 13 (before necropsy): All study animals
- At the end of the recovery period (before necropsy): All recovery animals
The urine was collected for 16 hours in an URIMAX funnel cage. The collection of urine was determined immediately prior to starting the blood withdrawals for the haematological and clinical biochemical examinations at study termination.
The following parameters were measured using the methods given below:
Parameter Units Method
- Volume mL graded measuring cylinder
- pH n/a using a digital pH meter type WTW InoLab pH 720
- Specific gravity g/mL using Atago Refractometer, type Uricon sample compared with water (nominal value of 1.000)
The following tests were also performed using qualitative indicators (Combur 9® Test, Roche Diagnostics GmbH, 68305 Mannheim, Germany) of analyte concentration:
Protein, g/L
Glucose, mmol/L
Bilirubin
Urobilinogen, µmol/L
Ketones
Haemoglobin (Hb, approx. values), ery/µL
Nitrite
Microscopic examinations of urine samples were carried out by centrifuging samples and spreading the resulting deposit on a microscopic slide. The deposits were examined for the presence of the following parameters:
- Epithelial cells (E)
- Leucocytes (L)
- Erythrocytes (R)
- Organisms (B)
- Further constituents (i.e. sperm, casts) (C)
- Crystalluria (A)
The colour and turbidity of the urine were examined visually.

Ophthalmological and auditory examinations
Examinations were performed on all animals before first dosing, at main study termina-tion and at the end of the recovery period.
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, the main study animals were dissected following a randomisation scheme. Animals not dissected on test day 91 were dosed again in test day 91 and dis-sected on test day 92.
- Necropsy of all animals allocated to the recovery period was per-formed on test day 123.
- 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)
heart
kidney (2)
liver
ovary (2)
pancreas
spleen
testicle (2)
thymus
as a whole: prostate, seminal vesicles with coagulating glands
uterus (incl. cervix)

Histopathology
- The following organs or parts of organs with the exception of the eyes and testicles of all animals were fixed in 7% buffered formalin.
- The eyes were preserved in Davidson’s solution and the testicles were preserved 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
lungs (with mainstem bronchi and bronchioles (preserved by inflation with fixative and then immersion))
lymph node (1, cervical)
lymph node (1, mesenteric)
mammary gland
muscle (skeletal, leg)
nerve (sciatic)
oesophagus
ovary (2) (and oviducts)
pancreas
pituitary
prostate and seminal vesicles with coagulating glands
salivary glands (mandibular, sublingual and parotid gland)
skin (left flank)
spinal cord (3 levels: 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.
- In addition, frozen sections of the heart, liver and one kidney were made, stained with Oil Red O and examined microscopically.
- Parathyroids cannot always be identified macroscopically; they were examined micro-scopically if in the plane of section and in all cases where they are noted as grossly en-larged.

Other examinations:

no other examinations

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 biochemistry / urinalysis / relative and absolute organ weights (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:
All numerical functional tests: Body temperature / hind leg splay / grip strength / spontaneous motility (p 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)

Exact test of R. A. FISHER : Histopathology (p
Possible adverse effects or possible variations in the test item-treated animals were compared with current control animals. If necessary, available historical animal data were also used for comparison.

Clinical signs:

no effects observed

Description (incidence and severity):

No changes in behaviour or external appearance were noted for the male and female animals treated orally with 100, 300 or 1000 mg test item/kg b.w./day for 90 days during the treatment period.
None of the animals treated orally with 100, 300 or 1000 mg test item/kg b.w./day for 90 days revealed any changes in external appearance, body posture, movement and coordination capabilities, or behaviour at the detailed clinical observations performed weekly.
No changes were noted for the male and female rats previously treated orally with 1000 mg test item/kg b.w./day during the recovery period.
The faeces of all animals were of normal consistency.

Mortality:

no mortality observed

Description (incidence):

None of the male and female rats treated orally with 100, 300 or 1000 mg test item/kg b.w./day for 90 days died during the treatment period.
None of the male and female rats previously treated orally with 1000 mg test item/kg b.w./day died during the recovery period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The body weight, body weight gain and body weight at autopsy were not influenced in the male and female animals treated with 100, 300 or 1000 mg test item/kg b.w./day in a test item-related way during the treatment period compared to the control group.
No influence was noted for the male and female rats previously treated with 1000 mg test item/kg b.w./day during the recovery period.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related influence was noted on the relative food consumption of the male and female animals treated with 100, 300 or 1000 mg test item/kg b.w./day during the treatment period compared to the control group.
No influence was noted for the male and female rats previously treated with 1000 mg test item/kg b.w./day during the recovery period.
However, slight increases in the relative food consumption were noted for the high dosed male and female animals by up to 10% for the males and by up to 11% for the females during the treatment period starting in test week 2 and by up to 13% for the male animals during the recovery period. The food consumption of the female high dose animals was in the range of the control group during the recovery period. These differences compared to the control group are considered to be only minor and therefore to be neither biologically relevant nor adverse.

Food efficiency:

not specified

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

no effects observed

Description (incidence and severity):

The visual appraisal of the drinking water consumption did not reveal any test item-related differences between the test item-treated animals and the control
animals throughout the treatment and the recovery period.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No changes of the eyes and the optic region, i.e. adnexa oculi, conjunctiva, cornea, ante-rior chamber, iris (pupil dilated), lens, vitreous body and fundus were noted in the male and female rats of the animals treated orally with 100, 300 or 1000 mg test item/kg b.w./day at the end of the treatment period.
No changes were noted for the male and female rats previously treated with 1000 mg test item/kg b.w./day at the end of the recovery period.
There was no indication of any impairment to auditory acuity.

Haematological findings:

no effects observed

Description (incidence and severity):

No test item-related influence on haematological parameters was noted for the male and female animals treated with 100, 300 or 1000 mg test item/kg b.w./day compared to the control group.
No influence was noted for the male and female rats previously treated with 1000 mg test item/kg b.w./day during the recovery period.
No test item-related influence was noted for the haemoglobin content (HGB), the number of erythrocytes (RBC), leucocytes (WBC), reticulocytes (Reti) and platelets (PLT), the haematocrit value (HCT), the relative and absolute differential blood count, the thrombo-plastin time (TPT), the activated partial thromboplastin time (aPTT), the mean corpuscu-lar volume (MCV), the mean corpuscular haemoglobin (MCH) and the mean corpuscular haemoglobin concentration (MCHC).

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The following test item-related changes were noted (Test day 91):
Changes in biochemical parameters compared to control group 1 (vehicle) [%]

Parameter Group 2 Group 3 Group 4
males females males females males females
Cholesterol -31** none -26** -35** -32** -28**
ALAT +12 +37 +21 +16 +27** +74**
ASAT +35** +23* +42** +14 +50** +59**
LDH none none none +18 none +84**

Despite a clear effect was observed for the plasma level of cholesterol and the plasma activities of alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT) and lactate dehydrogenase (LDH) compared to the control group, these values are still within the LPT background data.

No test item-related influence was noted for the albumin/globulin ratio, the plasma levels of albumin, globulin, bilirubin, creatinine, glucose, protein (total), triglycerides, urea, calcium, chloride, potassium and sodium and the serum level of bile acids. Further, the plasma activity of alkaline phosphatase (aP) was not influenced.
Recovery period
The plasma activity of aspartate aminotransferase (ASAT) was still statistically significantly increased (at p ≤ 0.05) by 24% for the male animals previously treated with 1000 mg test item/kg b.w./day on test day 123.
No effects related to the previous treatment were noted for the albumin/globulin ratio, the plasma levels of albumin, globulin, bilirubin, cholesterol, creatinine, glucose, protein (total), triglycerides, urea, calcium, chloride, potassium and sodium, and the serum level of bile acids. Further, the plasma activities of alanine aminotransferase (ALAT), alkaline phosphatase (aP) and lactate dehydrogenase (LDH) was not influenced

Urinalysis findings:

no effects observed

Description (incidence and severity):

No test item-related influence on the urinary status was noted for the male and female animals treated with 100, 300 or 1000 mg test item/kg b.w./day at the end of the treatment period.
No test item-related influence on the urinary status was noted for the male and female animals previously treated with 1000 mg test item/kg b.w./day at the end of the recovery period.
No test item-related changes were noted for the specific gravity and the pH value of the urine and the urine volume. The analyte concentrations of nitrite, protein, glucose, ketones, urobilinogen, bilirubin and haemoglobin were not influenced in male and female animals. No test item-related changes were observed in the urine colour and the microscopically analysed urine sediments.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

The neurological screening performed at the end of the treatment period in test week 13 (before any blood sampling for laboratory examinations) and at the end of the recovery period in test week 17 did not reveal any test item-related influence on the male and female rats treated orally with 100, 300 or 1000 mg test item/kg b.w./day for 90 days, neither on any of the parameters examined during the functional observation tests nor on the fore- and hind limb grip strength or on the spontaneous motility.
No changes were noted for the male and female rats previously treated with 1000 mg test item/kg b.w./day at the end of the recovery period.

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The following test item-related changes in relative and absolute organ weights were not-ed for the male and female animals treated with 100, 300 or 1000 mg test item/kg b.w./day on test day 91/92:
Changes in organ weights compared to control group 1 (vehicle) [%]
Organ Group 2 Group 3 Group 4
males females males females males females
Spleen - relative +27** none +30** +36** +34** +35**
- absolute +22* none +28** +35** +34** +32**
Liver - relative none none none +11* none +21**
- absolute none none none +10 none +18*

Despite a clear dose-related effect was observed for the relative and absolute spleen and liver weight compared to the control group, these values are still within the LPT background data.

Recovery period
The relative and absolute spleen weights of the animals previously treated with 1000 mg test item/kg b.w./day were still increased at the end of the recovery period on test day 123 as follows:

Changes in organ weights compared to control group 1 (vehicle) [%]
Organ Group 4
males females
Test day 123
Spleen - relative +27 +25**
- absolute +15 +25*

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related changes were noted for the male and female rats treated orally with 100, 300 or 1000 mg test item/kg b.w./day at the end of the treatment period.
No test item-related changes were noted for the male and female rats previously treated with 1000 mg test item/kg b.w./day at the end of the recovery period.
However, macroscopic changes were noted in the thyroid (reduced in size), liver (in-creased lobular pattern), kidneys (dark-red stained discoloured), ovary (reduced in size), pituitary (enlarged), uterus (dilated and/or filled with liquid) and stomach (yellowish coat-ing and single dark-red discoloured focus in the fundus region) in individual animals of the control and test item-treated groups at terminal or recovery sacrifice.
These changes are considered to be incidental findings.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment and recovery period
The histomorphological examination revealed mild morphological changes in form of a minimally to mildly increased number of macrophages in the lymphatic sinuses of the mesenteric lymph nodes of the animals treated with 1000 mg test item/kg b.w./day which are considered to be related to the administration of the test item.
A mild reversibility was noted after a recovery period of 32 days.
The coincidental findings from different organs in a small number of control and high dose animals are considered to be spontaneous organ changes and are thus not test item-related.

Description (incidence and severity):

see above

Other effects:

not specified

Details on results:

All the above mentioned changes and described observations, although statistically significant, were not considered of any biological or any toxicological significance since no correlating effects were found at the histopathological evaluation in particular in the liver. Moreover, most changes noted had subsided at the end of the 4-week recovery period.

The hepatic enzyme induction correlates with the increase in liver weight. These changes are typical responses to xenobiotic exposure as a result of elevation in workload demand to initiate metabolic clearance.
The increased number of macrophages in the mesenteric lymph nodes noted at histopathological examination is also considered to be due to the increased work- load and, hence, is considered not to be adverse.

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

not specified

Test item formulation analysis

The analysis was performed at LPT according to a HPLC-UV method validated by LPT.

The results of the analysis of the test item concentrations in the test item formulations confirmed that the test item formulations were correctly prepared. The actual concentrations of the test item within the formulations ranged from 93% to 104% of the nominal concentrations. These values were within the admissible limits of 90% to 110% indicating correctly prepared formulations.

Conclusions:

All the above mentioned changes and described observations, although statistically significant, were not considered of any biological or any toxicological significance since no correlating effects were found at the histopathological evaluation in particular in the liver. Moreover, most changes noted had subsided at the end of the 4-week recovery period.

The hepatic enzyme induction correlates with the increase in liver weight. These changes are typical responses to xenobiotic exposure as a result of elevation in workload demand to initiate metabolic clearance.
The increased number of macrophages in the mesenteric lymph nodes noted at histopathological examination is also considered to be due to the in creased work- load and, hence, is considered not to be adverse.
Under the present test conditions of this study, the no observed adverse effect level (NOAEL) was above 1000 mg test item/kg b.w./day.
 

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 at dose levels of 100, 300 or 1000 mg/kg b.w./day for 90 days and on the reversibility of any affects after a treatment-free recovery period.

None of the animals died prematurely.

Decreased plasma levels of cholesterol and increased plasma activities of alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT) and lactate dehydrogenase (LDH) with no histopathological correlate, in particular in the liver, heart or skeletal muscle, were noted for the male and female animals partly starting at 100 mg test item/kg b.w./day.

Increased relative and absolute spleen and/or liver weights were noted for the male and female animals starting at 100 mg test item/kg b.w./day.

The histomorphological examination revealed mild morphological changes in form of a minimally to mildly increased number of macrophages in the lymphatic sinuses of the mesenteric lymph nodes of the animals treated with 1000 mg test item/kg b.w./day.

No test item-related influence was noted on behaviour, external appearance or faeces.The neurological screening did not reveal any test item-related changes.No test item-related influence was noted on body weight and body weight gain, food and drinking water consumption, haematological parameters, the urinary status, the eyes or optic region and the auditory acuity. No test item-related macroscopic changes were observed at necropsy.

At the end of the recovery period, the plasma activity of aspartate aminotransferase (ASAT) was still increased for the previously high dosed male animals and the relative and absolute spleen weights of the previously high dosed male and female animals were still increased at the end of the recovery period. A mild reversibility of the histopathological changes in the lymph nodes was noted after a recovery period of 32 days. All other changes had subsided after 4 weeks of recovery.

Overall Conclusion

All the above mentioned changes and described observations, although statistically significant, were not considered of any biological or any toxicological significance since no correlating effects were found at the histopathological evaluation in particular in the liver. Moreover, most changes noted had subsided at the end of the 4-week recovery period.

The hepatic enzyme induction correlates with the increase in liver weight. These changes are typical responses to xenobiotic exposure as a result of elevation in workload demand to initiate metabolic clearance.

The increased number of macrophages in the mesenteric lymph nodes noted at histopathological examination is also considered to be due to the in creased work- load and, hence, is considered not to be adverse.

Under the present test conditions of this study, the no observed adverse effect level (NOAEL) was above 1000 mg test item/kg b.w./day.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The study is GLP-compliant and valid without restriction (Klimisch code 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:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Data waiving:

other justification

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-09-20 to 2011-10-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)

Version / remarks:

adopted May 12, 1981

Deviations:

yes

Remarks:

Because the present study was a concentration range finding study not all aspects of the guideline were implemented.

Qualifier:

according to guideline

Guideline:

EU Method B.28 (Sub-Chronic Dermal Toxicity Test: 90-Day Repeated Dermal Dose Study Using Rodent Species)

Version / remarks:

published in the Official Journal of the European Union L142, dated May 31, 2008

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Crl:CD

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ORGANISMS: 
- Strain: Crt:CD
-Source: Charles River Laboratories Germany GmbH, Sulzfeld
- Age: males: 71 days, females: 79 days
- Body weight: males: 353 - 442 g, females 218 - 286 g
- Number of animals: 20 male and 20 female rats
- Controls: Vehicle
- Diet: ad libitum, special diet for rats, SSniff R/M-H V 1534
- Water: ad libitum, tap water
- Adaption period: 27 days
ENVIRONMENTAL CONDITIONS
- Housing: single in MAKROLON cages
- Temperature (°C): 22 +/- 3 °C
- Humidity (%): 55% +/- 20 %
- Photoperiod (hrs dark / hrs light): 12 hours artificial light, 12 hours dark

Type of coverage:

semiocclusive

Vehicle:

water

Remarks:

Tap water

Details on exposure:

Area of exposure:
- Coverage: 10 % of body surface
- Type of wrap: non-occlusive sterile gauze dressing, held in place with non-irritating tape
- Time intervals for shaving: one day before 1st application and on test day 8

REMOVAL OF TEST SUBSTANCE
- Washing: After the 6-hour exposure period the dressing was removed. At the end of the exposure period, residual test item was removed where
practicable using water or some other appropriate method of cleansing the skin.
TEST MATERIAL
- Administration volume: 4 mL/kg b.w. day
- Concentration: 0, 30, 100, 1000 mg/kg b.w,/day
- Constant volume or concentration used: amount of the test item was adjusted to each animal's current body weight weekly
- The test item was suspended in the vehicle to the appropriate concentrations to allow the test item to be spread over the application site

VEHICLE
- Vehicle: tap water, control animals were handled in an identical manner, except for treatment with the test substance

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

14 consecutive days, 6 hours per day

Frequency of treatment:

once daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control

Dose / conc.:

30 mg/kg bw/day (nominal)

Remarks:

Low dose

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Mid dose

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

High dose

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

The dose levels for this preliminary study were selected based on available toxicity data.

Positive control:

not necessary

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS:
- Cageside observations included skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behaviour
patterns
CLINICAL OBSERVATIONS:
- Animals were observed individually before and after dosing at each time of dosing for any signs of behavioural changes, reaction to treatment or
illness. In addition, animals were checked regularly throughout the working day from 7.30 a.m. to 4.30 p.m. On Saturdays and Sundays, animals
were checked regularly from 8.00 a.m. to 12.00 noon with a final check performed at approximately 4.00 p.m.
DERMAL IRRITATION :
- Starting on test day 1 and daily thereafter, always at the end of the 6-hour exposure period, the administration sites were assessed based on
DRAIZE
BODY WEIGHT:
- starting on test day 1, day 8 and day 15
FOOD CONSUMPTION:
- quantity of food left by individual animals was recorded on a weekly basis throughout the experimental period. Food intake per rat (g/rat/week) was calculated using the total amount of food given to and left by each rat in each group on comple-tion of a treatment week
FOOD EFFICIENCY:
- The relative food consumption (in g/kg b.w./day) was determined
WATER CONSUMPTION:
- Drinking water consumption was monitored daily by visual appraisal throughout the study
MORTALITY
- Further checks were made early in the morning and again in the afternoon of each working day to look for dead or moribund animals. This would
have allowed post mortem examination to be carried out during the working period of that day. On Saturdays and Sundays a similar procedure was
followed except that the final check was carried out at approximately 4.00 p.m.

Sacrifice and pathology:

GROSS PATHOLOGY:
-On test day 15 (approx. 24 hours after the last administration) the animals were dissected following a randomisation scheme.
The animals were sacrificed under ether anaesthesia by cutting the aorta abdominalis, exsanguinated, 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
gastrointestinal tract was examined as a whole and the stomach and caecum were incised and examined. The lungs were removed and all pleural
surfaces were examined under suitable illumination. The liver and the kidneys were examined. Any abnormalities in the appearance and size of the
gonads, adrenal glands, uterus, intraabdominal lymph nodes and accessory reproductive organs were recorded.

Other examinations:

no other examinations

Statistics:

The test item-treated groups 2 to 4 were compared with the control group 1. The following statistical method was used:
Multiple t-test based on DUNNETT, C. W., New tables for multiple comparisons with a control Biometrics, 482-491 (Sept 1964)
Body weight / Food consumption (p >/= 0.01)
This statistical procedure was used for all data

Clinical signs:

no effects observed

Description (incidence and severity):

None of the rats revealed any clinical signs.

Dermal irritation:

no effects observed

Description (incidence and severity):

No signs of local intolerance were noted.

Mortality:

no mortality observed

Description (incidence):

None of the rats died prematurely.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No test item-related changes were noted.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No test item-related influence was noted.

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

No test item-related influence was noted.

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

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No changes were noted.

Details on results:

- Local tolerance
No local intolerance reactions were noted for the male and female animals treated dermally with 30, 100 or 1000 mg test item (solvent free)/kg b.w./day.
- Behaviour, external appearance, faeces, mortality
None of the male and female rats revealed any changes in behaviour or external appearance. The faeces of all animals were normally formed.
None of the animals died prematurely.
- Body weight
No test itemrelated changes in body weight and body weight gain were noted for the animals compared to the control group.
- Food and drinking water consumption
No test item related influence was observed on the relative food consumption. The visual appraisal of the drinking water consumption revealed no test item-related influence.
- Macroscopic post mortem systemic findings
No test item-related macroscopic systemic changes were noted for the male and female rats compared to the control group.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: There were no signs of local or systemic intolerance.

Critical effects observed:

not specified

no other information

Conclusions:

Dermal treatment with 30, 100 or 1000 mg test item (solvent free)/kg b.w./day for 14 days did not cause any signs of local or systemic intolerance.

Executive summary:

The aim of this 14-day dose-range-finding study was to select the dose levels for a possible later 90-day repeated dose toxicity study of test item by dermal administration to rats.

Dose levels of 30, 100 and 1000 mg test item /kg b.w. /day were administered in this study.

The appropriate dose of the test item was administered epicutaneously onto the shaved dorsum (approx. 10% of the body surface (semi-occlusive)) once daily for 14 consecutive days. The exposure time was 6 hours per day. The test item was suspended in the vehicle (tap water) to the appropriate concentrations to allow the test item to be spread over the application site.

The control animals were handled in an identical manner with concurrent vehicle. On test day 15 the animals were dissected following a randomisation sheme.

Conclusion:

Dermal treatment with test item for 14 days did not cause any signs of local or systemic intolerance.

None of the rats died prematurely.

No test item-related influence was noted on the body weight, food and drinking water consumption.

Macroscopic examination at necropsy revealed no test item-related changes at any of the tested groups.

Therefore, under the conditions of this study the highest concentration (1000 mg/kg bw /day) is considered to be the No Observed Adverse Effect Level (NOAEL).

 

After consideration of these data, the following dose levels were selected for the 90-day toxicity study by dermal administration to CD rats:

Group 1:       Control

Group 2:       30 mg test item (solvent free)/kg b.w./day

Group 3:     100 mg test item (solvent free))/kg b.w./day

Group 4:    1000 mg test item (solvent free)/kg b.w./day

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The study is GLP-compliant and valid without restriction (Klimisch code 1).

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-09-20 to 2011-10-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)

Version / remarks:

adopted May 12, 1981

Deviations:

yes

Remarks:

Because the present study was a concentration range finding study not all aspects of the guideline were implemented.

Qualifier:

according to guideline

Guideline:

EU Method B.28 (Sub-Chronic Dermal Toxicity Test: 90-Day Repeated Dermal Dose Study Using Rodent Species)

Version / remarks:

published in the Official Journal of the European Union L142, dated May 31, 2008

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Crl:CD

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ORGANISMS: 
- Strain: Crt:CD
-Source: Charles River Laboratories Germany GmbH, Sulzfeld
- Age: males: 71 days, females: 79 days
- Body weight: males: 353 - 442 g, females 218 - 286 g
- Number of animals: 20 male and 20 female rats
- Controls: Vehicle
- Diet: ad libitum, special diet for rats, SSniff R/M-H V 1534
- Water: ad libitum, tap water
- Adaption period: 27 days
ENVIRONMENTAL CONDITIONS
- Housing: single in MAKROLON cages
- Temperature (°C): 22 +/- 3 °C
- Humidity (%): 55% +/- 20 %
- Photoperiod (hrs dark / hrs light): 12 hours artificial light, 12 hours dark

Type of coverage:

semiocclusive

Vehicle:

water

Remarks:

Tap water

Details on exposure:

Area of exposure:
- Coverage: 10 % of body surface
- Type of wrap: non-occlusive sterile gauze dressing, held in place with non-irritating tape
- Time intervals for shaving: one day before 1st application and on test day 8

REMOVAL OF TEST SUBSTANCE
- Washing: After the 6-hour exposure period the dressing was removed. At the end of the exposure period, residual test item was removed where
practicable using water or some other appropriate method of cleansing the skin.
TEST MATERIAL
- Administration volume: 4 mL/kg b.w. day
- Concentration: 0, 30, 100, 1000 mg/kg b.w,/day
- Constant volume or concentration used: amount of the test item was adjusted to each animal's current body weight weekly
- The test item was suspended in the vehicle to the appropriate concentrations to allow the test item to be spread over the application site

VEHICLE
- Vehicle: tap water, control animals were handled in an identical manner, except for treatment with the test substance

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

14 consecutive days, 6 hours per day

Frequency of treatment:

once daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control

Dose / conc.:

30 mg/kg bw/day (nominal)

Remarks:

Low dose

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Mid dose

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

High dose

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

The dose levels for this preliminary study were selected based on available toxicity data.

Positive control:

not necessary

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS:
- Cageside observations included skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behaviour
patterns
CLINICAL OBSERVATIONS:
- Animals were observed individually before and after dosing at each time of dosing for any signs of behavioural changes, reaction to treatment or
illness. In addition, animals were checked regularly throughout the working day from 7.30 a.m. to 4.30 p.m. On Saturdays and Sundays, animals
were checked regularly from 8.00 a.m. to 12.00 noon with a final check performed at approximately 4.00 p.m.
DERMAL IRRITATION :
- Starting on test day 1 and daily thereafter, always at the end of the 6-hour exposure period, the administration sites were assessed based on
DRAIZE
BODY WEIGHT:
- starting on test day 1, day 8 and day 15
FOOD CONSUMPTION:
- quantity of food left by individual animals was recorded on a weekly basis throughout the experimental period. Food intake per rat (g/rat/week) was calculated using the total amount of food given to and left by each rat in each group on comple-tion of a treatment week
FOOD EFFICIENCY:
- The relative food consumption (in g/kg b.w./day) was determined
WATER CONSUMPTION:
- Drinking water consumption was monitored daily by visual appraisal throughout the study
MORTALITY
- Further checks were made early in the morning and again in the afternoon of each working day to look for dead or moribund animals. This would
have allowed post mortem examination to be carried out during the working period of that day. On Saturdays and Sundays a similar procedure was
followed except that the final check was carried out at approximately 4.00 p.m.

Sacrifice and pathology:

GROSS PATHOLOGY:
-On test day 15 (approx. 24 hours after the last administration) the animals were dissected following a randomisation scheme.
The animals were sacrificed under ether anaesthesia by cutting the aorta abdominalis, exsanguinated, 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
gastrointestinal tract was examined as a whole and the stomach and caecum were incised and examined. The lungs were removed and all pleural
surfaces were examined under suitable illumination. The liver and the kidneys were examined. Any abnormalities in the appearance and size of the
gonads, adrenal glands, uterus, intraabdominal lymph nodes and accessory reproductive organs were recorded.

Other examinations:

no other examinations

Statistics:

The test item-treated groups 2 to 4 were compared with the control group 1. The following statistical method was used:
Multiple t-test based on DUNNETT, C. W., New tables for multiple comparisons with a control Biometrics, 482-491 (Sept 1964)
Body weight / Food consumption (p >/= 0.01)
This statistical procedure was used for all data

Clinical signs:

no effects observed

Description (incidence and severity):

None of the rats revealed any clinical signs.

Dermal irritation:

no effects observed

Description (incidence and severity):

No signs of local intolerance were noted.

Mortality:

no mortality observed

Description (incidence):

None of the rats died prematurely.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No test item-related changes were noted.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No test item-related influence was noted.

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

No test item-related influence was noted.

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

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No changes were noted.

Details on results:

- Local tolerance
No local intolerance reactions were noted for the male and female animals treated dermally with 30, 100 or 1000 mg test item (solvent free)/kg b.w./day.
- Behaviour, external appearance, faeces, mortality
None of the male and female rats revealed any changes in behaviour or external appearance. The faeces of all animals were normally formed.
None of the animals died prematurely.
- Body weight
No test itemrelated changes in body weight and body weight gain were noted for the animals compared to the control group.
- Food and drinking water consumption
No test item related influence was observed on the relative food consumption. The visual appraisal of the drinking water consumption revealed no test item-related influence.
- Macroscopic post mortem systemic findings
No test item-related macroscopic systemic changes were noted for the male and female rats compared to the control group.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: There were no signs of local or systemic intolerance.

Critical effects observed:

not specified

no other information

Conclusions:

Dermal treatment with 30, 100 or 1000 mg test item (solvent free)/kg b.w./day for 14 days did not cause any signs of local or systemic intolerance.

Executive summary:

The aim of this 14-day dose-range-finding study was to select the dose levels for a possible later 90-day repeated dose toxicity study of test item by dermal administration to rats.

Dose levels of 30, 100 and 1000 mg test item /kg b.w. /day were administered in this study.

The appropriate dose of the test item was administered epicutaneously onto the shaved dorsum (approx. 10% of the body surface (semi-occlusive)) once daily for 14 consecutive days. The exposure time was 6 hours per day. The test item was suspended in the vehicle (tap water) to the appropriate concentrations to allow the test item to be spread over the application site.

The control animals were handled in an identical manner with concurrent vehicle. On test day 15 the animals were dissected following a randomisation sheme.

Conclusion:

Dermal treatment with test item for 14 days did not cause any signs of local or systemic intolerance.

None of the rats died prematurely.

No test item-related influence was noted on the body weight, food and drinking water consumption.

Macroscopic examination at necropsy revealed no test item-related changes at any of the tested groups.

Therefore, under the conditions of this study the highest concentration (1000 mg/kg bw /day) is considered to be the No Observed Adverse Effect Level (NOAEL).

 

After consideration of these data, the following dose levels were selected for the 90-day toxicity study by dermal administration to CD rats:

Group 1:       Control

Group 2:       30 mg test item (solvent free)/kg b.w./day

Group 3:     100 mg test item (solvent free))/kg b.w./day

Group 4:    1000 mg test item (solvent free)/kg b.w./day

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

7.4 mg/cm²

Study duration:

subacute

Species:

rat

Quality of whole database:

The study is GLP-compliant and valid without restriction (Klimisch code 1).

Additional information

Oral Toxicity:

For cyclohexane, 5 -isocyanato-1 -(isocyanatomethyl)-1,3,3 -trimethyl-, homopolymer, acetone oxime-blocked there is a 90 day oral toxicity study available.

The no observed adverse effect level (NOAEL) was above 1000 mg test item/kg b.w./day.

InhalationToxicity:

No results from repeated-dose toxicity tests for cyclohexane, 5 -isocyanato-1 -(isocyanatomethyl)-1,3,3 -trimethyl-, homopolymer, acetone oxime-blocked are available and necessary for the inhalation route of exposure. In accordance with ECHA decision inhalation route is not considered as relevant exposure route. Data waiver are claimed.

Dermal toxicity:

Under the conditions of the subacute dermal toxicity study withcyclohexane, 5-isocyanato-1-(isocyanatomethyl)-1,3,3-trimethyl-, homopolymer, acetone oxime-blockedno adverse effects occurred up to a concentration of 1000 mg/ kg bw.

Therefore, the concentration of 1000 mg/kg bw was a No-Observed-Adverse-Effect-Level (NOAEL) of the test item. The NOAEL can be converted into a NOAEL [mg/cm2] as follows (according to: Appendix R.8 -9 of TGD "Chapter R.8: Characterisation of dose [concentration]-response for human health", p. 112):

-       Dermal absorption rat= dermal absorption human (TGD Chapter 8, Appendix 8-2, Example A.1)

-       Average weight of rats in the study is 330 g (220 -440 g)

-    The dose applied over an area which is approx. 10% of the total body surface, and

-     The total body surface of rats is on the average 445 cm²(363 to 527 cm²)

The generic modification will be

 

modified NOAEL_{dermal; rep.dose}= rat NOAEL_{dermal; rep. dose}* 0,33 kg/44,5cm²

 

                                                          =1000 mg/kg bw*0,33/44,5

 

                                                         = 7,4 mg/cm²

 In coordination with results from available 90 day oral toxicity study this estimation can be concluded as very conservative.

Justification for classification or non-classification

Regarding repeated dose toxicity the substance cyclohexane, 5-isocyanato-1-(isocyanatomethyl)-1,3,3-trimethyl-, homopolymer, acetone oxime-blocked must not be classified according to the criteria of EC Regulation 1272/2008.