Isopropylcyclohexane

Endpoint summary

Description of key information

No repeated-dose toxicity tests are available for the oral and dermal route of exposure. Data waiver are claimed.

Under the conditions of the 90 -day inhalation toxicity study in rats, the no observed adverse effect level (NOAEL) for test item is considered to be 1.61 mg/L (CitoxLab, 2017).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

repeated dose toxicity: inhalation, other

Remarks:

This study is a 28-day dose range finding study (according to OECD 412). The results of the study will be used for dose level selection for a forthcoming 90-day subchronic inhalation toxicity study.

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2016-08-11 to 2016-09-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Deviations:

no

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633 Sulzfeld.
- Strain: Wistar Crl:WI (Han) rat
- Age: Young adult rats, 8 weeks old at the initiation of treatment
- Animals: 20 males and 20 females
- Acclimatisation: 7 days
- Body Weight at study initiation: male: 214 - 240 g, female: 155 - 186 g
- Housing: Group caging (in groups of 3 or 2 per cage, by sex)
- Diet: ad libitum, ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest, Germany
- Water: tap water ad libitum
- Temperature (°C): 19.5 – 24.8°C
- Humidity: 35 - 75 %
- Illumination: 12 hours of continuous artificial light in each twenty-four period (from 6.00 a.m. to 6.00 p.m.)
- Air exchange: At least 15 air exchanges per hour

Route of administration:

inhalation: vapour

Type of inhalation exposure:

nose only

Vehicle:

air

Remarks on MMAD:

Since the test item was vaporized, the test atmosphere did not contain any particles, therefore no particle size analysis was required.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The animals were treated by the inhalation route using a nose-only exposure unit, in a TSE Rodent Exposure System with each individual concentration or control group in a dedicated tower. Four identical, modular multilevel flow – past, nose only exposure units (towers) were used. The exposure unit consisted of two, concentric anodised aluminium cylinders, the inner plenum and the outer chamber with 20 circularly arranged exposure ports.
- Method of holding animals in test chamber: The animals were held in polycarbonate restraint tubes located around the chamber which allowed only the animals’ nares to enter the exposure port.
- Source and rate of air: The flow of air through each port was approximately 0.5 L/min. This flow rate is considered adequate to minimise re-breathing of the test atmosphere and maintained oxygen concentrations at greater than 19% and a carbon dioxide concentration not exceeding 1%.
- System of generating particulates/aerosols: For the test atmosphere generation, a stainless steel concentric jet nebuliser (TSE Systems GmbH, Bad Homburg, Germany) located at the top of the exposure chamber were used.
- Temperature, humidity, pressure in air chamber: Temperature of the test atmospheres and air used for the control group exposure were increased over the limit of 25ºC (up to 26.0 ºC) due to temporary malfunctioning of the cooling system. The relative humidity was lower than the optimal range of 30-70% due to the use of filtered, dry air for the dispersion of the test item. This deviation had no effect on the purpose and integrity of the study
- Air flow rate: approximately 0.5 L/min
- Method of particle size determination: Since the test item was vaporized, the test atmosphere did not contain any particles, therefore no particle size analysis was required.


TEST ATMOSPHERE
- Brief description of analytical method used: The actual (achieved) concentration of generated atmospheres was measured gravimetrically at regular intervals during an exposure by pulling a suitable, known volume of test atmosphere, from the exposure chamber, through Anasorb CSC sorbent tubes (SKC Inc., USA, or similar). Sampling was normally performed each day shortly after chamber equilibration and then at regular intervals during the exposure, and samples were collected from a vacant animal exposure port (animals breathing zone). The actual sampling schedule employed was dependent on the required sample volume in order to obtain adequate quantities of test item (exposure period).
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The exposure concentrations were monitored intermittently by gravimetrical analysis of the test item captured in charcoal filled sorbent tubes. The achieved test atmosphere concentrations were confirmed by an analytical method in the first and last week of the treatment using gas chromatography. No test item was detected in the control air.

Duration of treatment / exposure:

5 days per week for 4 weeks

Frequency of treatment:

5 days/week x 6 hours/day

Dose / conc.:

0 mg/L air (nominal)

Remarks:

Filtered Air Control

Dose / conc.:

0.1 mg/L air (nominal)

Remarks:

Low Dose

Dose / conc.:

1 mg/L air (nominal)

Remarks:

Mid Dose

Dose / conc.:

5 mg/L air (nominal)

Remarks:

High Dose

No. of animals per sex per dose:

5 males and 5 females per dose, 4 doses (incl. control)
20 males and 20 females

Control animals:

yes, concurrent vehicle

Details on study design:

The inhalation route is considered to be the predominant route for human exposure.
The concentration levels were determined based on the available experimental data from a previous acute inhalation dose study in agreement with the Sponsor.

Positive control:

not nesessary

Observations and examinations performed and frequency:

MORBIDITY/MORTALITY:
- Checks for mortality and/or morbidity were made twice daily, early and late during the normal working day.

DETAILED CLINICAL OBSERVATIONS:
- Individual clinical observations were performed prior to exposure and during exposure whilst the animals were still restrained (limited observation). Following exposure, clinical observations were performed twice (as soon as practicable after removal from restraint, and approximately one hour after completion of the exposure.
Detailed clinical observations were made on all animals outside the home cage in a standard arena once a week.
The animals were observed for changes in the skin and fur, eyes and mucous membranes and also respiratory, circulatory, autonomic and central nervous system, somatomotor activity and behaviour pattern. Particular attention was directed to observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep, coma.

BODY WEIGHT:
- The body weight of each animal were recorded with precision of 1 g on Day 1 (before the exposure), once a week thereafter and on Days 26 and 27 (prior to necropsy, fasted weight).

FOOD CONSUMPTION:
- Food consumption was recorded with precision of 1 g weekly and the weekly food consumption was calculated.


CLINICAL PATHOLOGY
- At the end of the treatment period, prior to scheduled necropsy on Day 27, blood samples for clinical pathology evaluation (haematology, coagulation, and clinical biochemistry) were collected by heart puncture under pentobarbital anaesthesia.
• After an overnight period of food deprivation of animals, 3 blood samples were collected into S-Monovette® tubes manufactured by Sarsted, Germany, as follows: for haematology approximately 1.2 mL blood in tubes with K3-EDTA as anticoagulant, 1.6 mg EDTA/mL blood
• for blood clotting times approximately 1.4 mL blood for APTT and PT measurements, in tubes with sodium citrate (0.14mL citrate solution) as anticoagulant
• for clinical chemistry approximately 1 mL blood in tubes with no anticoagulant to obtain serum.

HAEMATOLOGY:
The following parameters were evaluated in all animals on Day 27:
PARAMETERS (UNITS) METHODS EQUIPMENT
RBC Red Blood Cell (erythrocyte) count (1012/L) M/L Automatic laser cell count ADVIA 120
Hgb Haemoglobin concentration (g/dL) Determination of cyan-methemoglobin absorbance ADVIA 120
Hct Haematocrit (relative volume of erythrocytes) (%) Computed by equipment ADVIA 120
MCV Mean Corpuscular (erythrocyte) Volume (fL) Laser cell volume determination ADVIA 120
MCH Mean Corpuscular (erythrocyte) Haemoglobin (pg) Computed by equipment ADVIA 120
MCHC Mean Corpuscular (erythrocyte) Haemoglobin Concentration (g/dL) Computed by equipment ADVIA 120
RDW Red Cell (erythrocyte) volume Distribution Width (%) Distribution Width Laser detection ADVIA 120
Plt Platelet (thrombocyte) count (109/L) K/L Automatic laser cell count ADVIA 120
MPV Mean Platelet Volume (fL) Cell volume determination by laser ADVIA 120
RETIC % Reticulocyte count (%) Comparative value based on laser light detection ADVIA 120
WBC White Blood Cell (leukocyte) count (109/L) K/L Automatic laser cell count ADVIA 120
NE abs, % Neutrophil (absolute and %) Cell differentiation based on myeloperoxidase activityADVIA 120
LY abs, % Lymphocyte (absolute and %) Cell differentiation based on myeloperoxidase activityADVIA 120
MO abs, % Monocyte (absolute and %) Cell differentiation based on myeloperoxidase activityADVIA 120
EO abs, % Eosinophil (absolute and %) Cell differentiation based on myeloperoxidase activityADVIA 120
BA abs, % Basophil (absolute and %) Cell differentiation based on myeloperoxidase activityADVIA 120
LUC % Large Unstained Cells (%) Cell differentiation based on myeloperoxidase activityADVIA 120

The following blood clotting parameters were evaluated:

PARAMETERS (UNITS) METHODS EQUIPMENT
APTT Activated Partial Thromboplastin Time (sec) Micronized silica method AMAX Destiny Plus Coagulometer
PT Prothrombin Time (sec) Quick method (Biggs, R., and R.G. MacFarlane(1962)
Human Blood Coag.and its Disorders, Oxford) AMAX Destiny Plus Coagulometer
CLINICAL CHEMISTRY:

The following parameters were evaluated:

PARAMETERS (UNITS) METHODS EQUIPMENT
Glucose Blood sugar concentration (mmol/L) Colorimetric test (540 nm) VITROS 250/950
T-BIL Total Bilirubin concentration (μmol/L) End-point colorimetric (dual-wavelength) test (400 & 460nm) VITROS 250/950
Urea Urea concentration (mmol/L) Colorimetric test (670 nm) VITROS 250/950
Chol. Cholesterol concentration (mmol/L) Colorimetric test (540 nm) VITROS 250/950
Creat. Creatinine concentration (μmol/L) Two-point rate test (670 nm) VITROS 250/950
Trig. Triglycerides concentration (mmol/L) Colorimetric test (670 nm) VITROS 950
Phos. Phosphorus concentration (mmol/L) Colorimetric test (680 nm) VITROS 250/950
Na+ Sodium concentration (mmol/L) Potentiometric test VITROS 250/950
K+ Potassium concentration (mmol/L) Potentiometric test VITROS 250/950
Ca++Calcium concentration (mmol/L) Colorimetric test (680 nm) VITROS 250/950
Cl- Chloride concentration (mmol/L) Potentiometric test VITROS 250/950
Tot. Prot. Total Protein concentration (g/L) Colorimetric test (540 nm) VITROS 250/950
Alb. Albumin concentration (g/L) Colorimetric test (630 nm) VITROS 250/950
A/G Alb/glob ration Calculated value
AST/GOT Aspartate Aminotransferase activity (U/L) Multiple-point rate test (340 nm) VITROS 250/950
ALT/GPT Alanine Aminotransferase activity (U/L) Multiple-point rate test (340 nm) VITROS 250/950
ALKP Alkaline. Phosphatase – activity (U/L) Multiple-point rate test (400 nm) VITROS 250/950
GGT Gamma Glutamyltransferase -activity (U/L) *-Glutamyl-p-nitroanilid e+ Glycylglycine. Increase in p-nitroaniline-monitored at 400 nm VITROS 250/950



URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

TERMINAL PROCEDURES AND MACROSCOPIC EVALUATION
- All animals were euthanised under pentobarbital anaesthesia (Euthanimal 40%; Lot No.: 1409236-06; Expiry: 09-2017; Produced by Alfasan Nederland BV, Kulpersweg 9, Woerden, Netherlands) by exsanguination.
- After exsanguination the external appearance was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed macroscopically. All abnormalities were recorded.

Organ weight measurements
The following organs were weighed in all animals:
With precision of 0.01g:
Brain
Lungs
Epididymis
Spleen
Heart
Testes
Kidneys
Liver
Thymus
Uterus including cervix
With precision of 0.001g
Adrenals
Ovaries
Paired organs were weighed together. Absolute organ weights were measured, and relative organ weights to the body and brain weights were calculated.

Tissue preservation and microscopic evaluation
On completion of the macroscopic examination the following tissues and organs were retained from all animals.
Adrenal gland
Animal identification 1
Aorta
Brain 2
Bone marrow (smear)
Epididymis
Eye with optic nerve 6
Femur with joint
Gross findings
Heart 3
Kidneys
Large intestine 4
Lachrymal gland (extraorbital) and Harderian gland
Larynx 8
Liver
Lungs with bronchi 5
Lymph nodes 11
Nasal cavity 9
Ovary with oviduct
Oesophagus
Pancreas
Pituitary
Prostate
Salivary gland (including
mandibular and sublingual)
Sciatic nerve
Seminal vesicles & coagulating glands
Skeletal muscle (quadriceps)
Skin, subcutis and mammary gland area (inguinal)
Small intestine 7
Spinal cord (3 levels)
Spleen
Sternum with marrow
Stomach
Testis
Thymus
Thyroid with parathyroid gland 6
Tongue
Trachea 10
Urinary bladder
Uterus including cervix
Vagina

1. Fixation and preservation only
2. 7 section according to the NTP recommendations
3. Section including both ventricles and atria, septum with papillary muscle
4. Caecum, colon and rectum
5. Lungs after weighing were infused with formalin at a pressure of 20-30 cm of water.
6. Parathyroids and optic nerves were examined histologically only if present in routine sections.
7. Duodenum, ileum and jejunum with Peyer’s patches
8. 3 levels, Base of the Epiglottis, Ventral pouch, Cricoid cartilage
9. At least 4 levels, 1 level to include the nasopharyngeal duct and Nasal Associated Lymphoid Tissue (NALT)
10. Two levels, transverse section in the middle part, longitudinal horizontal including the carina of the bifurcation
11. Mediastinal, submandibular and mesenteric
Bone marrow smears (taken from the femur of all animals) were stained with May Grünwald – Giemsa but were not examined as there were no indicative haematology findings. The smears are stored/archived at CiToxLAB Hungary Ltd.
The eyes with the optic nerves and testes with epididymides were retained in modified Davidson’s fixative, all other organs in 10% buffered formalin solution.
The whole respiratory tract from nasal cavities to lung and associated lymphoid tissue (BALT) of animals from all dose groups including controls were investigated by histopathology examination.
Full histopathology was not performed.
The retained tissues and organs were embedded in paraffin wax, sections were cut at 4-6µm by microtome and transferred to slides. Tissue sections were stained with haematoxylin-eosin/phloxine and examined by light microscope.

Other examinations:

no other examinations

Statistics:

Data were collected using the software PROVANTIS v.9 or were recorded on the appropriate forms from the relevant SOPs of CiToxLAB Hungary Ltd., then tabulated using the Microsoft Office Word and/or Excel, as appropriate.
Group mean and standard deviation were calculated for numerical data. Statistical analysis was performed using SAS 9.2 (built in Provantis System).
The following decision tree was automatically applied within the validated Provantis system for statistical evaluation of numeric data:
The normality and heterogeneity of variance between groups were checked by Shapiro-Wilk and Levene tests using the most appropriate data format (log-transformed when justified). Where both tests showed no significant heterogeneity, an Anova / Ancova (one-way analysis of variance) test was carried out. If the obtained result was positive, Dunett (Multiple Range) test was used to assess the significance of inter-group differences; identifying differences of <0.05 or <0.01 as appropriate. This parametric analysis it the better option when the normality and heterogeneity assumptions implicit in the tests were adequate.
If either of the Shapiro-Wilk or Levene tests showed significance on the data, then the ANOVA type approach was not valid and a non-parametric analysis was required. A Kruskal-Wallis analysis of variance was used after Rank Transformation. If there was a positive result, the inter-group comparisons was performed using Dunn test; identifying differences of <0.05 or <0.01 as appropriate.
The frequency of clinical observations and necropsy and histopathology findings were calculated as applicable.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Incidental laboured respiration (slight) was observed in each treatment groups and in the control group, this was considered to be related to the test system and not to the test item. In the first 3 days of the study persistent tremors were observed for the first hour after exposure in all the females of the high dose group. This was a clear neurotoxic effect, common to many “solvent” type chemicals.
Red-brown staining was also recorded in each groups. These observations were considered to be related to the restraint and exposure procedures and, in isolation, were considered not to be of toxicological relevance.

Mortality:

no mortality observed

Description (incidence):

There was no mortality at any of the exposure level.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no significant difference in the body weight and there was no dose dependent change in the body weight gain of the animals during the treatment period.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The food consumption of the females in the High Dose group was transiently decreased in week one. There was no other dose dependent change in the food consumption.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The haemoglobin concentration and haematocrit values were slightly (7.2% and 7.1%, respectively), but significantly higher in the High dose males. In the Mid and High dose females the relative monocyte was significantly lower comparing to the control. All groups were within the expected historical control range hence these changes were considered to be incidental.
The Prothrombin Time (PTT) was significantly lower (4%) in the High dose females. This slight difference is not considered to be toxicologically relevant.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Occasionally, statistically significant differences were observed for some parameters between the Control and treated groups (e.g. higher creatinine in Low and Mid dose males, higher albumin in high dose males, while higher chloride and albumin in Mid dose females). All groups were within the expected control ranges hence the differences were regarded as incidental or individual findings, not related to treatment and were with no toxicological significance.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

In the male High dose group, the kidney and liver weight (relative to the body weight) was slightly but statistically significantly higher with 15.8% and 12.2% compared to the Control. The weight of the high dose male spleen was not different to control relative to body weight, but did show a statistical difference relative to the brain weight (-19.8%) but the relationship of this difference with treatment is not evident.
There were no other toxicologically significant differences among groups in the weights of other organs measured when compared to controls.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

In the animals, no test item-related macroscopic changes could be detected at the necropsy.
In one Mid dose rat (3504), in the uterine horns and body dilatation and clear fluid was observed, as incidental and unrelated to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

The test item, administered daily via inhalation route for 4 weeks to Wistar (Han) rats at 0.1, 1 and 5 mg/L atmosphere concentration, did not cause any test item related macroscopic changes or microscopic changes in examined tissues.

Histopathological findings: neoplastic:

no effects observed

Details on results:

Observed were tremors in all females for the first 3 days, with a slight food intake decrease; male liver and kidney weights were slightly higher than control when adjusted for Body Weight. There were no other effects of treatment observed. The histopathology of the respiratory tract was not affected by treatment.

Key result

Basis for effect level:

other: Based on the data obtained in this study, the following dose levels are suggested for the main 90-day repeated dose study: 0 mg/L, 0.5 mg/L, 1.5 mg/L and 5 mg/L

Mean achieved actual and nominal test atmosphere concentrations

Group No.	Group Designation	Target Concentration (mg/L)	Achieved Concentration (mg/L)			Nominal Concentration (mg/L)
			Gravimetry		GC analysis
			Mean of all samples	Mean of daily exposures	Mean of all investigated samples
2	Low	0.1	0.12 (SD: 0.02)	0.12 (SD: 0.01)	0.14 (SD: 0.04)	0.18 (SD: 0.04)
3	Mid	1.0	1.04 (SD: 0.14)	1.04 (SD: 0.11)	1.04 (SD: 0.1)	1.35 (SD: 0.16)
4	High	5.0	5.01 (SD: 0.25)	5.01 (SD: 0.12)	5.38 (SD: 0.58)	5.27 (SD: 0.24)

Conclusions:

The exposure to the test item in the form of vapour to Hannover Wistar rats for 4 weeks for 6 hours/day on a 5 day per week basis at dose levels up to 5 mg/L the only effects observed were tremors in all females for the first 3 days, with a slight food intake decrease; male liver and kidney weights were slightly higher than control when adjusted for Body Weight. There were no other effects of treatment observed. The histopathology of the respiratory tract was not affected by treatment.
The clear neurotoxic effect observed in the female High dose group in the first week suggests that the use of a significantly higher concentration in the Main study could be above an ethically acceptable level.
Based on the data obtained in this study, the following dose levels are suggested for the main 90-day repeated dose study: 0 mg/L, 0.5 mg/L, 1.5 mg/L and 5 mg/L.

Executive summary:

The objective of this 4-week dose range finding study is to determine the Maximum Tolerated Concentration (MTC) and to obtain information on the toxicity of the test item when administered to Wistar rats via inhalation route for 4 weeks with the aim of inducing toxic effects but no death or suffering at the highest concentration, and a NOAEL at the lowest concentration level.

The results of the study will be used for dose level selection for a forthcoming 90-day subchronic inhalation toxicity study.

 

Five male and 5 female Wistar rats Crl:WI (Han) in each group were exposed, by a 6 hour nose-only exposure, to filtered air (control), Low, Mid or High aerosol concentrations for consecutive 5 days/week to cover a wide range of doses. The duration of the study was 4 weeks, each week consisted of 5 consecutive treatment days and 2 days without treatment. The total number of exposures for each animal was 20. The animals were sacrificed on the day following the last exposure in Week 4.

The objective was to determine suitable concentrations which will be used for a 90-day repeated dose study (with 5 days per week administration). The dose levels were set based on the results of an acute study available, in discussion with the Sponsor; the High concentration was expected to cause some toxicity but without severe effects.

 

The animals were exposed to the test atmosphere in the form of vapour using a nose-only exposure system. The test item was vaporized using a jet nebulizer. The target concentrations were 0.1 mg/L, 1 mg/L and 5 mg/L, as the Low, Mid and High Dose, respectively. Analytical concentrations of 0.14 mg/L, 1.04 mg/L and 5.38 mg/Lwere achieved in the respective groups. The control animals were exposed to filtered air. The treatment was performed in 4 inhalation towers parallel, one tower for each treatment groups and an additional tower for the filtered air control group.

 

Parameters monitored for the animals during the study included mortality, clinical observations, body weight, food consumption and clinical pathology evaluation (haematology, coagulation and clinical chemistry). Gross macroscopic examination was performed at necropsy and selected organs were weighed.

Full histopathology of the whole respiratory tract from nasal cavities to lung and associated lymphoid tissue (BALT) was performed in each group.

The test atmosphere concentration was monitored based on the gravimetric analysis and by a validated GC method. The results of the test atmosphere characterization were considered suitable for the study purposes.

Results

There was no mortality during the study.

In the first 3 days of the study persistent tremors were observed for the first hour after exposure in all the females of the high dose group. This was a clear neurotoxic effect, common to many “solvent” type chemicals.

There was no significant difference in the body weight and there was no dose dependent change in the body weight gain of the animals during the treatment period.

The food consumption of the females in the High Dose group was transiently decreased in week one. There was no other dose dependent change in the food consumption.

There was no effect of treatment on haematology, blood coagulation and clinical chemistry parameters.

In the animals, no test item-related macroscopic changes could be detected at the necropsy.

In the male High dose group, the kidney and liver weight (relative to the body weight) was slightly but statistically significantly higher with 15.8% and 12.2% compared to the Control. The weight of the high dose male spleen was not different to control relative to body weight, but did show a statistical difference relative to the brain weight (-19.8%) but the relationship of this difference with treatment is not evident.

The test item administered daily via inhalation route for 4 weeks to Wistar (Han) rats at 0.1, 1 and 5 mg/L Atmosphere Concentration, did not cause any test item related macroscopic changes or microscopic changes in examined tissues.

Conclusion

The exposure to the test item in the form of vapour to Hannover Wistar rats for 4 weeks for 6 hours/day on a 5 day per week basis at dose levels up to 5 mg/L the only effects observed were tremors in all females for the first 3 days, with a slight food intake decrease; male liver and kidney weights were slightly higher than control when adjusted for Body Weight. There were no other effects of treatment observed. The histopathology of the respiratory tract was not affected by treatment.

The clear neurotoxic effect observed in the female High dose group in the first week suggests that the use of a significantly higher concentration in the Main study could be above an ethically acceptable level.

Based on the data obtained in this study, the following dose levels are suggested for the main 90-day repeated dose study: 0 mg/L, 0.5 mg/L, 1.5 mg/L and 5 mg/L.

 

Reference 2

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-05-11 to 2017-08-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633 Sulzfeld.
- Strain: Wistar Crl:WI (Han) rat
- Age: Young adult rats, 8 weeks old at the initiation of treatment
- Animals: 20 males and 20 females
- Acclimatisation: at least 5 days
- Body Weight at study initiation: male: 233 g - 276 g, female: 159 g - 195 g
- Housing: Group caging (in groups up to 3, by sex)
- Cage type: Polycarbonate solid floor cages (type II or III) with stainless steel mesh lids.
- Diet: ad libitum, ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest, Germany
- Water: tap water ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.8–28.5°C
- Humidity: 25–50% %
- Illumination: 12 hours of continuous artificial light in each twenty-four period (from 6.00 a.m. to 6.00 p.m.)
- Air exchange: 15-20 air exchanges/hour

Route of administration:

inhalation: vapour

Type of inhalation exposure:

nose only

Vehicle:

air

Remarks on MMAD:

Since the test item was vaporized, the test atmosphere did not contain any particles, therefore no particle size analysis was required.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The animals were treated by the inhalation route using a nose only exposure unit, in a TSE Rodent Exposure System with each individual concentration or control group in a dedicated tower. Four identical, modular multilevel flow – past, nose only exposure units (towers) will be used. The exposure unit consisted of two, concentric anodised aluminium cylinders, the inner plenum and the outer chamber with 20 circularly arranged exposure ports.
The equipment was supported by a computer control system incorporating pressure detectors, mass flow controllers as well as temperature/RH, O2 and CO2 sensors. These units were manufactured by TSE Systems GmbH, Bad Homburg, Germany and are similar to the inhalation system evaluated by Pauluhn. The exposure units were placed in closed hoods in order to avoid cross-contamination and contamination of the laboratory environment.
- Method of holding animals in test chamber: The animals were held in polycarbonate restraint tubes located around the chamber which allowed only the animals’ nares to enter the exposure port.
- Source and rate of air: The flow of air through each port was approximately 0.5 L/min. This flow rate is considered adequate to minimise re-breathing of the test atmosphere and maintained oxygen concentrations at greater than 19% and a carbon dioxide concentration not exceeding 1%.
- System of generating particulates/aerosols: For the test atmosphere generation, a stainless steel concentric jet nebuliser (TSE Systems GmbH, Bad Homburg, Germany) located at the top of the exposure chamber were used.
- Temperature, humidity, pressure in air chamber: Temperature of the test atmospheres and air used for the control group exposure were increased over the limit of 25ºC (up to 26.0 ºC) due to temporary malfunctioning of the cooling system. The relative humidity was lower than the optimal range of 30-70% due to the use of filtered, dry air for the dispersion of the test item. This deviation had no effect on the purpose and integrity of the study
- Air flow rate: approximately 0.5 L/min
- Method of particle size determination: Since the test item was vaporized, the test atmosphere did not contain any particles, therefore no particle size analysis was required.


TEST ATMOSPHERE
- Brief description of analytical method used: The actual (achieved) concentration of generated atmospheres was measured gravimetrically at regular intervals during an exposure by pulling a suitable, known volume of test atmosphere, from the exposure chamber, through Anasorb CSC sorbent tubes (SKC Inc., USA, or similar). Sampling was normally performed each day shortly after chamber equilibration and then at regular intervals during the exposure, and samples were collected from a vacant animal exposure port (animals breathing zone). The actual sampling schedule employed was dependent on the required sample volume in order to obtain adequate quantities of test item (exposure period).
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

ACTUAL TEST ATMOSPHERE CONCENTRATIONS
The actual (achieved) concentration of generated atmospheres was measured gravimetrically at regular intervals during an exposure by pulling a suitable, known volume of test atmosphere, from the exposure chamber, through Anasorb CSC sorbent tubes (SKC Inc., USA, or similar). Sampling was normally be performed each day shortly after chamber equilibration and then at regular intervals during the exposure and samples were collected from a vacant animal exposure port (animals breathing zone). The actual sampling schedule employed was dependent on the required sample volume in order to obtain adequate quantities of test item (exposure period).
The average concentration of the test item in the test atmosphere during filter sampling (CAS, in mg/L) was calculated by the following formula:
CAS = M / (tS x FS)
Where
M = Mass of the active substance on the filter (mg)
tS = Duration of sampling (min)
FS = Sampling flow rate (L/min)

From the samples collected during the animal exposure for gravimetric concentration measurement, a confirmatory analysis was performed using GC method validated under Study code: 15/204-316AN. The GC analysis was performed at regular interval during the exposure period, (i.e. every 4th week including the first and last weeks of the treatment).

NOMINAL TEST ATMOSPHERE CONCENTRATIONS
Nominal concentrations were calculated by dividing the total weight of test item disseminated into the test chamber by the total volume of air used during the same period.

PARTICLE SIZE ANALYSIS
Since the test item is vaporized the test atmosphere was not contain any particles, therefore no particle size analysis is required.

See table " any other information on results"

Duration of treatment / exposure:

5 days per week for 13 weeks

Frequency of treatment:

5 days/week x 6 hours/day

Dose / conc.:

0 mg/L air (nominal)

Remarks:

Filtered Air Control

Dose / conc.:

0.5 mg/L air (nominal)

Remarks:

Low Dose (0.536 mg/L, analytic)

Dose / conc.:

1.5 mg/L air (nominal)

Remarks:

Mid Dose (1.61 mg/L, analytic)

Dose / conc.:

5 mg/L air (nominal)

Remarks:

High Dose (5.36 mg/L, analytic)

No. of animals per sex per dose:

10 animals/group/sex:
40 male and 40 female rats for Main groups,
20 male and 20 female rats for Recovery groups,

Control animals:

yes, concurrent vehicle

Details on study design:

The inhalation route is a possible route for human exposure.

The dose levels were selected by the Sponsor in consultation with the Study Director, based on previous data available, including the results of a preliminary dose range finding study in the rat (CiToxLAB study code 15/204-212PE). In this preliminary study, the animals were exposed to a test item atmosphere at the target concentration of 0.1, 1 and 5 mg/L during 4-week period (6 hours/day, 5 days/week). The achieved concentrations measured by chemical analysis of the representative samples were 0.14, 1.04 and 5.38 mg/L.

In the preliminary study, a concentration of 5.38 mg/L was associated with persistent tremors for the first hour after exposure in all the females of the High dose group. This was a clear neurotoxic effect, common to many “solvent” type chemicals. In the male High dose group, the kidney and liver weight (relative to the body weight) was slightly but statistically significantly higher with 15.8% and 12.2% compared to the Control.

The concentration levels were selected by taking into account the clear neurotoxic effect in the dose range finding study at 5.38 mg/L and the fact that the expected human exposure level during manufacture and use of the test substance is far below 5 mg/L.

A control group was included and exposed to filtered air.

Positive control:

not nesessary

Observations and examinations performed and frequency:

MORBIDITY/MORTALITY:
- Checks for mortality and/or morbidity were made twice daily, early and late during the normal working day.

DETAILED CLINICAL OBSERVATIONS:
- Individual clinical observations were performed prior to exposure and during exposure whilst the animals were still restrained (limited observation). Following exposure, clinical observations were performed twice (as soon as practicable after removal from restraint, and approximately one hour after completion of the exposure.
Detailed clinical observations were made on all animals outside the home cage in a standard arena once a week.
The animals were observed for changes in the skin and fur, eyes and mucous membranes and also respiratory, circulatory, autonomic and central nervous system, somatomotor activity and behaviour pattern. Particular attention was directed to observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep, coma.
- All observations were recorded.
- The times of death were recorded as precisely as possible.


NEUROLOGICAL ASSESSMENT (Functional Observation Battery)
Towards the end of the treatment period on Day 84-85, each animal was subjected to the functional observation battery, including measurements of the landing foot splay, fore/hind grip strength and motor activity assessment, on one of the 2 days per week when there was no exposure.
Sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity were conducted and the general physical condition and behaviour of animals were tested. A modified Irwin test was performed.
A detailed assessment for neurotoxicity effects were made on the basis of these measurements (Irwin, S.: Comprehensive Observational Assessment: Ia. A systematic, Quantitative procedure for Assessing the Behavioral and Physiologic State of the Mouse, Psychopharmacologia (Berl) 13 222-257 1968).
Parameters such as body position, locomotor activity, respiration rate, respiration type, piloerection, head searching, compulsive biting or licking, circling, upright walking, retropulsion, jumping, exophthalmos, twitches, clonic convulsions, tonic convulsions, tremor, startle, transfer arousal, spatial locomotion, gait, posture, limb position, finger approach, finger withdrawal, touch escape response, diarrhoea, diuresis, visual placing, grip strength, body tone, corneal reflex, pinna reflex, toe pinch, grasping reflex, positional struggle, skin, mucous membrane colour, salivation, palpebral closure, lachrymation, limb tone, abdominal tone, tail pinch, righting reflex, and vocalisation were evaluated.


EXAMINATION OF VAGINAL SMEARS
Prior to necropsy, the oestrus cycle of all females were determined by taking vaginal smears, which will be prepared and stained with 1% aqueous methylene blue solution. The smear was examined with a light microscope, in order to provide information regarding the stage of oestrus cycle at the time of sacrifice and assist in histological evaluation of oestrogen sensitive tissues.

OPHTALMOLOGY EVALUATION
Ophthalmoscopic examination was conducted in all animals before treatment and in the Control (Group 1) and High dose (Group 4) females on Week 13. In males only the Recovery animals were examined on Week 13 to replace the Main Study males (not made due to technical oversight). Since no alterations were found, the Recovery animals were not examined at the end of the recovery period.
Mydriasis was produced after instillation of eye drops "Cicloplegicedol" (10 mg/mL ciclopentolato hydrochloride; Batch No.: 160515, exp.: May 2021) and “Humapent” (5 mg/mL ciclopentolate hydrochloride; Batch No.: 8680415, exp.: April 2017) into the conjunctival sac. The evaluation was performed by external examination and using a Gowllands ophthalmoscope.

BODY WEIGHT:
-Body weight of each animal was recorded with precision of 1 g at randomization, on Day 1 (before the exposure), and twice a week thereafter including Day 90 (last exposure day) and Day 104 (last day of recovery), and prior to necropsy (prior to necropsy, fasted on Days 91 and 105). The body weight of the Recovery animals were measured only at the end of the recovery period.
Body weights were recorded at death for animals found dead during the course of the study. Weekly body weight and body weight gain were reported.


FOOD CONSUMPTION:
- Food consumption was recorded with precision of 1 g weekly and the weekly food consumption was calculated. Daily food consumption was calculated for reporting purposes.


CLINICAL PATHOLOGY
At the end of the treatment period, prior to scheduled necropsy on Day 91 or Day 105, blood samples for clinical pathology evaluation (haematology, coagulation, and clinical biochemistry) were collected by heart puncture under pentobarbital anaesthesia.
After an overnight period of food deprivation of animals, 3 blood samples were collected, for haematology (approximately 1.2 mL blood in tubes with K3-EDTA as anticoagulant, 1.6 mg/mL blood), for blood clotting times (approximately 1.4 mL blood for APTT and PT measurements, in tubes with sodium citrate as anticoagulant) and one to obtain serum (approximately 1 mL blood as practical in tubes with no anticoagulant) for clinical chemistry.
Urine collection was conducted over approximately 16 hours, during an overnight period of food but not water deprivation, from each animal by placing the animals in metabolic cages.

HAEMATOLOGY:
The following parameters were evaluated in all animals euthanized at termination:
PARAMETERS (UNITS) METHODS EQUIPMENT
RBC Red Blood Cell (erythrocyte) count (1012/L) M/L Automatic laser cell count ADVIA 120
Hgb Haemoglobin concentration (g/dL) Determination of cyan-methemoglobin absorbance ADVIA 120
Hct Haematocrit (relative volume of erythrocytes) (%) Computed by equipment ADVIA 120
MCV Mean Corpuscular (erythrocyte) Volume (fL) Laser cell volume determination ADVIA 120
MCH Mean Corpuscular (erythrocyte) Haemoglobin (pg) Computed by equipment ADVIA 120
MCHC Mean Corpuscular (erythrocyte) Haemoglobin Concentration (g/dL) Computed by equipment ADVIA 120
RDW Red Cell (erythrocyte) volume Distribution Width (%) Distribution Width Laser detection ADVIA 120
Plt Platelet (thrombocyte) count (109/L) K/L Automatic laser cell count ADVIA 120
MPV Mean Platelet Volume (fL) Cell volume determination by laser ADVIA 120
RETIC % Reticulocyte count (%) Comparative value based on laser light detection ADVIA 120
WBC White Blood Cell (leukocyte) count (109/L) K/L Automatic laser cell count ADVIA 120
NE abs, % Neutrophil (absolute and %) Cell differentiation based on myeloperoxidase activityADVIA 120
LY abs, % Lymphocyte (absolute and %) Cell differentiation based on myeloperoxidase activityADVIA 120
MO abs, % Monocyte (absolute and %) Cell differentiation based on myeloperoxidase activityADVIA 120
EO abs, % Eosinophil (absolute and %) Cell differentiation based on myeloperoxidase activityADVIA 120
BA abs, % Basophil (absolute and %) Cell differentiation based on myeloperoxidase activityADVIA 120
LUC % Large Unstained Cells (%) Cell differentiation based on myeloperoxidase activityADVIA 120

Coagulation

PARAMETERS (UNITS) METHODS
APTT Activated Partial Thromboplastin Time (sec) Micronized silica method
PT Prothrombin Time (sec) Quick method (Biggs, R., and R.G. MacFarlane(1962)
Human Blood Coag.and its Disorders, Oxford)

CLINICAL CHEMISTRY
The following parameters were evaluated in all animals euthanized at termination:

PARAMETERS (UNITS) METHODS EQUIPMENT
Glucose Blood sugar concentration (mmol/L) Colorimetric test (540 nm) VITROS 250/950
T-BIL Total Bilirubin concentration (μmol/L) End-point colorimetric (dual-wavelength) test (400 & 460nm) VITROS 250/950
Urea Urea concentration (mmol/L) Colorimetric test (670 nm) VITROS 250/950
Chol. Cholesterol concentration (mmol/L) Colorimetric test (540 nm) VITROS 250/950
Creat. Creatinine concentration (μmol/L) Two-point rate test (670 nm) VITROS 250/950
Trig. Triglycerides concentration (mmol/L) Colorimetric test (670 nm) VITROS 950
Phos. Phosphorus concentration (mmol/L) Colorimetric test (680 nm) VITROS 250/950
Na+ Sodium concentration (mmol/L) Potentiometric test VITROS 250/950
K+ Potassium concentration (mmol/L) Potentiometric test VITROS 250/950
Ca++Calcium concentration (mmol/L) Colorimetric test (680 nm) VITROS 250/950
Cl- Chloride concentration (mmol/L) Potentiometric test VITROS 250/950
Tot. Prot. Total Protein concentration (g/L) Colorimetric test (540 nm) VITROS 250/950
Alb. Albumin concentration (g/L) Colorimetric test (630 nm) VITROS 250/950
A/G Alb/glob ration Calculated value
AST/GOT Aspartate Aminotransferase activity (U/L) Multiple-point rate test (340 nm) VITROS 250/950
ALT/GPT Alanine Aminotransferase activity (U/L) Multiple-point rate test (340 nm) VITROS 250/950
ALKP Alkaline. Phosphatase – activity (U/L) Multiple-point rate test (400 nm) VITROS 250/950
GGT Gamma Glutamyltransferase -activity (U/L) *-Glutamyl-p-nitroanilid e+ Glycylglycine. Increase in p-nitroaniline-monitored at 400 nm VITROS 250/950


URINALYSIS
The evaluation of the urine samples was performed by observation (e.g. colour, appearance) or test strips as applicable. The following parameters were evaluated:
 


PARAMETERS METHODS EQUIPMENT
LEU / Leukocyte Medi-Test Stick 10 URYXXON 300
NIT / Nitrite Medi-Test Stick 10 URYXXON 300
pH Medi-Test Stick 10 URYXXON 300
PRO / Protein Medi-Test Stick 10 URYXXON 300
GLU / Glucose Medi-Test Stick 10 URYXXON 300
UBG / Urobilinogen Medi-Test Stick 10 URYXXON 300
BIL / Bilirubin Medi-Test Stick 10 URYXXON 300
KET / Ketones Medi-Test Stick 10 URYXXON 300
BLD/ERY / Blood/Erythrocytes Medi-Test Stick 10 URYXXON 300
SG / Specific Gravity Medi-Test Stick 10 URYXXON 300
SED / Sediment Microscopic examination Leitz Microscope
Clarity Observation
Colour Observation
Volume Volumetric method

Sacrifice and pathology:

TERMINAL PROCEDURES AND MACROSCOPIC EVALUATION
- Gross necropsy was performed on each main study and recovery animal irrespective of the date of death, including the animals found dead. Terminally on Days 91 or 105 surviving animals were euthanized under pentobarbital anaesthesia by exsanguination.
- After exsanguination the external appearance was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate.


Organ weight measurements
The following organs were trimmed of fat and weighed in all animals:
With precision of 0.01g:
Brain
Epididymides
Heart
Kidneys
Liver
Prostate
Seminal vesicles with coagulating glands
Spleen
Testes
Thymus
Uterus including cervix


With precision of 0.001g
Adrenals
Ovaries
Thyroids with parathyroids
Pituitary

Paired organs were weighed together. Absolute organ weights were measured, and relative organ weights to the body and brain weights were calculated and reported.

Tissue preservation and microscopic evaluation
On completion of the macroscopic examination the following tissues and organs were retained from all animals.
Adrenal gland
Animal identification 1
Aorta 10
Brain 2
Bone marrow (smear)
Epididymis
Eye with optic nerve 7
Femur with joint
Gross findings
Heart 3
Kidneys
Large intestine 4
Lachrymal gland (extraorbital) and Harderian gland
Larynx 8
Liver 12
Lungs with bronchi 5
Lymph nodes 6
Ovary
Oviduct
Pancreas
Pituitary
Prostate
Salivary gland (including mandibular and sublingual)
Sciatic nerve
Seminal vesicles & coagulating glands
Skeletal muscle (quadriceps)
Skin, subcutis and mammary gland area (inguinal)
Small intestine 8
Spinal cord 11
Spleen
Sternum with marrow
Stomach
Testis
Thymus
Thyroid with parathyroid gland 6
Tongue
Trachea 10
Urinary bladder
Uterus 9
Vagina

1. Fixation and preservation only.
2. 7 section according to the NTP recommendations.
3. Section including both ventricles and atria, septum with papillary muscle.
4. Caecum, colon and rectum.
5. Lungs of euthanized animals were infused with formalin; 3 lobes, left, right cranial, right caudal.
6. Mandibular and mesenteric.
7. If applicable, parathyroids and optic nerves were examined histologically only if present in routine sections.
8. Duodenum, ileum and jejunum with Peyer’s patches.
9. Horns, body and cervix.
10. Aorta thoracic and abdominal
11. Transverse sections, 3 levels -cervical, thoracic and lumbar.
12. Liver, 3 lobes, left lateral, right medial, caudate


The eyes with the optic nerves and testes with epididymides were retained in modified Davidson’s fixative, all other organs in 10% buffered formalin solution.

Bone marrow smears (taken from the femur of all animals) were preserved.

Full histopathology was performed in Control and High dose groups and in animals found dead. In addition, lungs were investigated in Mid and Low dose groups. Examination of tissues from Low dose and/or Mid dose animals were performed where treatment-related changes are found in High dose group. Any organs or tissues with macroscopic abnormalities (except minor changes) were subjected to histological examination.

The retained tissues and organs for histological examination were embedded in paraffin wax, sections were cut at 4-6µ by microtome and transferred to slides. Tissue sections were stained with haematoxylin-eosin/phloxine and examined by light microscope.

Kidney samples from Control and High dose groups in FFPE blocks were sent out to CiToxLAB France for immunohistochemical investigation of renal pathology to determine if the pathology is mediated by alpha-2 microglobulin nephropathy.
Alpha(2)-microglobulin-induced nephropathy is a phenomenon which is exclusively found in adult male rats. Various chemicals are able to bind to alpha(2)-microglobulin thus inhibiting its proteolytic degradation in lysosomes of the P2 segment of the rat nephron. The accumulation of this protein in 'protein droplets' or 'hyaline droplets' leads to necrosis, followed by regeneration.
Alpha(2)-microglobulin expression was evaluated in the kidney from Control and High Dose treated male rats at the end of the treatment and the recovery period.

Left Kidney samples (longitudinal sections) embedded in paraffin wax (paraffin blocks) were used for α-2u globulin analysis. Sections were cut and transferred to slides. Tissue sections were immunostained for α-2u globulin and examined by Frédéric Gervais (Principal Investigator (PI)). The immunostaining methods are described in a specific SOP of CiToxLAB France (appended to the Phase Report).

Kidney blocks were stored in room temperature in the Test Site.

Other examinations:

no other examinations

Statistics:

Data were collected using the software PROVANTIS v.9 or were recorded on the appropriate forms from the relevant SOPs of CiToxLAB Hungary Ltd., then tabulated using the Microsoft Office Word and/or Excel, as appropriate.
Group mean and standard deviation were calculated for numerical data. Statistical analysis was performed using SAS 9.2 (built in Provantis System).
The following decision tree was automatically applied within the validated Provantis system for statistical evaluation of numeric data:
The normality and heterogeneity of variance between groups were checked by Shapiro-Wilk and Levene tests using the most appropriate data format (log-transformed when justified). Where both tests showed no significant heterogeneity, an Anova / Ancova (one-way analysis of variance) test was carried out. If the obtained result was positive, Dunett (Multiple Range) test was used to assess the significance of inter-group differences; identifying differences of <0.05 or <0.01 as appropriate. This parametric analysis it the better option when the normality and heterogeneity assumptions implicit in the tests were adequate.
If either of the Shapiro-Wilk or Levene tests showed significance on the data, then the ANOVA type approach was not valid and a non-parametric analysis was required. A Kruskal-Wallis analysis of variance was used after Rank Transformation. If there was a positive result, the inter-group comparisons was performed using Dunn test; identifying differences of <0.05 or <0.01 as appropriate.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No treatment related clinical signs were observed in the Control and Low dose groups.

Noisy respiration (slight) was observed in 1 male in Mid dose group on Day 9 and 1 female in the High dose group: This observation is not considered to be a clearly treatment related effect.
Laboured respiration (slight to moderate), hunched back, prostration, tremors (continuous – whole body) were observed in 1 female in the High dose group transiently from Day 16 to Day 20. This was considered to be an adverse treatment related effect, consistent with the preliminary study observations.

All other animals were clinically normal during the study.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One male in the High dose group (#4018) was found dead on Day 1 and replaced by animal #4118 and 1 female in the High dose group (#4503) was found dead on Day 51.
There deaths were not considered to be related to test item exposure.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no test item related adverse effects noted on body weight or body weight gain under the conditions of this study. All treated groups were comparable with the Control.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There were no Test Item related adverse effects noted on animal food consumption under the conditions of this study.

There were no consistent changes measured in the food consumption during the treatment period between the dose groups. However, there were sporadic differences in food consumption measurements in both sexes compared to the Control group with statistical significance, but these were considered unrelated to the Test Item.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No Test Item related changes compared to pre-treatment or/and the Control were noted at ophthalmoscopy examination. All examined animals were found to be normal.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

In the haematology parameters, there were a few statistical differences, but not consistent between sexes or clearly related to treatment. It is considered that there were no adverse haematology changes in the study.
See table "overall remarks".

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were Test Item related effects on the clinical chemistry parameters evaluated at the completion of the 90-day treatment period in High dose males (ALT/GPT).
See table "overall remarks".
In males the ALT/GPT level showed dose dependent increase reaching statistical significance in the High dose (the Mid dose value was below the recovery control value). After recovery, this parameter was still above control, but with no statistical significance. In females there was no statistical significance. In the absence of any associated changes in clinical pathology or tissues at histology, it is considered treatment related adaptive and not adverse effect.

Other statistical differences tended to be within the historical control range and/or without a dose response, therefore the changes were not considered to be related to treatment.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

In both sexes, significantly decreased pH was measured in High dose; in the recovery animals there was no effect. The pH difference may reflect the presence of metabolites (which would not be an adverse effect). In the absence of any kidney histopathology or other clinical chemistry effects, this difference was not considered to reflect an adverse effect.
See table "overall remarks".
The other urinalysis parameters were comparable to the controls in all dose groups. Variations occurred and consisted of minor differences to controls, these were unrelated to treatment and within the normal range.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

There was no effect in the Grip Strength, Landing Foot Splay or Irwin Test in any dose group.

There were no effects considered to be related to the test item on locomotor activity; some time points had statistical differences in locomotor distance in males, but overall the shape of the curve showed a normal form with an approximate plateau after about 20-30 minutes, for the main and recovery animals.

Immunological findings:

no effects observed

Description (incidence and severity):

IHC ANALYSIS OF ALPHA2 MICROGLOBULIN

There were no significant differences in α-2u globulin scoring between controls males or rats exposed to Hydrocumene at 5 mg/L.
See below Appendix 4 "IHC Report" in "Attached background material"

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Liver weights were higher than controls at the High dose, as absolute and relative to body weight in both sexes (18% p<0.01 and 12% p<0.05 for males and females respectively absolute weights). In males only the High dose liver weights relative to brain weight were also higher than control (no statistical difference in females). After recovery, liver weights of High dose males were also higher than control, by 11% (p<0.05) for absolute weights. In males only, there was a similar trend in the Mid dose group, but no statistical significance for the absolute liver weights, only when adjusted for body and brain weights. The liver weight differences were considered to be treatment related adaptive and not adverse, since there were no histological hepatic changes.

Kidney weights were higher than control in High dose males, only when adjusted relative to body weight in the main groups, and only when adjusted for brain weight in the recovery animals. There were no kidney weight effects in females. The kidney weight differences were no consistent, tended to be within the normal historical range and there were no histological changes in the organ; hence the differences were not considered to reflect an adverse effect.

Adrenal gland weights were above control in females only (p<0.05) as absolute and adjusted for body or brain weights. There were no statistical differences in recovery animals. The adrenal weight differences were no consistent, tended to be within the normal historical range and there were no histological changes in the organ; hence the differences were not considered to reflect an adverse effect.

Thymus weights were statistically significantly higher in High dose recovery males only as absolute and adjusted for body weight only, not for brain weight. The thymus weight differences were no consistent, tended to be within the normal historical range and there were no histological changes in the organ; hence the differences were not considered to reflect an adverse effect.

See table "overall remarks".

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Found dead animals
No test item-related macroscopic findings were detected.
In both found dead animals the lungs were dark red and collapsed. Additionally, dark red thymus was seen in male 4018. The observations were considered to be incidental for these dead animals.

Terminal (Day 91)
No test item-related macroscopic changes were seen during the necropsy in any organs or tissues.
Changes, such as dark red discoloration of the mandibular lymph node, thymus or gastric mucosa, small testes/seminal vesicles or medial lobe of the liver, or dilated uterine horns, were all considered as incidental, or background findings.

Recovery (Day 105)
No test item-related macroscopic changes were seen at necropsy.
Changes such as red focus in the gastric mucosa, small testes and epididymis, red discoloration of the thymus or dilated uterine horns, were considered as incidental, or background findings.

See below Appendix 3 "Pathology Report" in "Attached background material"

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Found dead animals
No test item-related microscopic findings were observed.
Agonal congestion was seen in the lungs and in the thymus (4018) of the found dead animals, these findings are common in dead animals and not considered to be treatment related.

Terminal (Day 91)
No test-item related microscopic changes were noted in the experimental rats for respiratory tract tissues, or in any other organs or tissues.
Findings including tubular degeneration/atrophy of the testes and reduced sperm content in the epididymis, the focal tubular basophilia, casts, or mineralization in the kidneys, congestion/haemorrhage in the mandibular lymph node and in the thymus. There was no relation with treatment group; the findings were all considered as incidental, based on the low occurrence through control and dosed animals.

Recovery (Day 105)
No test-item related microscopic changes were recorded in the experimental rats.
Findings, such as tubular degeneration/atrophy of the testes and reduced sperm content in the epididymis, the focal tubular basophilia, casts, mineralization or pyelonephritis in the kidneys, congestion/haemorrhage in the thymus, focal degeneration/necrosis of cardiomyocyte, inflammatory cell infiltrate in the prostate or in the urinary bladder and oestrus in the uterine horns, were considered as incidental or background, based on the low occurrence through control and dosed animals.

See below Appendix 3 "Pathology Report" in "Attached background material"

Histopathological findings: neoplastic:

not specified

Other effects:

no effects observed

Description (incidence and severity):

EXAMINATION OF VAGINAL SMEARES
There were no adverse or Test Item related observations in the animal oestrus cycle evaluated prior to necropsy and the animals showed the normal distribution of the oestrus phases.

Details on results:

The High dose concentration was considered to be an acceptable High dose in this study, since in the DRF there was a clear neurotoxic effect of persistent tremors for the first hour after exposure in all females at 5.38 mg/L. Similar effects was recorded in a High dose female in the 90 day study. Hence the High dose of 5 mg/L was very close to a significantly adverse neurotoxic effect level, and is much higher than any expected human exposure level during manufacture and use of the test substance.

In the 90-day GLP inhalation study, a neurological effect was observed in the High dose group, with prostration and tremors in 1 female; a similar observations had been seen in all females in the DRF study at 5.38 mg/L. Hence the neurological effect was considered a clear adverse effect of the test item at 5 mg/L.

There were no other adverse effects of treatment in body weight, food intake, clinical signs, neurological assessments, clinical pathology or in tissues or organs of treated animals. There were no effects on the respiratory tissues.

The only other consistent findings were restricted to the High dose group, with slightly reduced pH of urine (about one unit) and a liver weight increase (<20%) in both sexes, with slightly elevated ALT/GPT levels in males only. The urine and hepatic changes were considered to be non-adverse, with no histopathological changes in kidneys or liver.

Key result

Dose descriptor:

NOAEL

Effect level:

1.61 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: neurological effect

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

5.36 mg/L air (analytical)

System:

other: neurological effect

Organ:

other: clinical signs, like laboured respiration, tremors, prostration

Treatment related:

yes

Dose response relationship:

yes

The actual atmospheric concentrations were:

	Low Dose (mg/L)			Mid Dose (mg/L)			High Dose (mg/L)
	gravimetric	analytic	nominal	gravimetric	analytic	nominal	gravimetric	analytic	nominal
Mean	0.4564	0.5357	0.58	1.4784	1.61	1.57	5.0118	5.36	5.52
SD	0.0217	0.05	0.07	0.0392	0.04	0.13	0.1410	0.23	0.27

The achieved concentrations for all groups were considered to be fully acceptable for Concentration and Stability of the exposures.

Analysis of filter samples for concentration were performed and no Test Item was detected in the control samples.

 

Conclusions:

In conclusion, under the conditions of this inhalation study, the no observed adverse effect level (NOAEL) for test item is considered to be 1.61 mg/L.

Executive summary:

The objective of this 90-day study was to obtain information on the toxicity of the test item when administered to Wistar (Han) rats via inhalation route for at least 90 days with the aim of inducing toxic effects but no death or suffering at the highest concentration, and little or no evidence of toxicity at the lowest concentration level.

 

The animals were exposed to the test atmosphere at selected target concentrations of 0.5, 1.5 and 5 mg/L, as the Low, Mid and High Concentration, respectively, using a nose-only exposure system. The concentrations were selected by the Study Monitor in consultation with the Study Director, based on previous data available, including the results of a preliminary dose range finding study in the rat (CiToxLAB study code (15/204-212PE).

 

Male and female Wistar rats Crl:WI (Han) were exposed to the test atmosphere for 6 hour per day on a 5 day per week basis for a period of 13 weeks (total study duration of at least 90 days) according to the following Experimental Design.

 

Gr. No.	Group Designation	Target Test Atmosphere Concentration (mg/L)	Animal Numbers
			Male		Female
			Main	Recovery	Main	Recovery
1	Filtered Air Control	0	1001-1010	1011-1020	1501-1510	1511-1520
2	Low Dose	0.5	2001-2010	-	2501-2510	-
3	Mid Dose	1.5	3001-3010	-	3501-3510	-
4	High Dose	5	4001-4010	4011-4020	4501-4510	4511-4520

Start of the exposure for all 4 groups was Day 1.

 

A control group was exposed to filtered air.

 

The treatment was performed in 4 inhalation towers in parallel, one tower for each treatment groups and an additional tower for the filtered air control group.

 

Surviving Main Study animals including controls were terminated on the day following the last exposure on Day 91.

 

Rats assigned to Recovery groups (10 male and 10 female in control and High dose groups), following the completion of the 90-day treatment period were kept for a 14-day observation period and then euthanized.

 

For practical reasons, the start of treatment was staggered on different days.

 

The achieved concentrations for all groups were considered to be fully acceptable for Concentration and Stability of the exposures.

 

Analysis of filter samples for concentration were performed and no Test Item was detected in the control samples.

 

Results:

One HD male (#4018) was found dead on Day 1 and replaced by animal #4118, and 1 HD female (#4503) was found dead on Day 51. Deaths were considered not to be treatment related.

 

No treatment related clinical signs were observed in the Control and Low dose groups. Noisy respiration (slight) was observed in 1 male in Mid dose group on Day 9 and 1 female in the High dose group on Day 12. This observation is not considered to be a clearly treatment related effect. Laboured respiration (slight to moderate), hunched back, prostration, tremors (continuous – whole body) were observed in 1 female in the High dose group transiently from Day 16 to Day 20. This was considered to be an adverse treatment related effect, consistent with the preliminary study observations.

 

There was no significant difference between groups in the bodyweight or weight gain, or in the food consumption.

 

A few statistical differences were found in the haematology parameters, not consistent between sexes or clearly related to treatment. It is considered that there were no adverse haematology changes in the study.

 

In the clinical chemistry parameters, there was a dose dependent increase of ALT/GPT levels of males, statistically significant in High dose group.

 

In the urinalysis decreased pH was measured in both sexes in the High dose; in the recovery animals there was no effect. The pH difference may reflect the presence of metabolites (which would be not be an adverse effect). In the absence of any kidney histopathology or other clinical chemistry effects, this difference was not considered to reflect an adverse effect.

 

There was no effect in the Grip Strength, Landing Splay or Irwin Test in any dose group. There were no effects considered to be related to the test item on locomotor activity; some time points had statistical differences in locomotor distance in males, but overall the shape of the curve showed a normal form with an approximate plateau after about 20-30 minutes, for the main and recovery animals.

 

There was no treatment related effect in the ophthalmoscopy.

 

Increased liver weights were measured in the treated groups, but these differences were considered to be treatment related adaptive and not adverse, since there were no histological hepatic changes.

Increased relative kidney weights were measured in High dose males. The kidney weight differences were not consistent, tended to be within the normal historical range and there were no histological changes in the organ; hence the differences were not considered to reflect an adverse effect.

Absolute and relative adrenal gland weights were above control in High dose females. There were no statistical differences in recovery animals. The adrenal weight differences were not consistent, tended to be within the normal historical range and there were no histological changes in the organ; hence the differences were not considered to reflect an adverse effect.

Thymus weights were statistically significantly higher in High dose recovery males only as absolute and adjusted for body weight only, not for brain weight. The thymus weight differences were not consistent, tended to be within the normal historical range and there were no histological changes in the organ; hence the differences were not considered to reflect an adverse effect.

 

There were no treatment related macroscopic changes observed at necropsy in any organs or tissues.

 

Hydrocumene daily administered via inhalation route to Hannover Wistar rats for 90 days at 0.5, 1.5 and 5.0 mg/L target atmosphere concentration, did not cause any macroscopic or microscopic changes in the respiratory tract, or in any body organ or tissues in the experimental animals.

 

There were no significant differences in α-2u globulin scoring between the males under filtered air and those exposed to Hydrocumene at 5 mg/L.

 

Conclusion

The High dose concentration was considered to be an acceptable High dose in this study, since in the DRF there was a clear neurotoxic effect of persistent tremors for the first hour after exposure in all females at 5.38 mg/L. Similar effects was recorded in a High dose female in the 90 day study. Hence the High dose of 5 mg/L was very close to a significantly adverse neurotoxic effect level, and is much higher than any expected human exposure level during manufacture and use of the test substance.

 

In the 90-day GLP inhalation study, a neurological effect was observed in the High dose group, with prostration and tremors in 1 female; a similar observations had been seen in all females in the DRF study at 5.38 mg/L. Hence the neurological effect was considered a clear adverse effect of the test item at 5 mg/L.

 

There were no other adverse effects of treatment in body weight, food intake, clinical signs, neurological assessments, clinical pathology or in tissues or organs of treated animals. There were no effects on the respiratory tissues.

 

The only other consistent findings were restricted to the High dose group, with slightly reduced pH of urine (about one unit) and a liver weight increase (<20%) in both sexes, with slightly elevated ALT/GPT levels in males only. The urine and hepatic changes were considered to be non-adverse, with no histopathological changes in kidneys or liver.

 

 

In conclusion, under the conditions of this inhalation study, the no observed adverse effect level (NOAEL) for test item is considered to be 1.61 mg/L.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

1 610 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

The study is valid without restriction (Klimisch score 1).

System:

other: neurological effect

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Study duration:

subchronic

Species:

rat

Quality of whole database:

The study is valid without restriction (Klimisch score 1).

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The objective of the 90-day study was to obtain information on the toxicity of the test item when administered to Wistar (Han) rats via inhalation route for at least 90 days with the aim of inducing toxic effects but no death or suffering at the highest concentration, and little or no evidence of toxicity at the lowest concentration level (CiToxLab, 2017).

The animals were exposed to the test atmosphere at selected target concentrations of 0.5, 1.5 and 5 mg/L, as the Low, Mid and High Concentration, respectively, using a nose-only exposure system. Male and female Wistar rats Crl:WI (Han) were exposed to the test atmosphere for 6 hour per day on a 5 day per week basis for a period of 13 weeks (total study duration of at least 90 days).

Justification for classification or non-classification

According to the criteria of EC Regulation 1272/2008 and based on the results of the repeated dose inhalation study the test item is not classified.