N-methyl-N-[C18-(unsaturated)alkanoyl]glycine

Endpoint summary

Description of key information

Oral (90-day, OECD 408, rat): NOAEL (systemic) = 300 mg/kg bw/day; LOAEL (local, rat) = 100 mg/kg bw/day

Inhalation (28-day, OECD 412, rat): NOAEC = 60 mg/m3 air

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 Oct 2019 - 11 Feb 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 2018

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:

adopted 2008

GLP compliance:

yes (incl. QA statement)

Remarks:

Behörde für Gesundheit und Verbraucherschutz, Hamburg, Germany

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD (SD)

Details on species / strain selection:

Selected due to its proven suitability in toxicology studies and to comply with regulatory requirements for testing in a rodent animal species.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Germany GmbH, Sulzfeld, Germany
- Age at study initiation: 58 days (males), 59 days (females)
- Weight at study initiation: 290.7 g - 351.7 g (males), 207.4 g - 271.5 g (females)
- Housing: 3 animals/cage in MAKROLON cages (type III plus or type IV) with granulated textured wood (Granulat A2, J. Brandenburg, Goldenstedt, Germany) as bedding material.
- Diet: Commercial diet ssniff® R/M-H V1534 (ssniff Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: Tap water in bottles, ad libitum
- Acclimatisation period: 13 days (males), 14 days (females)

DETAILS OF FOOD AND WATER QUALITY:
Periodic analysis of the food for contaminants is conducted by Landwirtschaftliche Untersuchungs- und Forschungsanstalt, Institut für Tiergesundheit und Lebensmittelqualität GmbH (Kiel, Germany). Certificates of analysis of the composition and for contaminants are provided by the manufacturer. The drinking water is examined according to the "Deutsche Trinkwasserverordnung 2018" [German Regulations on drinking water 2018] by the Hamburger Wasser-werke, Hamburg, Germany, at least four times/year. In addition, once/year the drinking water is subjected to a bacteriological examination.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): 15 - 20
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 15 Oct 2019 To: 11 Feb 2020

Route of administration:

oral: gavage

Vehicle:

other: 0.5% aqueous hydroxylpropyl methyl-cellulose gel

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was suspended in the vehicle to the appropriate concentrations (w/v) and was administered by gavage at a constant volume of 10 mL/kg bw/day. The dose formulations were continuously stirred throughout the entire administration procedure to ensure homogeneity. The dose of the test item has been adapted to the animal's body weight daily up to and including test week 6, thereafter weekly.

VEHICLE
- Supplier: Fagron Services B.V., The Netherlands
- Lot/batch no. (if required): 18A17-B04

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical method (HPLC-UV) was developed and validated in a GLP study (LPT study no. 37647). A test item formulation of 100 mg/L was homogenous and stable for at least 8 h. The following parameters were determined: linearity, accuracy, precision, sensitivity, specificity and stability. The accuracy of all formulation samples obtained during the study was well within the acceptable limit of ±15% (percentage of the actual concentration of active ingredient in the formulation samples compared to the respective nominal concentration).

Duration of treatment / exposure:

90 days (control and test groups)
90 days and 28 days post-exposure observation period (recovery control and high-dose groups)

Frequency of treatment:

once daily, 7 days/week

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

The initial dose level of the high-dose group was reduced to 750 mg/kg bw as of test days 30 (males) an 29 (females) and further reduced to 600 mg/kg bw as of test days 37 (males) an 36 (female) due to observed morbidity/mortality.

Dose / conc.:

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

Remarks:

The initial dose level of the high-dose group was reduced to 750 mg/kg bw as of test days 30 (males) an 29 (females) and further reduced to 600 mg/kg bw as of test days 37 (males) an 36 (female) due to observed morbidity/mortality.

Dose / conc.:

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

Remarks:

The initial dose level of the high-dose group was reduced to 750 mg/kg bw as of test days 30 (males) an 29 (females) and further reduced to 600 mg/kg bw as of test days 37 (males) an 36 (female) due to observed morbidity/mortality.

No. of animals per sex per dose:

Main study: 10 (40 males and 40 females)
Recovery animals: 5 (10 males and 10 females), only control and high-dose groups

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were based on the results of a foregoing 28-day dose range finding study, in which animals were orally exposed to 100, 300 and 1000 mg/kg bw/day for 28 days (LPT study no. 37715). Only slightly reduced body weights and body weight gains were observed at 300 and 1000 mg/kg bw/day but no severe signs of toxicity were detected. Therefore, 100, 300 and 1000 mg/kg bw/day were selected as the dose levels for the main study.
- Rationale for selecting satellite groups: Investigation of the reversibility of any effects after a treatment-free recovery period.
- Post-exposure recovery period in satellite groups: 14 days
- Fasting period before blood sampling for clinical biochemistry: yes, overnight
- Anaesthesia for blood sampling: yes, isoflurane

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily from 7:30 a.m. to 4:30 p.m., on Saturdays and Sundays from 8:00 a.m. to 12:00 a.m.
- Cage side observations include skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behavior patterns.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the first exposure and once/week thereafter

BODY WEIGHT: Yes
- Time schedule for examinations: at the time of group allocation, daily from the day of commencement of treatment up to and including test week 6 for dose adjustment, thereafter weekly throughout the experimental period.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before first dosing, at the end of treatment period and at the end of the recovery period.
- Dose groups that were examined: performed on all animals.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of test week 13 (main study); at the end of recovery period (recovery control and high-dose groups)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all main study and all recovery groups animals
- Parameters checked: differential blood count (relative and absolute), Erythrocytes (RBC), Leucocytes (WBC), Haematocrit value, Haemoglobin content, Platelets, Reticulocytes, Mean corpuscular volume (MCV), Mean corpuscular haemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC), Prothrombin time (PT), Activated partial thromboplastin time (aPTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of test week 13 (main study); at the end of recovery period (recovery control and high-dose groups)
- Animals fasted: Yes
- How many animals: all main study and all recovery groups animals
- Parameters checked: Sodium, Potassium, Calcium, Chloride, Albumin, Total bilirubin, Total cholesterol, High density lipoprotein (HDL), Low density lipoprotein (LDL), Glucose, Total protein, Blood urea nitrogen (BUN), Creatinine, Bile acids, Aspartate aminotransferase (ASAT/GOT), Alanine amino-transferase (ALAT/GPT), Alkaline phosphatase (aP), Lactate dehydrogenase (LDH), Thyroid hormones (T3, T4, TSH)

URINALYSIS: Yes
- Time schedule for collection of urine: at the end of test week 13 (main study); at the end of recovery period (recovery control and high-dose groups)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked : Colour and the turbidity, Volume, pH, Specific gravity, Protein, Glucose, Bilirubin, Urobilinogen, Ketones, Hemoglobin (Hb), Nitrite

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at the end of test week 13 (main study); at the end of recovery period (recovery control and high-dose groups)
- Dose groups that were examined: all main study and all recovery groups animals
- Battery of functions tested: sensory activity and grip strength and motor activity

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- Organs to be weighed: Adrenal gland, Brain, Epididymis, Heart, Kidney, Liver, Ovary, Spleen, Pituitary, Prostate and seminal vesicles with coagulating glands, Testicle, Thymus, Thyroid, Uterus (including cervix)
- Tissues to be preserved: Adrenal gland, Aorta abdominalis, Bone (os femoris with joint), Bone marrow (os femoris), Brain (cerebrum, cerebellum, medulla/pons), Epididymis, Eye with optic nerve, Heart (left and right ventricle, septum), Intestine, small (duodenum, jejunum, ileum, including Peyer’s patches), Intestine, large (colon, rectum), Kidney and ureter, Liver (2 lobes), Lungs (with mainstem bronchi and bronchioles, Lymph node (1, cervical and 1, mesenteric), Mammary gland, Muscle (skeletal, leg), Nerve (sciatic), Oesophagus, Ovary with oviducts, Pancreas, Pituitary, Prostate and seminal vesicles with coagulating glands, Salivary glands (mandibular, parotid, sublingual), Skin (left flank), Spinal cord (3 sections: cervical, mid-thoracic, lumbar), Spleen, Stomach, Testicle, Thymus, Thyroid (including parathyroids), Tissue masses or tumours (including regional lymph nodes), Trachea (including larynx), Urinary bladder, Uterus (including cervix), Vagina

HISTOPATHOLOGY: Yes
- Animals: all main study animals of the control and high-dose groups and all animals of the recovery and high-dose groups.
- Organs and tissues to be examined: the same as summarised under 'GROSS PATHOLOGY'

Statistics:

Multiple t-test based on DUNNETT: Body weight / Haematology / Coagulation / Clinical chemistry / Relative and absolute organ weights (p <= 0.05 and p <= 0.01)
STUDENT's t-test: Body temperature / Hind leg splay / Grip strength / Spontaneous motility (p <= 0.05 and p <= 0.01)
Exact test of FISHER: Histopathology (p <= 0.05)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Salivation and breathing sounds occurred during the treatment period in the mid- and high-dose groups. The detailed clinical observations performed once per week confirmed the clinical signs. For details please refer to Tables 2 and 3 under 'Attached background material'.

Mortality:

mortality observed, treatment-related

Description (incidence):

Deaths/sacrifices caused by local changes to the stomach occurred at the low-dose level (1 of 10 males sacrificed) and at the high-dose level (2 of 15 males and 2 of 15 females). No further deaths were noted after the dose reduction to 600 mg/kg bw/day.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males of the mid-dose and males and females of the high-dose groups revealed a slightly reduced body weight gain during the treatment period, resulting in a reduced absolute body weight of the animals compared to the control group. For details please refer to Tables 7, 8 and 9 under 'Attached background material'.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Treatment with 1000/750/600 mg/kg bw/day led to a slightly decreased absolute food intake of male and female animals in several test weeks during the treatment period compared to the control group (by up to 16% in males and 20% in females). A significant decrease of the corresponding relative food intake was only observed in test week 1 (by 9% in males and 17% in females compared to the control group). For details please refer to Tables 10 and 11 under 'Attached background material'.

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

no effects observed

Description (incidence and severity):

For details please refer to Table 16 under 'Attached background material'.

Haematological findings:

no effects observed

Description (incidence and severity):

For details please refer to Table 12 under 'Attached background material'.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The following changes were noted in males and females of the mid- and high-dose groups at the end of the treatment period: decreased plasma levels of total cholesterol and HDL, increased plasma levels of LDL and increased enzyme activities of ALAT and aP. In addition, decreased plasma levels of total protein, chloride and sodium were observed for the animals of the high-dose group. For details please refer to Table 13 under 'Attached background material'.

Urinalysis findings:

no effects observed

Description (incidence and severity):

For details please refer to Table 15 under 'Attached background material'.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

For details please refer to Tables 4, 5 and 6 under 'Attached background material'.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The females of the mid- and high-dose groups revealed increased relative and absolute liver weights at terminal sacrifice. For details please refer to Tables 18 and 19 under 'Attached background material'.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Treatment with 300 or 1000/750/600 mg/kg bw/day led to a thickened mucosa in the stomachs (partly associated with yellow or white deposits on the mucosa) of male and female animals. For details please refer to Table 17 under 'Attached background material'.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In the stomach of males and females of the low-, mid- and high-dose groups squamous cell hyperplasia with hyperkeratosis, minimal to slight erosions/ulcerations and submucosal oedema was observed. Further, changes were noted in the liver of males and females treated with 300 or 1000/750/600 mg/kg bw/day in form of an increase in cytoplasmic eosinophilic granulation (resulting in an increase in overall cellular eosinophilia).

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Thyroid hormone (T3, T4 and TSH): Increased serum levels of T4 (thyroxine) at the end of the treatment period compared to the control animals was observed in animals of the mid- and high-dose groups. For details please refer to Table 14 under 'Attached background material'.

Details on results:

A complete recovery was noted for the following parameters during or at the end of the recovery period: all clinical signs, the food intake, the plasma levels of total protein, chloride, sodium and LDL, the serum level of T4, and the relative and absolute liver weights of the male and female animals were within the range of the control group values. The macroscopic inspection at necropsy did not reveal any visible changes related to the previous treatment with the test item. The histopathological examination of the recovery animals revealed a complete recovery of the changes noted for the liver of male and female animals.
A trend towards recovery was noted for the decreased body weight gain of male and female animals, and for the total cholesterol and HDL plasma levels of the male animals of the high-dose group. The microscopic changes noted in the forestomach of males and females in form of squamous hyperplasia were still observed in male and female animals of the high-dose group; however, at a lower severity, indicating a trend towards recovery.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

gross pathology

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Key result

Dose descriptor:

LOAEL

Remarks:

local

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

gross pathology

histopathology: non-neoplastic

Key result

Critical effects observed:

no

Table 1: Noteworthy observations of the prematurely deceased/sacrificed animals of the high dose group

Animal no./ Sex	Noteworthy observations
71 m (PS, TD 36)	Clinical signs	Salivation (slight to moderate), breathing sounds; no particular premortal symptoms
	Body weight gain	Body weight gain reduced by approx. 50% compared to control group (test days 1-36)
	Macroscopy	Stomach: mucosa thickened
	Histopathology	Slight squamous cell hyperplasia with minimal hyperkeratosis in the forestomach

Animal no./ Sex	Noteworthy observations
80 m (PS, TD 35	Clinical signs	Salivation (slight to moderate), breathing sounds; no particular premortal symptoms
	Body weight gain	Body weight loss of 22 g (i.e. 6%) from test days 22 to 29; body weight gain reduced by approx. 50% compared to control group (test days 1-29)
	Macroscopy	Stomach: mucosa thickened and indurated Intestine (small): dilated Adrenals: enlarged
	Histopathology	Moderate squamous cell hyperplasia with hyperkeratosis, erosions and submucosal oedema in the forestomach, and minimal hepatocellular cytoplasmic alteration (eosinophilic granulation) in the liver
87 f (PD, TD 28)	Clinical signs	Salivation (slight to moderate), breathing sounds; no particular premortal symptoms
	Body weight gain	Body weight loss of 55 g (i.e. 20%) from test days 22 to 27; body weight gain reduced by approx. 40% compared to control group (test days 1-22)
	Macroscopy	External observations: nose haemorrhagic, abdomen dilated Stomach: mucosa thickened Intestines: dilated Adrenals: enlarged, dark red discoloured All inner organs: autolytic
	Histopathology	Assessment not possible due to severe autolysis

Animal no./ Sex	Noteworthy observations
94 f (PS, TD 24)	Clinical signs	Salivation (slight to moderate), breathing sounds; no particular premortal symptoms
	Body weight gain	Body weight loss of 23 g (i.e. 9%) from test days 22 to 24; body weight gain reduced by approx. 40% compared to control group (test days 1-22)
	Macroscopy	External observations: abdomen dilated Stomach: cardia region: mucosa with white deposits, enlarged; dilated Intestines: dilated Duodenum: mucosa with white deposits and thickened Liver (left medial lobe): periphery with white focus (approx. 0.5 x 6 mm, sharp-edged) Liver (left lateral lobe): white focus (approx. 2 x 12 mm) Liver (right medial lobe): white focus (approx. 0.5 x 8 mm) Adrenals: enlarged, dark red discoloured
	Histopathology	Slight squamous cell hyperplasia in the forestomach, moderate multifocal necrosis in the liver, slight hepatocellular cytoplasmic alteration (eosinophilic granulation), hypertrophy of the duodenal mucosa, multifocal acute bronchiolitis in the lung and adrenal cortical hypertrophy

m: male

f:: female

PD: premature death

PS: premature sacrifice

TD: test day

For the following tables, please refer to the attached pdf documents under 'Attached background material':

Table 2: Clinical Signs - Summary and individual data

Table 3: Detailed Clinical Observations - Summary and individual data

Table 4: Functional Observations - Summary and individual data

Table 5: Grip Strength - Summary and individual data

Table 6: Spontaneous Motility - Summary and individual data

Table 7: Body Weight - Summary and individual data

Table 8: Body Weight Gain - Summary and individual data

Table 9: Body Weights at Autopsy

Table 10: Absolute Food Consumption - Summary and individual data

Table 11: Relative Food Consumption - Summary and individual data

Table 12: Haematological Parameters - Summary and individual data

Table 13: Biochemical Parameters - Summary and individual data

Table 14: Thyroid Hormone Levels - Summary and individual data

Table 15: Urinalysis - Summary and individual data

Table 16: Ophthalmological and auditory examinations

Table 17: Macroscopic Post Mortem Findings

Table 18: Relative Organ Weights - Summary and individual data

Table 19: Absolute Organ Weights - Summary and individual data

Table 20: Oestrus Cycle

Conclusions:

The subchronic (90-day) repeated dose toxicity after oral administration by gavage was investigated according to OECD guideline 408 under GLP conditions. Based on the results, the following effect levels were determined: Systemic No-Observed-Adverse-Effect-Level (NOAEL) = 300 mg/kg bw/day and local Lowest-Observed-Adverse-Effect-Level (LOAEL) = 100 mg/kg bw/day.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The available study is reliable without restrictions (Klimisch score 1), adequate and was performed with the registered substance under GLP conditions. It is thus sufficient to fulfil the standard information requirements set out in Annex IX, Section 8.6.2. of Regulation (EC) No. 1907/2006 (REACH).

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 Mar - 9 Jul 2010

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:

yes (incl. QA statement)

Remarks:

Bundesamt für Sicherheit im Gesundheitswesen, Wien, Austria

Limit test:

no

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Sulzfeld, Germany
- Age at study initiation: 8 weeks
- Weight at study initiation:
- Fasting period before study: animals were fasted overnight prior to administration.
- Housing: animals were housed in groups of 3 per sex in the dose range finding study. In the main study, animals were housed in groups of 2-3 per cage per sex in Makrolon cages Type IV with wire mesh lids.
- Diet: Ssniff R/M-H maintenance diet for rats and mice (V1534-300, Ssniff Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap water, acidified with HCL to pH ≥ 3, ad libitum
- Acclimation period: about 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.7 (average dose range finding study), 19.8 (average main study)
- Humidity (%): 59.1 (average dose range finding study), 56.6 (average main study)
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

other: ethanol

Remarks on MMAD:

MMAD / GSD: MMAD: 1.11, 1.15 and 1.22 µm (low-, mid- and high-dose)
GSD: 1.68-2.57

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: head nose only exposure unit (TSE-Systems GmbH, Bad Homburg, Germany)
- Method of holding animals in test chamber: animal exposure cage
- Source and rate of air: compressed air supply or compressed air generator
- Method of particle size determination: the size distribution of the aerosol particles was analysed with a cascade impactor (Berner-Impaktor, Type LPI40,06/2, Hauke KG, Gmunden, Austria)

TEST ATMOSPHERE
- Brief description of analytical method used: the aerosol concentration in the breathing zone was determined gravimetically.
- Samples taken from breathing zone: yes

VEHICLE
- Justification for use and choice of vehicle: the original high viscous liquid could not be pressed through the aerosol nozzle. Therefore the test substance was mixed with ethanol to decrease the viscosity. In the high-dose group the concentration was 10%. It was the target to have the same concentration of ethanol in all dose groups and in the control group. Therefore different concentrations of ethanol in the test substance were used.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The aerosol concentration in the breathing zone was determined gravimetrically. An accurately measured volume of air from the inhalation devices was sucked through a pre-weighed filter with cotton wool. The filters were dried before and after the sampling by pressing dry air through them. From the weight difference and the volume the actual aerosol concentration was calculated.

Duration of treatment / exposure:

4 weeks (main study)
4 weeks and 2 weeks post-exposure observation period (satellite control and high-dose test group)

Frequency of treatment:

6 h/day, 5 days/week

Dose / conc.:

1.2 other: mg/kg bw/day (calculated doses male animals according to Guyton´s equation)

Dose / conc.:

4 other: mg/kg bw/day (calculated doses male animals according to Guyton´s equation)

Dose / conc.:

11.6 other: mg/kg bw/day (calculated doses male animals according to Guyton´s equation)

Dose / conc.:

1.3 other: mg/kg bw/day (calculated doses female animals according to Guyton´s equation)

Dose / conc.:

4.3 other: mg/kg bw/day (calculated doses female animals according to Guyton´s equation)

Dose / conc.:

12.9 other: mg/kg bw/day (calculated doses female animals according to Guyton´s equation)

Dose / conc.:

0.006 mg/L air (nominal)

Dose / conc.:

0.02 mg/L air (nominal)

Dose / conc.:

0.06 mg/L air (nominal)

Dose / conc.:

6 mg/m³ air (nominal)

Dose / conc.:

20 mg/m³ air (nominal)

Dose / conc.:

60 mg/m³ air (nominal)

No. of animals per sex per dose:

3 (preliminary study)
5 (main study)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose ranges are based on the results of the dose range finding study. In the high-dose group (0.6 mg/L) and mid-dose group (0.19 mg/L) all animals died on the day of administration. Therefore, the dose of 0.06 mg/L, which was the low-dose in the dose range finding study, was chosen as high-dose for the main study. The low dose was set at 10% of the high dose; the mid-dose was interpolated geometrically.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were carefully observed for general signs and the health status before, during and after the exposure; on days without exposure once a day.

BODY WEIGHT: Yes
- Time schedule for examinations: Twice per week

FOOD CONSUMPTION:
- Food consumption per cage in weekly intervals in all animals. Yes

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 29 (main study groups) and Day 43 (satellite groups)
- Anaesthetic used for blood collection: Yes (slight ether anaesthesia)
- Animals fasted: Yes
- How many animals: All animals of main study and satellite groups.
- Parameters checked: red blood cell count (RBC), haemoglobin concentration (Hb), haematrocrit (Hct), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), mean cell volume (MCV), reticulocyte count (% of erythrocytes), white blood cell count (WBC), platelet count, differential white blood cell count (% of the different cell species), prothombin time (Quick) as indicator of blood clotting capacity

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 29 (main study groups) and Day 43 (satellite groups)
- Anaesthetic used for blood collection: Yes (slight ether anaesthesia)
- Animals fasted: Yes
- How many animals: All animals of main study and satellite groups.
- Parameters checked: aspartate aminotransferase, alanine aminotrasferase, albumin, alkaline phosphatase, bilirubin, calcium, chloride, cholesterol, creatinine, gamma glytamyl transferase, globulin, glucose, phosphorus, potassium (K+), sodium (NA+), total protein, triglycerides, urea

Sacrifice and pathology:

GROSS PATHOLOGY: Yes. Animals were killed and subjected to gross pathological examination immediately after death.
HISTOPATHOLOGY: Yes. Gross lesions, tissue masses or tumours, adrenal glands, brain (including cerebrum, cerebellum and pons), eyes, heart, kidneys, larynx (3 levels, including the base of the epiglottis), liver, lungs (all lobes at one level, including main bronchi), lymph nodes (hilar region of the lungs), nasopharyngeal tissues (4 levels), oesophagus, ovaries, seminal vesicles, spinal cord (cervical, thoracal, lumbar), spleen, sternum with bone marrow, stomach, testes, thymus, thyroid glands, trachea (longitudinal section through the carna and 1 transverse section), uterus

Statistics:

Analysis of variance followed by the Scheffe-test was used for all data with means and standard deviations with comparison of more than two groups. The t-test was used for all data with means and standard deviations for comparison of two groups only. H-test of Kruskal and Wallis followed by the test of Nemenyi counted events with scoring or in cases where the requirements for the analysis of variance were not fulfilled. Chi2-test was used for counted events. Fisher´s exact test was used for counted events, if the Chi2-test was not applicable.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

mid- and high dose males and females and female control group: chromodakryorrhoea; high dose satellite group males and low- and high-dose group females: reduced well-being

Mortality:

mortality observed, treatment-related

Description (incidence):

mid- and high dose males and females and female control group: chromodakryorrhoea; high dose satellite group males and low- and high-dose group females: reduced well-being

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

high-dose groups: generally lower body weight gain

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

high-dose males: increased MCV, high-dose males after recovery: reduced WBC, lymphocytes; mid-dose females: decreased reticulocyte count, non adverse

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

low-dose females: decreased chloride, increased phosphor; high-dose females increased AP and ALT; low-dose females: increased ALT, all females: decreased CHOL during dosing, increased at the end of the recovery period

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

significant changes in organ weights are summarized in Table 3 under "Any other information on results incl. tables".

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

adaptive responses to the deposits of the test substance and the resulting irritation (squamous metaplasia and epithelium proliferation and submucous acute inflammation at the basis of the epiglottis).

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
All animals survived until the end of the study period. Chromodakryorrhoe occurred in male animals of the mid and high dose groups. This finding was also observed in females of the mid- and high-dose and control group. The findings may be caused by the stress during the administration procedure.
Signs of reduced well-being were noted in male animals of the high dose satellite group and in females of the low and high dose group. Stress can also be the cause for these findings. The effects were seen during the administration period and were not observed during the recovery period.

BODY WEIGHT AND WEIGHT GAIN
Body weight and body weight gain were lower in the high dose groups. In the mid dose group an effect was observed only in male animals and only at the end of the administration period (see Table 1 under “Any other information on results incl. tables”).

FOOD CONSUMPTION
No significant effects on food consumption were noted during the study period.

HAEMATOLOGY
Changes in the MCV, WBC and lymphocytes in male animals of the high dose groups were observed. In female animals of the mid-dose group reticulocyte counts were significantly reduced. The effects may be random events, because no other parameters are impaired (see Table 2 under “Any other information on results incl. tables”).

CLINICAL CHEMISTRY
No significant effects on clinical chemistry parameters were observed in male animals.
In females of the low-dose group increased phosphor, decreased cholesterol and increased AST was apparent. In the mid-dose group the cholesterol level was decreased as well. In high-dose females chloride level was decreased and normal at the end of the recovery period. Cholesterol was decreased during the dosing period and increased at the end of the recovery period in high-dose females. AP and ALT were statistically significant increased in the high dose group and normal after the recovery period. The changes of cholesterol in all females dosing groups may be caused by the test substance. However, corresponding damages of the liver were not found in the animals at necropsy, histopathology and in organ weights.
All other parameters and statistically calculated significances do not show a dose response relationship and/or are very small changes and are therefore assumed to be random events without toxicological importance (see Table 2 under “Any other information on results incl. tables”).

ORGAN WEIGHTS
Different significant changes in organs weights in the mid- and high-dose male animals were observed (see Table 3 under “Any other information on results incl. tables”). The changes in testes were also seen in the high-dose satellite group and can therefore be considered as not reversible. Changes in spleen were noted in the high-dose satellite group only. The author assumed that such an effect may be test substance related, and occur only after the end of the administration period.
In female animals relevant changes were noted in the high-dose group only. The changed parameters and statistically calculated significances in female animals do not show a dose-relationship. Therefore, the changes are considered to be random without toxicological importance.
In general the increase in relative organ weights may be associated with the reduced body weight and body weight gain.

GROSS PATHOLOGY
No test substance related changes were observed at the gross examination.

HISTOPATHOLOGY: NON-NEOPLASTIC
The test substance aerosol caused several effects in the respiratory system, in lungs and bronchi. Most effects were adaptive responses to the deposits of the test substance and the resulting irritation (squamous metaplasia and epithelium proliferation and submucous acute inflammation at the basis of the epiglottis). In addition focal early stage of fibrosis was noted at the alveoli walls mainly in the groups treated with the test substance. Due to the high amount of the test substance deposits in the lungs especially in the mid and high dose groups these changes may be explained by overloading the tissue and not necesarrily imply an intrinsic toxicity of the test material. The author considered an intrinsic toxicity as unlikely as the test material is insoluble and the shape of the particles is not fibrous.
Some other findings were considered as unspecific and of no toxicological relevance. However, most findings were observed in the high-dose groups.

OTHER FINDINGS
RESULTS OF THE DOSE RANGE FINDING STUDY
All animals of the mid and high dose group died on Day 1 acutely still during the exposition and/or directly thereafter (aerosol concentration 0.19 mg/L and 0.6 mg/L, respectively). No indication for a test substance related alteration in animals of the low dose group was noted (aerosol concentration 0.06 mg/L)). Body weight was lower in the high dose groups. In the mid dose group an effect was observed only in male animals and only at the end of the administration period. There were no statistically significant changes in the low dose groups in body weight, feed consumption and at necropsy.

AEROSOL CONCENTRATION
The mean actual aerosol concentrations were 0.006, 0.02 and 0.059 mg aerosol per liter air for the low, mid- and high-dose groups. The values were converted to the inhaled amount (dose) using the equation: minute_volume (L/min) = body weight (kg)^0.75 x 0.373 (Guyton´s equation) for the breathing volume of the animals.
The calculation gave doses of 11.6/12.9, 4.0/4.3 and 1.2/1.3 mg/kg/day for the high-, mid- and low-dose groups (males/females).
The inhaled amount of ethanol in every group is expected to be about 1/10 of the test substance amount (1 mg/kg/day in the high-dose group).

Dose descriptor:

NOEC

Effect level:

< 0.006 mg/L air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: effects on the respiratory system with indications for a local irritation

Key result

Dose descriptor:

NOAEC

Effect level:

> 0.06 mg/L air

Based on:

test mat.

Sex:

male

Basis for effect level:

other: effects on organ weight without corresponding effects in histopathological examination and without dose-relationship

Critical effects observed:

not specified

Table 1. Body weight (g) presented as mean ± SD (males and females).

Dose Level	Body weight (g) on Days / Males
	1	12	15	19	22	26	28	33	36	40	42
Control	205 ± 8.73	221 ± 13.3	218 ± 14.1	232 ± 14.0	229 ± 13.2	240 ± 13.0	237 ± 14.6	-	-	-	-
Control satellite	203 ± 3.21	245 ± 2.45	215 ± 2.92	227 ± 2.88	225± 1.82	236 ± 1.95	232 ± 0.84	246 ± 3.11	254 ± 5.39	265 ± 5.89	265 ± 6.06
Low-dose	200 ± 9.18	212 ± 9.42	210 ± 8.5	221 ±10.4	216 ± 7.29	226 ± 7.76	221 ± 8.79	-	-	-	-
Mid-dose	200 ± 6.46	210 ± 5.07	204 ± 4.76	219 ± 4.83	212 ± 4.47	225 ± 4.97	216 ± 5.63*	-	-	-	-
High-dose	200 ± 9.5	206 ± 8.7	200 ± 9.07	209 ± 9.18*	200 ± 8.41*	211 ± 8.94*	202 ± 7.23*	-	-	-	-
High-dose satellite	202 ± 7.96	207 ± 8.83	199 ± 9.36±	212 ± 9.64*	202 ± 8.58*	213 ± 10.8*	203 ± 8.29*	223 ± 10.4*	234 ± 12.1*	247 ± 13.1*	247 ± 16.4

Dose Level	Body weight (g) on Days / Females
	1	22	26	28	33	36	40	42
Control	140 ± 3.13	149 ± 4.49	153 ± 4.51	152 ± 5.41	-	-	-	-
Control satellite	137 ± 3.16	146 ± 4.1	152 ± 5.59	150 ± 4.87	158 ± 5.5	162 ± 6.38	168 ± 7.8	166 ± 9.13
Low-dose	138 ± 4.27	142 ± 9.15	146 ± 9.2	143 ± 9.02	-	-	-	-
Mid-dose	141 ± 4.82	145± 2.95	151 ± 2.7	147 ± 4.04	-	-	-	-
High-dose	138 ± 4.87	136 ± 5.1*	142 ± 4.3*	136 ± 4.97*	-	-	-	-
High-dose satellite	140 ± 6.19	138 ± 5.85*	141 ± 8.17*	138 ± 5.97*	148 ± 6.91*	153 ± 6.8	164 ± 7.3	162 ± 6.73

*: significant different from control group p < 0.05

Table 2. Mean haematological data and clinical chemistry (only significant changes are given).

Parameter		Results of statistical calculation
	Males						Females
	Control	Control satellite	High dose	High dose satellite	Control	Control satellite	Low dose	Mid dose	High dose	High dose satellite
MCV (mean corpuscular volume fL)	47.3 ± 0.95	46.9 ± 0.17	49.3 ± 1.33*	47.1 ± 0.83	-	-	-	-	-	-
WBC (white blood cell count, 1/nL)	7.82 ± 1.73	6.12 ± 1.3	5.66 ± 0.49	4.89 ± 0.49*	-	-	-	-	-	-
Lymphocytes (% of leukocytes or n 1/nL)	5.37 ± 1.28	4.42 ± 1.32	3.56 ± 0.47	3.21 ± 0.24*	-	-	-	-	-	-
Reticulocyte count (%), males	-	-	-	-	3.69 ± 0.36	-	-	2.58 ± 0.33*	-	-
Chlorid (mmol/L)	-	-	-	-	99.9 ± 0.72	99.7 ± 1.19	-	-	98.2 ± 0.77*	100 ± 1.07
Phosphor (mmol/L)	-	--	-	-	2.53 ± 0.23	-	3.09 ± 0.43*	-	-	-
Cholesterol (mmol/L)	-	-	-	-	2.37 ± 0.15	2.34 ± 0.06	1.90 ± 0.26*	1.87 ± 0.27*	1.83 ± 0.14*	2.6 ± 0.2*
AP (alkaline phosphatase, U/L)	-	-	-	-	118 ± 5.34	100 ± 7.6	-	-	138 ± 7.97*	103 ± 4.04
AST (aspartate aminotransferase, U/L)	-	-	-	-	92.2 ± 2.68	91.4 ± 7.47	105 ± 6.88*	-	-	-
ALT (alanin aminotransferase, U/L)	-	-	-	-	61.4 ± 6.43	64.4 ± 2.88	-	-	75.4 ± 7.99*	62.2 ± 1.79

*: significant different from control group p < 0.05

Table 3. Mean organ weights. Only significant changes are given.

Parameter	Results of statistical calculation
	Males				Females
	Control / Control satellite	Mid dose	High dose	High dose satellite	Control / Control satellite	High dose	High dose satellite
Absolute organ weights (mg)
Testes	2283 ±185 1966 ± 162	2716 ± 123*	2771 ± 42.4 *	2651 ± 204*	-	-	-
Spleen	512 ± 66.7 645 ± 48.4	-	-	531 ± 59.2*	-	-	-
Lung	-	-	-	-	1173 ± 118 926 ± 76.9	-	1104 ±154*
Thymus	291 ± 60.7 313 ± 51.5	186 ± 17.9*	188 ± 17.9*	261 ± 51.9	-	-	-
Organ weight/body weight ratios (mg/g)
Brain	7.86± 0.55 7.42 ± 0.19	5.66 ± 0.37*	9.0 ± 0.23*	7.61 ± 0.44	11.2 ± 0.74 10.4 ± 0.31	12.9 ± 0.46*	10.6 ± 0.46
Lung	-	-	-	-	7.69 ± 0.68 5.57 ± 0.47	-	6.79 ± 0.86*
Heart	3.42 ± 0.43 3.51 ± 0.22	-	3.97 ± 0.23*	3.68 ± 0.35	3.88 ± 0.11 3.38 ± 0.26	4.27 ± 0.18*	3.81 ± 0.25
Testes	9.64 ± 0.43 7.41 ± 0.19	12.6 ± 0.76*	13.7 ± 0.3*	10.8 ± 1.13*	-	-	-
Spleen	2.17 ± 0.29 2.43 ± 0.16	-	-	2.14 ± 0.18*	-	-	-
Kidneys	6.86 ± 0.65 6.65 ± 0.62	-	8.01 ± 0.53*	6.6 ± 0.52	8.08 ± 0.41 6.43 ± 0.35	-	7.28 ± 0.66*
Thymus	1.22 ± 0.18	0.86 ± 0.06*	-	-	-	-	-
Organ weight/brain weight ratios (mg/g)
Heart	-	-	-	-	-	-	-
Spleen	277 ± 41.1 325 ± 23.5	-	-	282 ± 24.5*	-	-	-
Thymus	157 ± 32.7 159 ± 24.9	99.4 ± 8.15*	103 ± 23.9*	139 ± 29.0	-	-	-
Kidneys	-		-	-	719 ± 31.3 618 ± 20.1	-	685 ± 53.9*
Testes	1231 ± 92.5 1000 ± 93.8	1456 ± 75.4*	1522 ± 41.8*	1415 ± 47*	-	-	-

*: significant different from control group p < 0.05

Conclusions:

The used concentration range of the study was chosen due to a range finding test. The aerosol of the test substance caused effects at several sites of the respiratory system at concentrations of 0.006 to 0.060 mg/L with indications for a local irritation. The alterations may be explained by the amounts of inert material deposited within the respiratory system. In the lungs and bronchi the most prominent finding was a focal early stage of fibrosis. Due to the high amount of test substance deposits in the lungs especially in the mid and high dosed groups these changes may be explained by simply overloading the tissue and do not necessarily imply an intrinsic toxicity of the test material. An intrinsic toxicity is unlikely as the test material is insoluble and the shape of the particles is not fibrous.
Alterations in clinical biochemistry were only seen in female animals. Beside this and the increased absolute and relative organ weights of testes in male animals there was no other substantially sex difference in the response to the test substance. Furthermore, the differences did not show a dose relationship and no correlating effects on the respective organ were seen in the histopathological examinations.
The observed effects were at most mild in severity, never became life threatening, but persisted partly until the end of the recovery period. Furthermore, due to the self-warning characteristics (immediate irritant reactions to the test substance if inhaled), high substance concentrations of inhalation may be avoided.
The No-observed-effect-concentration (NOEC) for local irritation of the test material via inhalation was smaller than 0.006 mg/L. There was however no life threatening systemic effect present up to the highest concentration tested. Therefore, the systemic NOAEC for the test material will be > 0.06 mg/L.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

60 mg/m³

Study duration:

subacute

Species:

rat

Quality of whole database:

The available study is reliable without restrictions (Klimisch score 1), adequate and was performed with the registered substance under GLP conditions. It is thus sufficient to fulfil the standard information requirements set out in Annex VIII, Section 8.5, of Regulation (EC) No. 1907/2006 (REACH).

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 Mar - 9 Jul 2010

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:

yes (incl. QA statement)

Remarks:

Bundesamt für Sicherheit im Gesundheitswesen, Wien, Austria

Limit test:

no

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Sulzfeld, Germany
- Age at study initiation: 8 weeks
- Weight at study initiation:
- Fasting period before study: animals were fasted overnight prior to administration.
- Housing: animals were housed in groups of 3 per sex in the dose range finding study. In the main study, animals were housed in groups of 2-3 per cage per sex in Makrolon cages Type IV with wire mesh lids.
- Diet: Ssniff R/M-H maintenance diet for rats and mice (V1534-300, Ssniff Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap water, acidified with HCL to pH ≥ 3, ad libitum
- Acclimation period: about 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.7 (average dose range finding study), 19.8 (average main study)
- Humidity (%): 59.1 (average dose range finding study), 56.6 (average main study)
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

other: ethanol

Remarks on MMAD:

MMAD / GSD: MMAD: 1.11, 1.15 and 1.22 µm (low-, mid- and high-dose)
GSD: 1.68-2.57

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: head nose only exposure unit (TSE-Systems GmbH, Bad Homburg, Germany)
- Method of holding animals in test chamber: animal exposure cage
- Source and rate of air: compressed air supply or compressed air generator
- Method of particle size determination: the size distribution of the aerosol particles was analysed with a cascade impactor (Berner-Impaktor, Type LPI40,06/2, Hauke KG, Gmunden, Austria)

TEST ATMOSPHERE
- Brief description of analytical method used: the aerosol concentration in the breathing zone was determined gravimetically.
- Samples taken from breathing zone: yes

VEHICLE
- Justification for use and choice of vehicle: the original high viscous liquid could not be pressed through the aerosol nozzle. Therefore the test substance was mixed with ethanol to decrease the viscosity. In the high-dose group the concentration was 10%. It was the target to have the same concentration of ethanol in all dose groups and in the control group. Therefore different concentrations of ethanol in the test substance were used.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The aerosol concentration in the breathing zone was determined gravimetrically. An accurately measured volume of air from the inhalation devices was sucked through a pre-weighed filter with cotton wool. The filters were dried before and after the sampling by pressing dry air through them. From the weight difference and the volume the actual aerosol concentration was calculated.

Duration of treatment / exposure:

4 weeks (main study)
4 weeks and 2 weeks post-exposure observation period (satellite control and high-dose test group)

Frequency of treatment:

6 h/day, 5 days/week

Dose / conc.:

1.2 other: mg/kg bw/day (calculated doses male animals according to Guyton´s equation)

Dose / conc.:

4 other: mg/kg bw/day (calculated doses male animals according to Guyton´s equation)

Dose / conc.:

11.6 other: mg/kg bw/day (calculated doses male animals according to Guyton´s equation)

Dose / conc.:

1.3 other: mg/kg bw/day (calculated doses female animals according to Guyton´s equation)

Dose / conc.:

4.3 other: mg/kg bw/day (calculated doses female animals according to Guyton´s equation)

Dose / conc.:

12.9 other: mg/kg bw/day (calculated doses female animals according to Guyton´s equation)

Dose / conc.:

0.006 mg/L air (nominal)

Dose / conc.:

0.02 mg/L air (nominal)

Dose / conc.:

0.06 mg/L air (nominal)

Dose / conc.:

6 mg/m³ air (nominal)

Dose / conc.:

20 mg/m³ air (nominal)

Dose / conc.:

60 mg/m³ air (nominal)

No. of animals per sex per dose:

3 (preliminary study)
5 (main study)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose ranges are based on the results of the dose range finding study. In the high-dose group (0.6 mg/L) and mid-dose group (0.19 mg/L) all animals died on the day of administration. Therefore, the dose of 0.06 mg/L, which was the low-dose in the dose range finding study, was chosen as high-dose for the main study. The low dose was set at 10% of the high dose; the mid-dose was interpolated geometrically.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were carefully observed for general signs and the health status before, during and after the exposure; on days without exposure once a day.

BODY WEIGHT: Yes
- Time schedule for examinations: Twice per week

FOOD CONSUMPTION:
- Food consumption per cage in weekly intervals in all animals. Yes

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 29 (main study groups) and Day 43 (satellite groups)
- Anaesthetic used for blood collection: Yes (slight ether anaesthesia)
- Animals fasted: Yes
- How many animals: All animals of main study and satellite groups.
- Parameters checked: red blood cell count (RBC), haemoglobin concentration (Hb), haematrocrit (Hct), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), mean cell volume (MCV), reticulocyte count (% of erythrocytes), white blood cell count (WBC), platelet count, differential white blood cell count (% of the different cell species), prothombin time (Quick) as indicator of blood clotting capacity

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 29 (main study groups) and Day 43 (satellite groups)
- Anaesthetic used for blood collection: Yes (slight ether anaesthesia)
- Animals fasted: Yes
- How many animals: All animals of main study and satellite groups.
- Parameters checked: aspartate aminotransferase, alanine aminotrasferase, albumin, alkaline phosphatase, bilirubin, calcium, chloride, cholesterol, creatinine, gamma glytamyl transferase, globulin, glucose, phosphorus, potassium (K+), sodium (NA+), total protein, triglycerides, urea

Sacrifice and pathology:

GROSS PATHOLOGY: Yes. Animals were killed and subjected to gross pathological examination immediately after death.
HISTOPATHOLOGY: Yes. Gross lesions, tissue masses or tumours, adrenal glands, brain (including cerebrum, cerebellum and pons), eyes, heart, kidneys, larynx (3 levels, including the base of the epiglottis), liver, lungs (all lobes at one level, including main bronchi), lymph nodes (hilar region of the lungs), nasopharyngeal tissues (4 levels), oesophagus, ovaries, seminal vesicles, spinal cord (cervical, thoracal, lumbar), spleen, sternum with bone marrow, stomach, testes, thymus, thyroid glands, trachea (longitudinal section through the carna and 1 transverse section), uterus

Statistics:

Analysis of variance followed by the Scheffe-test was used for all data with means and standard deviations with comparison of more than two groups. The t-test was used for all data with means and standard deviations for comparison of two groups only. H-test of Kruskal and Wallis followed by the test of Nemenyi counted events with scoring or in cases where the requirements for the analysis of variance were not fulfilled. Chi2-test was used for counted events. Fisher´s exact test was used for counted events, if the Chi2-test was not applicable.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

mid- and high dose males and females and female control group: chromodakryorrhoea; high dose satellite group males and low- and high-dose group females: reduced well-being

Mortality:

mortality observed, treatment-related

Description (incidence):

mid- and high dose males and females and female control group: chromodakryorrhoea; high dose satellite group males and low- and high-dose group females: reduced well-being

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

high-dose groups: generally lower body weight gain

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

high-dose males: increased MCV, high-dose males after recovery: reduced WBC, lymphocytes; mid-dose females: decreased reticulocyte count, non adverse

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

low-dose females: decreased chloride, increased phosphor; high-dose females increased AP and ALT; low-dose females: increased ALT, all females: decreased CHOL during dosing, increased at the end of the recovery period

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

significant changes in organ weights are summarized in Table 3 under "Any other information on results incl. tables".

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

adaptive responses to the deposits of the test substance and the resulting irritation (squamous metaplasia and epithelium proliferation and submucous acute inflammation at the basis of the epiglottis).

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
All animals survived until the end of the study period. Chromodakryorrhoe occurred in male animals of the mid and high dose groups. This finding was also observed in females of the mid- and high-dose and control group. The findings may be caused by the stress during the administration procedure.
Signs of reduced well-being were noted in male animals of the high dose satellite group and in females of the low and high dose group. Stress can also be the cause for these findings. The effects were seen during the administration period and were not observed during the recovery period.

BODY WEIGHT AND WEIGHT GAIN
Body weight and body weight gain were lower in the high dose groups. In the mid dose group an effect was observed only in male animals and only at the end of the administration period (see Table 1 under “Any other information on results incl. tables”).

FOOD CONSUMPTION
No significant effects on food consumption were noted during the study period.

HAEMATOLOGY
Changes in the MCV, WBC and lymphocytes in male animals of the high dose groups were observed. In female animals of the mid-dose group reticulocyte counts were significantly reduced. The effects may be random events, because no other parameters are impaired (see Table 2 under “Any other information on results incl. tables”).

CLINICAL CHEMISTRY
No significant effects on clinical chemistry parameters were observed in male animals.
In females of the low-dose group increased phosphor, decreased cholesterol and increased AST was apparent. In the mid-dose group the cholesterol level was decreased as well. In high-dose females chloride level was decreased and normal at the end of the recovery period. Cholesterol was decreased during the dosing period and increased at the end of the recovery period in high-dose females. AP and ALT were statistically significant increased in the high dose group and normal after the recovery period. The changes of cholesterol in all females dosing groups may be caused by the test substance. However, corresponding damages of the liver were not found in the animals at necropsy, histopathology and in organ weights.
All other parameters and statistically calculated significances do not show a dose response relationship and/or are very small changes and are therefore assumed to be random events without toxicological importance (see Table 2 under “Any other information on results incl. tables”).

ORGAN WEIGHTS
Different significant changes in organs weights in the mid- and high-dose male animals were observed (see Table 3 under “Any other information on results incl. tables”). The changes in testes were also seen in the high-dose satellite group and can therefore be considered as not reversible. Changes in spleen were noted in the high-dose satellite group only. The author assumed that such an effect may be test substance related, and occur only after the end of the administration period.
In female animals relevant changes were noted in the high-dose group only. The changed parameters and statistically calculated significances in female animals do not show a dose-relationship. Therefore, the changes are considered to be random without toxicological importance.
In general the increase in relative organ weights may be associated with the reduced body weight and body weight gain.

GROSS PATHOLOGY
No test substance related changes were observed at the gross examination.

HISTOPATHOLOGY: NON-NEOPLASTIC
The test substance aerosol caused several effects in the respiratory system, in lungs and bronchi. Most effects were adaptive responses to the deposits of the test substance and the resulting irritation (squamous metaplasia and epithelium proliferation and submucous acute inflammation at the basis of the epiglottis). In addition focal early stage of fibrosis was noted at the alveoli walls mainly in the groups treated with the test substance. Due to the high amount of the test substance deposits in the lungs especially in the mid and high dose groups these changes may be explained by overloading the tissue and not necesarrily imply an intrinsic toxicity of the test material. The author considered an intrinsic toxicity as unlikely as the test material is insoluble and the shape of the particles is not fibrous.
Some other findings were considered as unspecific and of no toxicological relevance. However, most findings were observed in the high-dose groups.

OTHER FINDINGS
RESULTS OF THE DOSE RANGE FINDING STUDY
All animals of the mid and high dose group died on Day 1 acutely still during the exposition and/or directly thereafter (aerosol concentration 0.19 mg/L and 0.6 mg/L, respectively). No indication for a test substance related alteration in animals of the low dose group was noted (aerosol concentration 0.06 mg/L)). Body weight was lower in the high dose groups. In the mid dose group an effect was observed only in male animals and only at the end of the administration period. There were no statistically significant changes in the low dose groups in body weight, feed consumption and at necropsy.

AEROSOL CONCENTRATION
The mean actual aerosol concentrations were 0.006, 0.02 and 0.059 mg aerosol per liter air for the low, mid- and high-dose groups. The values were converted to the inhaled amount (dose) using the equation: minute_volume (L/min) = body weight (kg)^0.75 x 0.373 (Guyton´s equation) for the breathing volume of the animals.
The calculation gave doses of 11.6/12.9, 4.0/4.3 and 1.2/1.3 mg/kg/day for the high-, mid- and low-dose groups (males/females).
The inhaled amount of ethanol in every group is expected to be about 1/10 of the test substance amount (1 mg/kg/day in the high-dose group).

Dose descriptor:

NOEC

Effect level:

< 0.006 mg/L air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: effects on the respiratory system with indications for a local irritation

Key result

Dose descriptor:

NOAEC

Effect level:

> 0.06 mg/L air

Based on:

test mat.

Sex:

male

Basis for effect level:

other: effects on organ weight without corresponding effects in histopathological examination and without dose-relationship

Critical effects observed:

not specified

Table 1. Body weight (g) presented as mean ± SD (males and females).

Dose Level	Body weight (g) on Days / Males
	1	12	15	19	22	26	28	33	36	40	42
Control	205 ± 8.73	221 ± 13.3	218 ± 14.1	232 ± 14.0	229 ± 13.2	240 ± 13.0	237 ± 14.6	-	-	-	-
Control satellite	203 ± 3.21	245 ± 2.45	215 ± 2.92	227 ± 2.88	225± 1.82	236 ± 1.95	232 ± 0.84	246 ± 3.11	254 ± 5.39	265 ± 5.89	265 ± 6.06
Low-dose	200 ± 9.18	212 ± 9.42	210 ± 8.5	221 ±10.4	216 ± 7.29	226 ± 7.76	221 ± 8.79	-	-	-	-
Mid-dose	200 ± 6.46	210 ± 5.07	204 ± 4.76	219 ± 4.83	212 ± 4.47	225 ± 4.97	216 ± 5.63*	-	-	-	-
High-dose	200 ± 9.5	206 ± 8.7	200 ± 9.07	209 ± 9.18*	200 ± 8.41*	211 ± 8.94*	202 ± 7.23*	-	-	-	-
High-dose satellite	202 ± 7.96	207 ± 8.83	199 ± 9.36±	212 ± 9.64*	202 ± 8.58*	213 ± 10.8*	203 ± 8.29*	223 ± 10.4*	234 ± 12.1*	247 ± 13.1*	247 ± 16.4

Dose Level	Body weight (g) on Days / Females
	1	22	26	28	33	36	40	42
Control	140 ± 3.13	149 ± 4.49	153 ± 4.51	152 ± 5.41	-	-	-	-
Control satellite	137 ± 3.16	146 ± 4.1	152 ± 5.59	150 ± 4.87	158 ± 5.5	162 ± 6.38	168 ± 7.8	166 ± 9.13
Low-dose	138 ± 4.27	142 ± 9.15	146 ± 9.2	143 ± 9.02	-	-	-	-
Mid-dose	141 ± 4.82	145± 2.95	151 ± 2.7	147 ± 4.04	-	-	-	-
High-dose	138 ± 4.87	136 ± 5.1*	142 ± 4.3*	136 ± 4.97*	-	-	-	-
High-dose satellite	140 ± 6.19	138 ± 5.85*	141 ± 8.17*	138 ± 5.97*	148 ± 6.91*	153 ± 6.8	164 ± 7.3	162 ± 6.73

*: significant different from control group p < 0.05

Table 2. Mean haematological data and clinical chemistry (only significant changes are given).

Parameter		Results of statistical calculation
	Males						Females
	Control	Control satellite	High dose	High dose satellite	Control	Control satellite	Low dose	Mid dose	High dose	High dose satellite
MCV (mean corpuscular volume fL)	47.3 ± 0.95	46.9 ± 0.17	49.3 ± 1.33*	47.1 ± 0.83	-	-	-	-	-	-
WBC (white blood cell count, 1/nL)	7.82 ± 1.73	6.12 ± 1.3	5.66 ± 0.49	4.89 ± 0.49*	-	-	-	-	-	-
Lymphocytes (% of leukocytes or n 1/nL)	5.37 ± 1.28	4.42 ± 1.32	3.56 ± 0.47	3.21 ± 0.24*	-	-	-	-	-	-
Reticulocyte count (%), males	-	-	-	-	3.69 ± 0.36	-	-	2.58 ± 0.33*	-	-
Chlorid (mmol/L)	-	-	-	-	99.9 ± 0.72	99.7 ± 1.19	-	-	98.2 ± 0.77*	100 ± 1.07
Phosphor (mmol/L)	-	--	-	-	2.53 ± 0.23	-	3.09 ± 0.43*	-	-	-
Cholesterol (mmol/L)	-	-	-	-	2.37 ± 0.15	2.34 ± 0.06	1.90 ± 0.26*	1.87 ± 0.27*	1.83 ± 0.14*	2.6 ± 0.2*
AP (alkaline phosphatase, U/L)	-	-	-	-	118 ± 5.34	100 ± 7.6	-	-	138 ± 7.97*	103 ± 4.04
AST (aspartate aminotransferase, U/L)	-	-	-	-	92.2 ± 2.68	91.4 ± 7.47	105 ± 6.88*	-	-	-
ALT (alanin aminotransferase, U/L)	-	-	-	-	61.4 ± 6.43	64.4 ± 2.88	-	-	75.4 ± 7.99*	62.2 ± 1.79

*: significant different from control group p < 0.05

Table 3. Mean organ weights. Only significant changes are given.

Parameter	Results of statistical calculation
	Males				Females
	Control / Control satellite	Mid dose	High dose	High dose satellite	Control / Control satellite	High dose	High dose satellite
Absolute organ weights (mg)
Testes	2283 ±185 1966 ± 162	2716 ± 123*	2771 ± 42.4 *	2651 ± 204*	-	-	-
Spleen	512 ± 66.7 645 ± 48.4	-	-	531 ± 59.2*	-	-	-
Lung	-	-	-	-	1173 ± 118 926 ± 76.9	-	1104 ±154*
Thymus	291 ± 60.7 313 ± 51.5	186 ± 17.9*	188 ± 17.9*	261 ± 51.9	-	-	-
Organ weight/body weight ratios (mg/g)
Brain	7.86± 0.55 7.42 ± 0.19	5.66 ± 0.37*	9.0 ± 0.23*	7.61 ± 0.44	11.2 ± 0.74 10.4 ± 0.31	12.9 ± 0.46*	10.6 ± 0.46
Lung	-	-	-	-	7.69 ± 0.68 5.57 ± 0.47	-	6.79 ± 0.86*
Heart	3.42 ± 0.43 3.51 ± 0.22	-	3.97 ± 0.23*	3.68 ± 0.35	3.88 ± 0.11 3.38 ± 0.26	4.27 ± 0.18*	3.81 ± 0.25
Testes	9.64 ± 0.43 7.41 ± 0.19	12.6 ± 0.76*	13.7 ± 0.3*	10.8 ± 1.13*	-	-	-
Spleen	2.17 ± 0.29 2.43 ± 0.16	-	-	2.14 ± 0.18*	-	-	-
Kidneys	6.86 ± 0.65 6.65 ± 0.62	-	8.01 ± 0.53*	6.6 ± 0.52	8.08 ± 0.41 6.43 ± 0.35	-	7.28 ± 0.66*
Thymus	1.22 ± 0.18	0.86 ± 0.06*	-	-	-	-	-
Organ weight/brain weight ratios (mg/g)
Heart	-	-	-	-	-	-	-
Spleen	277 ± 41.1 325 ± 23.5	-	-	282 ± 24.5*	-	-	-
Thymus	157 ± 32.7 159 ± 24.9	99.4 ± 8.15*	103 ± 23.9*	139 ± 29.0	-	-	-
Kidneys	-		-	-	719 ± 31.3 618 ± 20.1	-	685 ± 53.9*
Testes	1231 ± 92.5 1000 ± 93.8	1456 ± 75.4*	1522 ± 41.8*	1415 ± 47*	-	-	-

*: significant different from control group p < 0.05

Conclusions:

The used concentration range of the study was chosen due to a range finding test. The aerosol of the test substance caused effects at several sites of the respiratory system at concentrations of 0.006 to 0.060 mg/L with indications for a local irritation. The alterations may be explained by the amounts of inert material deposited within the respiratory system. In the lungs and bronchi the most prominent finding was a focal early stage of fibrosis. Due to the high amount of test substance deposits in the lungs especially in the mid and high dosed groups these changes may be explained by simply overloading the tissue and do not necessarily imply an intrinsic toxicity of the test material. An intrinsic toxicity is unlikely as the test material is insoluble and the shape of the particles is not fibrous.
Alterations in clinical biochemistry were only seen in female animals. Beside this and the increased absolute and relative organ weights of testes in male animals there was no other substantially sex difference in the response to the test substance. Furthermore, the differences did not show a dose relationship and no correlating effects on the respective organ were seen in the histopathological examinations.
The observed effects were at most mild in severity, never became life threatening, but persisted partly until the end of the recovery period. Furthermore, due to the self-warning characteristics (immediate irritant reactions to the test substance if inhaled), high substance concentrations of inhalation may be avoided.
The No-observed-effect-concentration (NOEC) for local irritation of the test material via inhalation was smaller than 0.006 mg/L. There was however no life threatening systemic effect present up to the highest concentration tested. Therefore, the systemic NOAEC for the test material will be > 0.06 mg/L.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEC

6 mg/m³

Study duration:

subacute

Species:

rat

Quality of whole database:

The available study is reliable without restrictions (Klimisch score 1), adequate and was performed with the registered substance under GLP conditions. It is thus sufficient to fulfil the standard information requirements set out in Annex VIII, Section 8.5, of Regulation (EC) No. 1907/2006 (REACH).

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

Repeated dose toxicity oral

The subchronic (90-day) repeated dose toxicity of N-methyl-N-(C18-(unsaturated)alkanoyl)glycine (EC No. 701-177-3) was investigated in a study according to OECD guideline 408 and observing GLP provisions (LPT, 2020a). Groups of 10 Sprague-Dawley Crl:CD (SD) rats per dose and sex were administered the test substance at doses of 100, 300 and 1000/750/600 mg/kg bw/day for a period of 90 consecutive days by gavage. The initial dose level of the high-dose group was reduced to 750 mg/kg bw in a first step and then to 600 mg/kg bw due to observed morbidity/mortality. The doses were based on an appropriate dose-range finding study. 0.5% aqueous hydroxylpropyl methyl-cellulose gel was used as vehicle and doses were administered at a constant volume of 10 mL/kg bw/day. Additional satellite control and high-dose groups with 5 animals per sex were included in the study in order to investigate the reversibility of effects after a 28-day post-exposure recovery period.

Test substance-related morbidity/mortality was observed for 1/20 animals in the low-dose and for 4/30 animals of the high-dose groups as a result of local changes to the stomach. A decreased body weight gain was noted for the mid- and high-dose group animals. Treatment with 1000/750/600 mg/kg bw/day led to a decreased food intake during the treatment period. The following test substance-related changes were noted for males and females in the mid- and high-dose groups: clinical signs (salivation and breathing sounds), changes in biochemical parameters, an increased T4 serum level and increased liver weights. The macroscopic inspection at necropsy revealed visible changes in the stomach of male and female animals in form of a thickened mucosa (partly associated with yellow or white deposits on the mucosa). The histopathological examination revealed local test substance-related microscopic changes in the stomach of male and female animals of all treatment groups in form of squamous cell hyperplasia with hyperkeratosis, minimal to slight erosions/ulcerations and submucosal oedema. An increase in cytoplasmic eosinophilic granulation (resulting in an increase in overall cellular eosinophilia) was observed in the liver of males and females of the mid- and high-dose groups. Since this cytoplasmic change was not accompanied by related clinical and/or other histopathologic changes, the change is considered not to be adverse. No test substance-related changes were observed during the neurological screening, for any of the haematological and urinary parameters, the eyes or optic region, and the auditory acuity at any tested dose level. A complete recovery was noted from the changes observed for all clinical signs, food intake, plasma levels of total protein, chloride, sodium and LDL, the serum level of T4 and the relative and absolute liver weights of male and female animals. A trend towards recovery was noted for the decreased body weight gain of male and female animals, and for the total cholesterol and HDL plasma levels of the male animals previously treated with 1000/750/600 mg/kg bw/day. The macroscopic inspection at necropsy of the recovery animals did not reveal any visible changes related to the previous treatment with the test item. The histopathological examination of the recovery animals revealed a complete recovery of the changes noted for the liver of male and female animals. However, the changes noted in the forestomach of males and females in form of squamous hyperplasia were still observed in the high-dose group animals but at a lower severity, indicating a trend towards recovery.

Based on the results, the following effect levels were determined: Systemic no-observed-adverse-effect level (NOAEL) = 300 mg/kg bw/day; local lowest-observed-adverse-effect level (LOAEL) = 100 mg/kg bw/day.

Repeated dose toxicity inhalation

A short-term (28-day) repeated dose toxicity study via the inhalation route with N-methyl-N-(C18-(unsaturated)alkanoyl)glycine (EC No. 701-177-3) was carried out according to OECD guideline 412 under GLP conditions (Seibersdorf, 2010). Groups of 5 Fischer rats per sex and dose were exposed to 0.006, 0.02 and 0.06 mg/L air of the test material for 6 h/day nose only, 5 days/week for 4 weeks. A concurrent negative control group receiving the vehicle ethanol only was included. Furthermore, additional satellite control and high-dose groups with 5 animals per sex were included in the study for investigating the reversibility of possible effects after a 14-day post-exposure recovery period. The concentration range used in the study was chosen based on the results of a previous range finding test.

All animals survived until the end of the study period. Body weight and body weight gain were lower in the high dose groups. In the mid dose male group an effect was observed at the end of the administration period. No significant effects on food consumption were noted during the study period. Alterations in clinical biochemistry were only seen in female animals; the cholesterol value was decreased during the dosing period and increased at the end of the recovery period in high-dose females. The changes of cholesterol in all females of the dosing groups may be caused by the test substance. However, corresponding damages of the liver were not found in the animals at necropsy, histopathology and in organ weights. Increased absolute and relative organ weights of testes in male animals were observed. However, the differences did not show a dose relationship and no correlating effects on the respective organ were seen in the histopathological examinations. In addition, no test substance related changes were observed at the gross examination. The aerosol of the test substance caused effects at several sites of the respiratory system at concentrations of 0.006 to 0.06 mg/L air with indications for a local irritation. The alterations may be explained by the amounts of inert material deposited within the respiratory system. In the lungs and bronchi the most prominent finding was a focal early stage of fibrosis. Due to the high amount of test substance deposits in the lungs especially in the mid and high dosed groups these changes may be explained by overloading the tissue and do not necessarily imply an intrinsic toxicity of the test material. An intrinsic toxicity is unlikely as the test material is insoluble and the shape of the particles is not fibrous.

In summary, the observed effects were at mostly mild in severity, never became life threatening, but persisted partly until the end of the recovery period. Furthermore, due to the self-warning characteristics (immediate irritant reactions to the test substance if inhaled), high substance concentrations of inhalation may be avoided.

The No-observed-effect-concentration (NOEC) for local irritation of the test material via inhalation was smaller than 0.006 mg/L air. There was, however, no life threatening systemic effect present up to the highest concentration tested. Therefore, the systemic NOAEC for the test material will be > 0.06 mg/L air.

Justification for classification or non-classification

The available data on subchronic (90-day) oral and short-term (28-day) inhalation repeated dose toxicity obtained with N-methyl-N-(C18-(unsaturated)alkanoyl)glycine (EC No. 701-177-3) do not meet the criteria for classification according to Regulation (EC) No. 1272/2008 (CLP) and are therefore conclusive but not sufficient for classification.