Reaction mass of N,N’-ethane-1,2-diylbis(12-hydroxyoctadecan-1-amide), Octadecanamide, 12-hydroxy-N-[2-[(1-oxooctadecyl)amino]ethyl]- and Octadecanoic acid, 12-hydroxy-, 1-hexyl-12-[[2-[(12-hydroxy-1-oxooctadecyl)amino]ethyl]amino]-12-oxododecyl ester

Endpoint summary

Description of key information

The substance did not produce adverse effects in a reliable 90 day subchronic repeat dose toxicity study in which the substance was administered by oral gavage. The NOAEL was 1000 mg/kg bw/day (highest dose tested).

The read-across substance bisamide (UVCB) was evaluated in two repeated-dose toxicity studies performed by the oral and inhalation routes. By the oral route, rats treated for 14 days showed no remarkable effects up to 1,000 mg/kg bw/day. By inhalation, no systemic effects were observed in rats exposed up to 2.02 mg/L air for 14 days while local respiratory effects were noted even at the lowest dose of 0.1 mg/L air.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

other: range-finding study

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Study period:

From 14 August 2012 to 19 November 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Read across study hence maximum reliability rating of 2 assigned according to ECHA guidance.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Principles of method if other than guideline:

The objective of this study was to evaluate the potential toxicity of the test substance following daily oral administration (gavage) to Crl:CD(SD) rats for 14 days, in order to select the dose-levels for an OECD 421 reproductive/developmental toxicity screening study.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: 10 weeks
- Weight at study initiation: 353 to 394 g (males) and 234 to 263 (females)
- Fasting period before study: no
- Housing: Four of one sex per cage. The cages were made of a polycarbonate body with a stainless steel mesh lid.
- Diet: Ad libitum, standard rodent diet (SDS VRF1 Certified). This diet contained no added antibiotic or other chemotherapeutic or prophylactic agent
- Water: Ad libitum, potable water taken from the public supply.
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19 to 23°C
- Humidity: 40 to 70%
- Air changes (per hr): Each animal room was kept at positive pressure with respect to the outside by its own supply of filtered fresh air, which was passed to atmosphere and not re-circulated
- Photoperiod (hrs dark / hrs light): 12 h continuous light and 12 h continuous dark/24 h.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): homogeneous and stable suspensions were obtained with corn oil as a vehicle
- Concentration in vehicle: 20, 60 and 200 mg/ml
- Amount of vehicle (if gavage): 5 ml/kg bw

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

No test substance formulation analysis was performed in this study. However, the homogeneity and stability of the formulation were determined. Formulations were confirmed stable for up to 24 hours at ambient temperature (nominally 21°C) and fifteen days when refrigerated (nominally 4°C).

Duration of treatment / exposure:

14 days

Frequency of treatment:

7 days/week

Remarks:

Doses / Concentrations:
0, 100, 300 and 1000 mg/kg bw/day
Basis:
other: nominal doses

No. of animals per sex per dose:

4

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The high dose (1000 mg/kg bw/day) for this study was the maximum dose level required for the subsequent main study (reproductive/developmental toxicity screening study). The low (100 mg/kg bw/day) and intermediate (300 mg/kg bw/day) dose levels were chosen to allow determination of a dose response.

Positive control:

none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- A detailed physical examination was performed on Days 1, 4, 8, 11 and 15 for each animal to monitor general health.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each rat was recorded during acclimatization, on Days -3, 1, 4, 8, 11 and 15 (before necropsy).

FOOD CONSUMPTION :
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
The weight of the food supplied to each cage, food remaining and an estimate of any spillage was recorded at Day -3 to -1, 1 to 3, 4 to 7, 8 to 10 and 11 to 14. From these records the mean daily consumption per animal (g/animal/d) was calculated for each cage.

WATER CONSUMPTION: Yes
-Daily by visual observation.

FOOD EFFICIENCY: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All animals were subject to a detailed necropsy. All external features and orifices were examined visually. After ventral mid-line incision, the neck and associated tissues and the thoracic, abdominal and pelvic cavities and their viscera were exposed and examined in situ. Any abnormal position, morphology or interaction was recorded.

HISTOPATHOLOGY:
Testes were fixed in modified Davidson's fluid. Epididymides, ovaries, kidneys, spleen and liver from all animals were preserved in 10% neutral buffered formalin but no microscopic examinations of these tissues were performed.

Other examinations:

Yes
The following organs were dissected free of adjacent fat and other contiguous tissue and the weights were recorded: epididymides, ovaries, kidneys, spleen, liver, testes, bilateral organs were weighed individually.

Statistics:

none

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY:
There were no premature deaths and no clinical signs.

BODY WEIGHT AND WEIGHT GAIN:
Mean bodyweight gain for females at 1,000 mg/kg bw/day was markedly lower than those at 100 or 300 mg/kg bw/day over the 2 week treatment period. However, this difference was mainly due to one female which showed overall slight bodyweight loss.The weight gain of the other 3 females was unremarkable.The overall bodyweight gain in males was similar in all groups.

FOOD CONSUMPTION:
Food consumption in males was unaffected by treatment and there was no conclusive effect on the food intake of females.

WATER CONSUMPTION:
The visual assessment of water intake did not reveal any dose-related effect up to the end of treatment.

ORGAN WEIGHTS:
Liver weights of females at 1000 mg/kg/day were slightly lower than those at 100 or 300 mg/kg bw/day groups. However, due to the absence of any similar finding in the males, the significance of this difference was unclear.

GROSS PATHOLOGY:
No effects

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: Except a slightly lower liver weight in females at 1,000 mg/kg bw/day when compared to controls, no other effects were observed.

Critical effects observed:

not specified

Conclusions:

Except a slightly lower liver weight in females at 1,000 mg/kg bw/day when compared to controls, no other effects were observed when the test substance was administered by gavage for fourteen days to male and female rats.

Executive summary:

In a dose range-finding study performed in compliance with good Laboratory Practices, the test substance was administered daily by gavage to CD rats for 14 days.

Three groups, each comprising four male and four female rats received the test substance at doses of 100, 300 or 1,000 mg/kg bw/day. During the study, the animals were checked at least twice daily for mortality and clinical condition.Bodyweight and food consumption were recorded twice weekly. Water consumption was assessed by daily visual observation. Animals were sacrificed on completion of the treatment period (day 15) and a complete macroscopic post-mortem examination was performed. The kidneys, liver , spleen, ovaries, testes and epididymides were weighted and preserved although no microscopic examination was performed.

There were no mortalities and the clinical condition of the animals was unaffected by treatment. Three females receiving 1000 mg/kg/day showed unremarkable bodyweight gains, but one female showed overall slight weight loss of uncertain relationship to treatment. Overall bodyweight gain in males was similar in all groups. Food consumption in males was unaffected by treatment and there was no conclusive effect on the food intake of females. There were no effects of treatment on the organ weights of animals which received 100, 300 or 1000 mg/kg/day except a slightly lower liver weight in females that received 1,000 mg/kg bw/day. Macroscopic examination at necropsy after 14 days of treatment did not reveal any abnormalities.  Based on these results, it could be concluded that the NOAEL for the test substance was 1000 mg/kg bw/day and dose levels of 100, 300 and 1000 mg/kg/day would be suitable for use in an associated main OECD 421 reproductive/developmental toxicity screening study.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 December 2017 - 19 April 2018

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 21 September 1998

Deviations:

yes

Remarks:

All females on Day 50 received a higher dose amount than required due to technician error. Dose amounts were correct for remainder of study. There were no adverse effects in any female on this day and no treatment related findings overall on the study.

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Physical State/Appearance: Off white powder
Purity: 100%
Batch Number: S/99/17
Storage Conditions: Stored in ambient temperature in darkness, used/formulated in light
Expiry Date: 30 March 2021

Species:

rat

Strain:

Wistar

Remarks:

Wistar Han™:RccHan™:WIST

Sex:

male/female

Details on test animals or test system and environmental conditions:

The animals were acclimatized for eight days during which time their health status was assessed. At the start of treatment the males weighed 197 to 232g, the females weighed 146 to 175g, and were approximately six weeks old. The animals were housed in groups of three or four by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding. The animals were allowed free access to food and water. A pelleted diet was used. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness.

Route of administration:

oral: gavage

Vehicle:

arachis oil

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of the substance formulations were taken on four occasions and analyzed for concentration. The results indicate that the prepared formulations were within acceptable ranges for the purpose of this study.

Duration of treatment / exposure:

90 consecutive days

Frequency of treatment:

Once daily

Dose / conc.:

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

Remarks:

Control

Dose / conc.:

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

Remarks:

Low

Dose / conc.:

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

Remarks:

Intermediate

Dose / conc.:

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

Remarks:

High

No. of animals per sex per dose:

10/sex/dose

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

Clinical Observations: All animals were examined for overt signs of toxicity, ill-health or behavioral change immediately before dosing, up to thirty minutes post dosing and one hour after dosing. All observations were recorded.

Body Weight: Individual body weights were recorded on Day 1 (prior to dosing) and at weekly intervals thereafter. Body weights were also recorded at terminal kill.

Food Consumption: Food consumption was recorded for each cage group at weekly intervals throughout the study. Food conversion efficiency was calculated retrospectively.

Water Consumption: Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes.

Functional Observations: Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioral toxicity. During Week 12 functional performance tests were also performed on all animals together with an assessment of sensory reactivity to different stimuli.

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

Functional Performance Tests: Motor Acivity, Forelimb/Hindlimb Grip Strength, Sensory Reactivity, Ophthalmoscopic Examination.

In-Life Sampling and Analysis included:
Haematology:
Hemoglobin (Hb)
Erythrocyte count (RBC)
Hematocrit (Hct)
Erythrocyte indices
- mean corpuscular hemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular hemoglobin concentration (MCHC)
Total leukocyte count (WBC)
Differential leukocyte count
- neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count (PLT)
Reticulocyte count (Retic)
- Methylene blue stained slides were prepared but reticulocytes were not assessed
Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/L).

Blood Chemistry:
Urea
Inorganic phosphorus (P)
Glucose
Aspartate aminotransferase (ASAT)
Total protein (Tot.Prot.)
Alanine aminotransferase (ALAT)
Albumin
Alkaline phosphatase (AP)
Albumin/Globulin (A/G) ratio (by calculation)
Creatinine (Creat)
Sodium (Na+)
Total cholesterol (Chol)
Potassium (K+)
Total bilirubin (Bili)
Chloride (Cl-)
Bile acids
Calcium (Ca++)

Sacrifice and pathology:

Necropsy: On completion of the dosing period all surviving animals were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination. All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Organ Weights:
Adrenals
Ovaries
Brain
Spleen
Epididymides
Testes
Heart
Thymus
Kidneys
Uterus
Liver

Histopathology:
Adrenals
Ovaries
Aorta (thoracic)
Pancreas
Bone & bone marrow (femur including stifle joint) (Retained only and not processed)
Pituitary
Bone & bone marrow (sternum)
Prostate
Brain (including cerebrum, cerebellum and pons)
Rectum
Caecum
Salivary glands (submaxillary)
Colon
Sciatic nerve
Duodenum
Seminal vesicles
Epididymides (preserved in Modified Davidson’s fluid)
Skin
Esophagus
Spinal cord (cervical, mid-thoracic
Eyes
and lumbar)
Gross lesions (where applicable)
Spleen
Heart
Stomach
Ileum (including Peyer’s patches)
Testes (preserved in Modified Davidson’s fluid)
Jejunum
Thymus
Kidneys
Thyroid/Parathyroid
Liver
Tongue (Retained only and not processed)
Lungs (with bronchi)
Trachea
Lymph nodes (mandibular and mesenteric)
Urinary bladder
Mammary glands
Uterus (with cervix)
Muscle (skeletal) (Retained only and not processed)
Vagina

Pathology: Microscopic examination was conducted by the Study Pathologist.

Statistics:

Statistical analysis was performed on the following parameters: Grip Strength, Motor Activity, Body Weight Change, Hematology, Blood Chemistry, Absolute Organ Weights, Body Weight-Relative Organ Weights.

Where appropriate, data transformations were performed using the most suitable method. The homogeneity of variance from mean values was analyzed using Bartlett’s test. Intergroup variances were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covariates. Any transformed data were analyzed to find the lowest treatment level that showed a significant effect using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found but the data shows non-homogeneity of means, the data were analyzed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests was performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).

Probability values (p) are presented as follows:
p<0.01 **
p<0.05 *
p>0.05 (not significant)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Incidences of increased salivation were evident in five males treated with 1000 mg/kg bw/day throughout the majority of the treatment period and in one male treated with 100 mg/kg bw/day on two occasions. An observation of this nature is commonly observed following the oral administration of an unpalatable substance formulation and is considered not to represent an adverse effect of treatment.

Noisy respiration was also evident in three males treated with 1000 mg/kg bw/day between Days 52 and 90, in one female treated with 1000 mg/kg bw/day on two occasions, in one male and two females treated with 100 mg/kg bw/day on one occasion and in one male and one female treated with 10 mg/kg bw/day on one occasion only. At these low frequencies and distributions, this is considered to indicate possible difficulties in dosing particular animals on isolated occasions and not indicative of systemic toxicity.

Incidental observations, showing no dose related response and considered to be unrelated to test item administration included one control male had noisy respiration on Day 43, one male treated at 100 mg/kg bw/day with an open wound on the left shoulder between Days 9 and 11, which then became a scab between Days 12 and 39 and a male treated with 10 mg/kg bw/day was observed to have misaligned posture, holding its head tilted to the left from Day 13 to termination.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One control female was found dead on Day 75. At necropsy this female had black colored contents in the cecum, colon, duodenum, ileum, jejunum and stomach and a pale liver with dark patches. There were no changes at histopathology to account for the death or the macroscopic changes, however, lymphoid necrosis/atrophy/depletion was present along with cortical hypertrophy in the adrenal glands which indicate poor clinical condition/stress.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Males treated with 1000 and 100 mg/kg bw/day showed statistically significantly higher (p<0.05-0.01) body weight gains during Week 2, with males treated at 1000 mg/kg bw/day also showing statistically significantly higher body weight gains during Weeks 4 (p<0.01), 5 (p<0.05) and 7 (p<0.01). An increase in body weight gain is considered not to be an adverse effect of treatment.

Food consumption and compound intake (if feeding study):

no effects observed

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Ophthalmoscopic examination of animals of both sexes from the control and 1000 mg/kg bw/day dose groups during Week 12 of the treatment period did not indicate any treatment-related differences.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Total leukocyte count was statistically significantly lower (p<0.05) in all treated males. With the exception of one individual male treated at 100 mg/kg bw/day, all individual values for treated males were within the historical control range. Mean corpuscular hemoglobin concentration and lymphocytes for males treated with 1000 mg/kg bw/day were statistically significantly lower (p<0.05) compared to controls. For mean corpuscular hemoglobin concentration, four of the individual control values exceeded the historical control range, whereas all of the individual values for males treated with 1000 mg/kg bw/day were within the historical control range. For lymphocytes, although all individual control values were within the historical control range, only one individual value for males at 1000 mg/kg bw/day was below this range. As the majority of values were within the historical control normal ranges, and in the absence of any histopathological correlates, these intergroup differences were considered to be of no toxicological significance.

Eosinophils were statistically significantly lower (p<0.05) than controls for males treated with 10 mg/kg bw/day. All individual values were within the historical control range and in the absence of any similar findings in males treated with 100 or 1000 mg/kg bw/day, the intergroup difference was considered to be of no toxicological significance.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Males treated at 1000 mg/kg bw/day showed a statistically significant increase (p<0.05) in calcium concentration. All of the individual values were within the historical control range and in the absence of any associated histopathological correlates the intergroup difference was considered not to be of toxicological significance. Bilirubin levels were also statistically significantly higher (p<0.05) than controls in males treated with 100 and 1000 mg/kg bw/day. However, two individual control values were below the historical control range and only one individual value for males at 1000 mg/kg bw/day was above the historical control range. All values for males treated with 100 mg/kg bw/day were within the historical control range. In the absence of any associated histopathological correlates the intergroup differences were considered not to be of toxicological significance.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Occasional incidences of noisy respiration were evident in one male treated with 1000 mg/kg bw/day during the final two weeks of treatment, but this is likely to be due to slight difficulties in dosing this particular animal on these occasions and is not indicative of true systemic toxicity.

The incidental clinical finding of mis-aligned posture was evident in one male treated with 10 mg/kg bw/day from Week 2 assessments onwards.

Motor activity assessment did not indicate any effect of treatment for either sex at 10, 100 or 1000 mg/kg bw/day. There was no effect of treatment at 10, 100 or 1000 mg/kg bw/day on forelimb or hindlimb grip strength.

There were no treatment-related changes in sensory reactivity. Intergroup differences observed in the scores for sensory reactivity did not indicate any effect of treatment for either sex at 10, 100 or 1000 mg/kg bw/day.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Males treated with 100 and 10 mg/kg bw/day showed statistically significant increases (p<0.05) in absolute and body weight relative kidney weights in comparison with controls. The majority of individual values were within historical control ranges and in the absence of any similar findings in males treated with 1000 mg/kg bw/day or any associated histopathological correlates, the intergroup differences were considered not to be of toxicological significance.

Both absolute and body weight relative liver weights were statistically significantly higher (p<0.05) for males treated with 100 and 1000 mg/kg bw/day compared to controls. However, the majority of these individual values were within historical control normal ranges, a true dose related response for relative weights was not evident and there were no histopathological correlates. The intergroup differences were therefore considered not to be of toxicological significance.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Red or dark coloration of the lungs was observed for one female at 10 mg/kg bw/day, three males and one female treated with 100 mg/kg bw/day and two males treated with 1000 mg/kg bw/day. Such findings are common in this type of study and were considered to be unrelated to treatment with the substance.

Increased pelvic space in both kidneys was observed in one control female and mottled kidneys was observed in one control male. In the absence of treatment, these were considered to be incidental findings. One female treated with 100 mg/kg bw/day had a dark liver and one female treated with 10 mg/kg bw/day had a dark patch on the liver. In the absence of a similar effect at 1000 mg/kg bw/day or any associated histopathological correlates, these findings were considered incidental and unrelated to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Centrilobular hypertrophy was noted in the liver of one male treated with 1000 mg/kg bw/day, however, due to the low incidence and minimal severity after ninety days of treatment this is considered not to be related to the administration of the substance. No changes were noted which could account for the weight increase noted in the liver of males and all are considered to be incidental.

Histopathological findings: neoplastic:

not specified

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

mortality

ophthalmological examination

organ weights and organ / body weight ratios

Key result

Critical effects observed:

no

Conclusions:

The oral (gavage) administration of the substance, for ninety consecutive days, to Wistar rats of both sexes at dose levels of 10, 100 or 1000 mg/kg bw/day did not result in any adverse treatment-related effects. The ‘No Observed Adverse Effect Level’ (NOAEL) was therefore considered to be >1000 mg/kg bw/day.

Executive summary:

Male and female Wistar rats (10/sex/dose) were administered the substance (dissolved in Arachis oil BP) by oral gavage at doses of 0 (vehicle only), 10, 100 or 1000 mg/kg bw/day once daily for 90 consecutive days. The study was conducted according to OECD Guideline 408 (Subchronic Oral Toxicity - Rodent: 90 Day Study) and in accordance with GLP. Clinical signs, functional observations, body weight change, dietary intake and water consumption were monitored during the study. Haematology and blood chemistry were evaluated for all animals at the end of the study. Ophthalmoscopic examination was also performed on control group and high dose animals. All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues from high dose and control animals was performed. No mortality occurred and no treatment-related clinical signs were observed during the study. Incidences of increased salivation were evident in males treated with 1000 mg/kg bw/day throughout the majority of the treatment period and in males treated with 100 mg/kg bw/day albeit to a lesser extent. Noisy respiration was also evident in three males treated with 1000 mg/kg bw/day between Days 52 and 90 and in one female treated with 1000 mg/kg bw/day, one male and two females treated with 100 mg/kg bw/day and one male and one female treated with 10 mg/kg bw/day on one occasion only. There were no toxicologically significant changes in the behavioral parameters measured. There were no treatment-related changes in functional performance. There were no treatment-related changes in sensory reactivity. No adverse effect on body weight development was evident in treated animals when compared to controls. No effect on food consumption or food efficiency was evident in treated animals when compared to controls. Visual inspection of water bottles did not reveal any intergroup differences. Ophthalmoscopic examination of animals of both sexes from the control and surviving 1000 mg/kg bw/day dose groups during Week 12 of the treatment period did not indicate any treatment-related differences. There were no toxicologically significant effects detected in the hematological or blood chemistry parameters examined. No treatment-related findings were reported at post mortem macroscopic observations and histopathological examination. No toxicologically significant effects were detected in the organ weights measured. In conclusion, no treatment-related changes, which could be considered adverse, were observed in male and female rats following dosing with the substance, when administered by oral gavage for 90 consecutive days at the dosages of 10, 100 and 1000 mg/kg bw/day. The No Observed Adverse Effect Level (NOAEL) for this study was >1000 mg/kg bw/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Sufficient to address requirements.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From 06 November 2012 to 14 February 2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Read across study hence maximum reliability rating of 2 assigned according to ECHA guidance.

Qualifier:

equivalent or similar to guideline

Guideline:

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

Principles of method if other than guideline:

This pilot study was performed in order to obtain preliminary information about the local and systemic toxicity of the substance when administered by nose-only inhalation exposure for 6 hours per day (5 days per week) to Wistar rats. The results of the study are intended to be used for dose level selection for a the future 90-day inhalation 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
- Age at study initiation: Young adult rats, 9 weeks old
- Weight at study initiation: 171 to 299 g (males: 268-299 g; females: 171-207g)
- Housing: group caging (5 animals/sex/cage)
- Bedding: Lignocel bedding for laboratory animals was available to animals during the study
- Diet (e.g. ad libitum): Ssniff SM R/M-Z+H “Autoclavable Complete Feed for Rats and Mice – Breeding and Maintenance" ad libitum
- Water (e.g. ad libitum): Tap water ad libitum
- Acclimation period: 11 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22±3 °C
- Humidity: 30–70 %
- Air changes: 15-20 air exchanges/h
- Photoperiod (hrs dark / hrs light): 12 h dark/12 h light

Deviation:The temperature (22±3ºC) and the humidity (30–70%) values deviated from the required range during the acclimation period in the animal room. However, these deviations had no effect on the purpose and integrity of the study.

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose only

Vehicle:

air

Remarks on MMAD:

MMAD / GSD: The Mass Median Aerodynamic Diameter (MMAD) of the test atmosphere of all groups was in the range of 1.97-2.73 µm with Geometric Standard Deviation (GSD) of 2.90-3.22. Due to the test item physical properties the Geometric Standard Deviation (GSD) of the test atmosphere was slightly above 3, but was kept in range of 3±10%.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus:TSE Rodent Exposure System (TSE Systems GmbH, Bad Homburg, Germany) which is a flow-past, nose-only exposure unit. This system consists of two, concentric anodised aluminium cylinders, the inner plenum and the outer chamber with 20 circularly arranged exposure ports.The equipment is supported by a computer control system incorporating pressure detectors, mass flow controllers as well as temperature, O2 and CO2 sensors.

- Method of holding animals in test chamber: animals were held in polycarbonate restraint tubes located around the chamber which allow only the animals’ nares to enter the exposure port.

- Source of air: compressed air

- Method of conditioning air: the compressed air was passed through a respiratory quality filter train and condensate separator prior to use.

- System of generating particulates: rotating brush powder disperser (Palas GmbH, Karlsruhe, Germany) located at the top of the exposure chamber

- Temperature, humidity, pressure in air chamber: 22±3 °C, 30–70 % and pressure in air chamber not reported

- Air flow rate: the flow of air through each port was at least 0.5 L/min.

- Method of particle size determination: Particle size analysis of generated atmospheres was performed using a 7-stage cascade impactor of Mercer style (TSE Systems GmbH, Bad Homburg, Germany). Samples were collected at least once during each week of exposure for each concentration tested. Samples were collected from a vacant animal exposure port (animals breathing zone). The collection substrates and the backup filter were weighed before and after sampling and the weight of test item, collected at each stage, calculated by this difference.The total amount collected for each stage was used to determine the cumulative amount below each cut-off point size. In this way, the proportion (%) of aerosol less than < 0.550, 0.550, 0.960, 1.550, 2.105, 3.555, 6.655 and 10.550 µm was calculated. From these data, using the software supplied with the impactor (TSE Systems GmbH, Bad Homburg, Germany), the Mass Median Aerodynamic Diameter (MMAD), and Geometric Standard Deviation (GSD) were calculated. In addition, the proportion (%) of aerosol less than 3 µm was determined.

- Treatment of exhaust air: After passing through the animal’s breathing zone, spent aerosol enters the outer cylinder from where it was exhausted through a suitable filter system.

TEST ATMOSPHERE
- Brief description of analytical method used: The actual 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 GF10 glass fibre filters (Whatman, Germany, ref. no. 10370302). Sampling was normally performed shortly after chamber equilibration and then at regular intervals (approximately hourly intervals) during the exposure and samples were collected from a vacant animal exposure port (animals breathing zone). The difference in the pre- and post-sampling weights, divided by the volume of atmosphere sampled, was equal to the actual achieved test atmosphere concentration.

- Samples taken from breathing zone: yes, samples were collected from a vacant animal exposure port.

VEHICLE (if applicable)
- none

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 deposited on a sampling filter. Sometimes, marked fluctuations in sample concentration occurred. These fluctuations can be explained by the short duration of the sampling, however the average concentration was equal to the target concentrations. The nominal concentration (mass of the test item dispersed into the exposure system in total air flow used for exposure) deviates significantly from mean achieved concentration due to loss of particles in pre-separation devices used for particle size optimization.
The mean achieved concentrations were 0.10; 0.50 and 2.02 mg/L and corresponded to 100; 100 and 101% of the target concentrations respectively.

Duration of treatment / exposure:

14 days

Frequency of treatment:

5 days/ week

Remarks:

Doses / Concentrations:
0.1; 0.5 and 2.0 mg/L
Basis:
other: target concentrations

Remarks:

Doses / Concentrations:
0.10 ; 0.50 and 2,02 mg/L
Basis:
analytical conc.

No. of animals per sex per dose:

5

Control animals:

yes, sham-exposed

Details on study design:

- Dose selection rationale: The target concentration levels were 0.10, 0.50 and 2.0 mg/L of test item. They were set on the basis of available data and information from previous experimental work, including the results of an acute inhalation toxicity study (see section 7.2.2). In this 4–hour exposure study, no death occurred in group of six male and female rats exposed to a mean achieved atmosphere of 5.05 mg/l.

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Clinical observations were performed prior to exposure on the working days, and at least twice during the exposure whilst the animals were still restrained. Following exposure clinical observation was performed at least twice (as soon as practicable after removal from restraint, and approximately one hour after completion of the exposure. During the weekend (no exposure) the clinical signs were recorded once per day only.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were made on all animals outside the home cage weekly, on Day 1, 7, 14.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight of each animal was recorded with precision of 1 g at randomization, then on Day 1 and at least on Days 4, 8, 11, 14 and 15 (prior to necropsy, fasted before the scheduled euthanasia).

FOOD CONSUMPTION: Yes
- Food was recorded on Day 1 and at least on Days 4, 8, 11 and 14. The remaining, non-consumed food given was weighed with a precision of 1g for each cage. The mean individual daily food consumption was calculated per animal.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 15
- Anaesthetic used for blood collection: Yes (pentobarbital)
- Animals fasted: Yes
- How many animals: all animals of all groups
- Parameters checked in table 7.5.2/1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 15
- Animals fasted: Yes
- How many animals: all animals of all groups
- Parameters checked in table 7.5.2/2 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Gross necropsy and macroscopic examination were performed on all animals. After exsanguination, the external appearance was examined, cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed macroscopically. Any abnormality were recorded with details of the location, colour, shape and size, as appropriate.

HISTOPATHOLOGY: Yes
Initial processing was limited to larynx, trachea, lungs, tracheobronchial lymph nodes, nasal cavity and any gross abnormalities for all animals of all groups.

Other examinations:

Yes
The following organs were weighed : Brain, heart, kidneys, liver, lungs, spleen, testes, thymus, adrenals and ovaries.

Statistics:

The heterogeneity of variance between groups were checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance was carried out. If the obtained result was positive, Duncan’s Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorov-Smirnov test. If the data was not normal distributed, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was used. If there was a positive result, the inter-group comparisons was performed using Mann-Whitney U-test.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In the animals exposed to the concentrations of 2.02 mg/L, the following clinical signs were observed:
Slight increase in respiratory rate was observed in 4/5 females on Day 1 and in 2/5 males on Days 3-4,
Slight and in few occasion moderate laboured respiration was noted throughout the study in females. In males, a slight laboured respiration was observed on Day 2 in 3/5 animals,
Slight noisy respiration turning to moderate in two occasions, was observed from Day 2 to Day 10 in 1 to 2 females,
Hunched posture was noted from Day 4 to Day 12 in 1 to 3 females and on Days 10-11 in 1 male,
Sneezing was noted for 2 females on Days 10-11.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In the animals exposed to the concentrations of 2.02 mg/L,, a very slight mean bodyweight loss was recorded during the first days of treatment in both sexes. In comparison with controls, the decrease in mean bodyweight during days 1 to 4 was -2.2% and -0.43% in males and females respectively. In addition, females showed a statistically significant decrease in mean bodyweight gain on the overall period of treatment (-36% compared with controls).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In the animals exposed to the concentrations of 2.02 mg/L, there was a significant decrease in food consumption in both males and females. The lower food consumption was in accordance with the effect on body weight.

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):

An increase in White Blood Count (WBC) was observed in both sexes at all doses but without dose-dependency. In parallel, increase of relative Neutrophils and concomitant decrease of relative Lymphocytes were observed during the hematology analysis. Slight decreased of Red Blood Count and slight increase of Mean Cell Haemoglobin were observed in males. However the changes were slight in magnitude, and were within the physiological range. Therefore these changes were not considered toxicologically significant (see table 7.5.2/1).

Clinical biochemistry findings:

no effects observed

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):

The mean absolute and relative lungs weights, adjusted for both brain and terminal body weight, were significantly higher in all treated groups. They increased in a dose dependent manner up to 2 times in the 2.02 mg/L Group (see table 7.5.2/2).

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

There was evidence of test item-related macroscopic changes in the lungs at the dose levels of 0.50 and 2.02 mg/L.
Enlargement/pale discoloration were observed in 5/5 males and 5/5 females in the 2.02 mg/L group. Pale foci were seen in 2/5 females of the 0.50 mg/L group.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Substance-related findings were noted in the lungs and the tracheobronchial lymph nodes at all dose levels. There was clear relationship of these microscopic findings to the dose.
The alterations in the lungs were characterized as presence of white/pink foreign material in the alveoli, granular in consistency. The presence of foreign alveolar material was generally minimal at low dose and moderate at high dose.
The inflammatory alterations in the lungs ranged from minimal to moderate severity, with the presence of mixed cell population (prominent feature of lymphocytes, macrophages and neutrophils) in the bronchiolar and alveolar compartments. The incidence and severity of the inflammatory responses were generally increased consistently to dose. Additionally, the formation of microgranulomas was appeared by light microscopy in the alveoli of animals treated at 2.02 mg/L. A similar pattern of dose relationship was recorded for the accumulation of foamy macrophages in the tracheobronchial lymph nodes. The low severity (minimal) at0.1 mg/L, increased (mild) at 0.50 mg/L and was moderateat 2.02 mg/L (see table 7.5.2/3).

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
There was no mortality observed during the study.
In the animals exposed to the concentrations of 2.02 mg/L, the following clinical signs were observed:
• Slight increase in respiratory rate was observed in 4/5 females on Day 1 and in 2/5 males on Days 3-4,
• Slight and in few occasion moderate laboured respiration was noted throughout the study in females. In males, a slight laboured respiration was observed on Day 2 in 3/5 animals,
• Slight noisy respiration turning to moderate in two occasions, was observed from Day 2 to Day 10 in 1 to 2 females,
• Hunched posture was noted from Day 4 to Day 12 in 1 to 3 females and on Days 10-11 in 1 male,
• Sneezing was noted for 2 females on Days 10-11.

In the animals exposed to the concentrations of 0.50 mg/L, no significant clinical signs were noted except for 1 male where initially slight subcutaneous mass in the left axillary was recorded from Day 5 which continuously grew until the end of the study.

In the animals exposed to the concentrations of 0.10 mg/L, slightly laboured respiration was noted in 1 female on day 5. No significant clinical signs were noted for the other animals in this group during the exposure period.

No clinical signs were recorded for the air control animals.


BODY WEIGHT AND WEIGHT GAIN
In the animals exposed to the concentrations of 2.02 mg/L,, a very slight mean bodyweight loss was recorded during the first days of treatment in both sexes. In comparison with controls, the decrease in mean bodyweight during days 1 to 4 was -2.2% and -0.43% in males and females respectively. In addition, females showed a statistically significant decrease in mean bodyweight gain on the overall period of treatment (-36% compared with controls).

No effects on bodyweight and bodyweight gain were noted in the animals exposed to 0.50 or 0.10 mg/L when compared with controls.

FOOD CONSUMPTION
In the animals exposed to the concentrations of 2.02 mg/L, there was a significant decrease in food consumption in both males and females. The lower food consumption was in accordance with the effect on body weight.

No effects on food consumption were noted in the animals exposed to 0.50 or 0.10 mg/L when compared with controls.

HAEMATOLOGY
An increase in White Blood Count (WBC) was observed in both sexes at all doses but without dose-dependency. In parallel, increase of relative Neutrophils and concomitant decrease of relative Lymphocytes were observed during the hematology analysis. Slight decreased of Red Blood Count and slight increase of Mean Cell Haemoglobin were observed in males. However the changes were slight in magnitude, and were within the physiological range. Therefore these changes were not considered toxicologically significant (see table 7.5.2/1).

CLINICAL CHEMISTRY

There was no effect of treatment on clinical chemistry.

ORGAN WEIGHTS
The mean absolute and relative lungs weights, adjusted for both brain and terminal body weight, were significantly higher in all treated groups. They increased in a dose dependent manner up to 2 times in the 2.02 mg/L Group (see table 7.5.2/2).

GROSS PATHOLOGY
There was evidence of test item-related macroscopic changes in the lungs at the dose levels of 0.50 and 2.02 mg/L.
Enlargement/pale discoloration were observed in 5/5 males and 5/5 females in the 2.02 mg/L group. Pale foci were seen in 2/5 females of the 0.50 mg/L group.

HISTOPATHOLOGY: NON-NEOPLASTIC
Test item-related findings were noted in the lungs and the tracheobronchial lymph nodes at all dose levels. There was clear relationship of these microscopic findings to the dose.
The alterations in the lungs were characterized as presence of white/pink foreign material in the alveoli, granular in consistency. The presence of foreign alveolar material was generally minimal at low dose and moderate at high dose.
The inflammatory alterations in the lungs ranged from minimal to moderate severity, with the presence of mixed cell population (prominent feature of lymphocytes, macrophages and neutrophils) in the bronchiolar and alveolar compartments. The incidence and severity of the inflammatory responses were generally increased consistently to dose. Additionally, the formation of microgranulomas was appeared by light microscopy in the alveoli of animals treated at 2.02 mg/L. A similar pattern of dose relationship was recorded for the accumulation of foamy macrophages in the tracheobronchial lymph nodes. The low severity (minimal) at0.1 mg/L, increased (mild) at 0.50 mg/L and was moderateat 2.02 mg/L (see table 7.5.2/3).

Key result

Dose descriptor:

LOAEC

Effect level:

0.1 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: At 0.1 mg/L, slight increase in neutrophils and slight concomitant decrease in lymphocytes were observed during the Hematology analysis. These effects were associated with a slight inflammation process in the respiratory tract of the animals

Key result

Critical effects observed:

no

Table 7.5.2/1 Mean values in affected haematologic parameters

GROUP	1 (Control)	2 (Low)	3 (Mid)	4 (High)	Remarks
Concentration (mg/L)	0	0.1	0.5	2.02
Males
White Blood Count – WBC (K/mL)	2.776	3.244	3.664	3.314	NS
Red Blood Count – RBC (M/mL)	8.726	8.384	8.272	8.096*	DN
Neutrophils –NEU, Relative (%)	25.160	21.480	28.320	34.720**	DN
Lymphocytes – LYMPH, Relative (%)	69.620	74.080	66.780	59.040**	DN
Mean Cell Haemoglobin – MCH, pg	18.18	19.00**	19.12**	19.06**	DN
Females
White Blood Count – WBC (K/mL)	1.216	2.394*	2.130*	1.948*	U
Red Blood Count – RBC (M/mL)	7.932	8.108	7.986	8.034	NS
Neutrophils –NEU, Relative (%)	14.660	20.920*	20.420*	26.360**	DN
Lymphocytes – LYMPH, Relative (%)	80.460	73.920*	74.480*	67.900**	DN
Mean Cell Haemoglobin – MCH, pg	18.94	19.32	19.18	19.62	NS

NS = Not Significant

DN =Duncan's Multiple Range Test

U = Mann- Whitney U-Test

*= p<0.05

** = p<0.01

Table 7.5.2/2 Mean values of lungs weights following the 14-day exposure period

GROUP	1 (Control)	2 (Low)	3 (Mid)	4 (High)	Remarks
Concentration (mg/L)	0	0.1	0.5	2.02
Males
Lungs Weight (g)Absolute	1.318	1.620**	2.092**	2.884**	U
Relative to Brain Weight (%)	69.52	83.78**	109.68**	145.41**	U
Relative to Body Weight (%)	0.44	0.54**	0.68**	0.99**	DN
Females
Lungs Weight (g)Absolute	1.014	1.286**	1.804**	2.256**	DN
Relative to Brain Weight (%)	55.01	73.04**	98.92**	124.25**	DN
Relative to Body Weight (%)	0.51	0.64**	0.88**	1.17**	DN

DN =Duncan's Multiple Range Test

U = Mann- Whitney U-Test

*= p<0.05

** = p<0.01

Table 7.5.3/3 Summary of histopatology data for lungs and tracheobronchial lymph nodes

Sex	male	male	male	male	female	female	female	female
Concentration (mg/l)	0	0.10	0.50	2.02	0	0.10	0.50	2.02
Number of Animals on Study	5	5	5	5	5	5	5	5
LUNGS;
Examined	(5)	(5)	(5)	(5)	(5)	(5)	(5)	(5)
WithinLimits	5	0	0	0	5	0	0	0
Foreign Material; alveolar; all lobes, Glanular, white/pink	(0)	(5)	(5)	(5)	(0)	(5)	(5)	(5)
minimal	0	4	1	0	0	5	0	0
mild	0	1	4	0	0	0	3	0
Foreign Material; alveolar; all lobes, Glanular, white/pink; correlated with necropsy.	(0)	(5)	(5)	(5)	(0)	(5)	(5)	(5)
mild	0	0	0	1	0	0	2	2
moderate	0	0	0	4	0	0	0	3
Inflammation, Bronchioloalveolar; mixed; all lobes	(0)	(0)	(5)	(5)	(0)	(0)	(5)	(5)
minimal	0	0	5	0	0	0	5	0
mild	0	0	0	4	0	0	0	1
moderate	0	0	0	1	0	0	0	1
Inflammation, Bronchioloalveolar; mixed; all lobes correlated with necropsy	(0)	(0)	(5)	(5)	(0)	(0)	(5)	(5)
mild	0	0	0	4	0	0	0	3
Microgranuloma; alveolar; multifocal	(0)	(0)	(0)	(3)	(0)	(0)	(0)	(3)
minimal	0	0	0	1	0	0	0	2
mild	0	0	0	2	0	0	0	1
Cell Infiltrate, Mixed Cellular; bronchiole; alveolus, all lobes	(0)	(5)	(0)	(0)	(0)	(5)	(0)	(0)
minimal	0	3	0	0	0	5	0	0
mild	0	2	0	0	0	0	0	0
LYMPH NODE, LUNG-ASSOCIATED;
Examined	(5)	(5)	(5)	(5)	(4)	(5)	(5)	(5)
WithinLimits	5	0	0	0	4	2	0	0
Not Examined: NOT PRESENT	0	0	0	0	1	0	0	0
Accumulation, Foamy Macrophage; medulla; paracortex	(0)	(5)	(5)	(5)	(0)	(3)	(5)	(5)
minimal	0	2	1	0	0	3	2	0
mild	0	3	4	0	0	0	3	1
moderate	0	0	0	5	0	0	0	4

Conclusions:

Exposure to the test substance at gravimetrically determined dose levels of 0.10, 0.50 and 2.02 mg/L air resulted in findings in the lungs and tracheobronchial lymph nodes. In the lungs, they consisted of inflammatory reactions ranging from minimal to moderate severity, with the presence of mixed cell population. Also, foreign material was seen in the alveoli. The incidence and severity of the inflammatory responses and presence of foreign alveolar material were generally increased consistently to dose. A similar pattern of dose relationship was recorded for the accumulation of macrophages in the tracheobronchial lymph nodes. The low severity (minimal) in Low dose, increased (mild) in Mid dose and was moderate in High dose animals.

Executive summary:

In a pilot study performed in compliance with Good Laboratory Practices, the test substance was administered by nose-only inhalation exposure for 6 hours per day (5 days per week) to Wistar rats.

The test item was administered as a dry aerosol. The animals were exposed on 10 occasions during a 14-day period. Three groups, each comprising five male and five female rats received the test substance at target exposure levels of of 0.1, 0.5 and 2.00 mg/L. A similarly constituted Control group received air at the same operating conditions as the other groups.

The mortality observations were performed twice daily. The general clinical observations were recorded five times for the exposed animals on the days of exposure. Detailed clinical examination was performed on Days 1, 7 and 14 prior to the 6-hour exposure. Body weight and food consumption were measured on Days 1, 4, 8, 11 and 14. Blood samples were collected for clinical pathology at necropsy. Gross macroscopic examination was performed at necropsy one day after the last treatment. Selected organs were weighed. Histopathology investigation was performed on selected tissues.

The achieved aerosol concentrations were 0.10, 0.50 and 2.02 mg/L (100, 100 and101% of target). The Mass Median Aerodynamic Diameters for all treated groups were within the ideal range (1-3 µm) for a repeat dose inhalation study. 

There was no mortality during the study. Clinical signs related to treatment were noted only in animals given 2.02 mg/L. All but one female and only 2 males had a transient slight increase in respiratory rate. In females, slight and in few occasions moderate laboured and/ or noisy respiration was observed throughout the study. In males, slight laboured respiration was noted only on day 2 in 3 animals. Hunched posture was observed from day 4 to day 10 in 1 to 3 females and on days 10-11 in 1 male. In addition, sneezing was noted for two females on Day 10 and 11. 

Changes in bodyweight and bodyweight gain were noted only in animals that received 2.02 mg/L . Temporary mean bodyweight loss was observed at the beginning of the study in both sexes on (Days 1-4) ( -2.2% and -0.43% in males and females respectively). This bodyweight loss correlated with the significant decrease in food consumption noted during the same treatment-period. In addition, females, showed a statistically significant decrease in mean bodyweight gain on the overall period of treatment (-36% compared with controls).

Following 2 weeks of treatment, an increase in White Blood Count was observed in both sexes at all doses but without dose-dependency. In addition, statistically significant increase of relative Neutrophils and concomitant decrease of relative Lymphocytes were observed in males given 2.02 mg/l and in all treated females. These changes in haematology might be contributed to changes in the lungs. Slight decreased of Red Blood Count and slight increase of Mean Cell Haemoglobin were also observed in males. However the changes were slight in magnitude, and were within the physiological range. Therefore these changes were not considered toxicologically significant. There was no effect of treatment noted during the evaluation of the clinical chemistry parameters.

Macroscopic examination revealed enlargement and/or pale discoloration of the lungs in animals given 0.50 or 2.02 mg/L. Treatment related histopathological changes were seen in lungs and tracheobronchial lymph nodes at all doses levels.There was clear relationship of these microscopic findings to the dose. The alterations in the lungs were characterized as presence of white/pink foreign material in the alveoli. The presence of foreign alveolar material was generally minimal at low dose and moderate at high dose. The inflammatory reactions in the lungs ranged from minimal to moderate severity, with the presence of mixed cell population in the bronchiolar and alveolar compartments. The incidence and severity of the inflammatory responses were generally increased consistently with dose. The formation of microgranulomas was observed microscopically in the alveoli of animals treated at a dose level of 2.02 mg/L. A similar pattern of dose relationship was recorded for the accumulation of foamy macrophages in the tracheobronchial lymph nodes with low severity (minimal) in Low dose, increased (mild) in Mid dose and moderate in the High dose group. The mean absolute and relative lungs weights adjusted for both brain and terminal body weight were significantly higher in males and females at dose levels of 0.1, 0.5 and 2.02 mg/L compared to controls.

Based on these findings, it was anticipated that a target exposure level of 100 µg/L at maximum would be tolerated for a 90 day exposure in the same species.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

2 020 mg/m³

Study duration:

subacute

Species:

rat

Quality of whole database:

One study available.

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From 06 November 2012 to 14 February 2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Read across study hence maximum reliability rating of 2 assigned according to ECHA guidance.

Qualifier:

equivalent or similar to guideline

Guideline:

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

Principles of method if other than guideline:

This pilot study was performed in order to obtain preliminary information about the local and systemic toxicity of the substance when administered by nose-only inhalation exposure for 6 hours per day (5 days per week) to Wistar rats. The results of the study are intended to be used for dose level selection for a the future 90-day inhalation 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
- Age at study initiation: Young adult rats, 9 weeks old
- Weight at study initiation: 171 to 299 g (males: 268-299 g; females: 171-207g)
- Housing: group caging (5 animals/sex/cage)
- Bedding: Lignocel bedding for laboratory animals was available to animals during the study
- Diet (e.g. ad libitum): Ssniff SM R/M-Z+H “Autoclavable Complete Feed for Rats and Mice – Breeding and Maintenance" ad libitum
- Water (e.g. ad libitum): Tap water ad libitum
- Acclimation period: 11 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22±3 °C
- Humidity: 30–70 %
- Air changes: 15-20 air exchanges/h
- Photoperiod (hrs dark / hrs light): 12 h dark/12 h light

Deviation:The temperature (22±3ºC) and the humidity (30–70%) values deviated from the required range during the acclimation period in the animal room. However, these deviations had no effect on the purpose and integrity of the study.

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose only

Vehicle:

air

Remarks on MMAD:

MMAD / GSD: The Mass Median Aerodynamic Diameter (MMAD) of the test atmosphere of all groups was in the range of 1.97-2.73 µm with Geometric Standard Deviation (GSD) of 2.90-3.22. Due to the test item physical properties the Geometric Standard Deviation (GSD) of the test atmosphere was slightly above 3, but was kept in range of 3±10%.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus:TSE Rodent Exposure System (TSE Systems GmbH, Bad Homburg, Germany) which is a flow-past, nose-only exposure unit. This system consists of two, concentric anodised aluminium cylinders, the inner plenum and the outer chamber with 20 circularly arranged exposure ports.The equipment is supported by a computer control system incorporating pressure detectors, mass flow controllers as well as temperature, O2 and CO2 sensors.

- Method of holding animals in test chamber: animals were held in polycarbonate restraint tubes located around the chamber which allow only the animals’ nares to enter the exposure port.

- Source of air: compressed air

- Method of conditioning air: the compressed air was passed through a respiratory quality filter train and condensate separator prior to use.

- System of generating particulates: rotating brush powder disperser (Palas GmbH, Karlsruhe, Germany) located at the top of the exposure chamber

- Temperature, humidity, pressure in air chamber: 22±3 °C, 30–70 % and pressure in air chamber not reported

- Air flow rate: the flow of air through each port was at least 0.5 L/min.

- Method of particle size determination: Particle size analysis of generated atmospheres was performed using a 7-stage cascade impactor of Mercer style (TSE Systems GmbH, Bad Homburg, Germany). Samples were collected at least once during each week of exposure for each concentration tested. Samples were collected from a vacant animal exposure port (animals breathing zone). The collection substrates and the backup filter were weighed before and after sampling and the weight of test item, collected at each stage, calculated by this difference.The total amount collected for each stage was used to determine the cumulative amount below each cut-off point size. In this way, the proportion (%) of aerosol less than < 0.550, 0.550, 0.960, 1.550, 2.105, 3.555, 6.655 and 10.550 µm was calculated. From these data, using the software supplied with the impactor (TSE Systems GmbH, Bad Homburg, Germany), the Mass Median Aerodynamic Diameter (MMAD), and Geometric Standard Deviation (GSD) were calculated. In addition, the proportion (%) of aerosol less than 3 µm was determined.

- Treatment of exhaust air: After passing through the animal’s breathing zone, spent aerosol enters the outer cylinder from where it was exhausted through a suitable filter system.

TEST ATMOSPHERE
- Brief description of analytical method used: The actual 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 GF10 glass fibre filters (Whatman, Germany, ref. no. 10370302). Sampling was normally performed shortly after chamber equilibration and then at regular intervals (approximately hourly intervals) during the exposure and samples were collected from a vacant animal exposure port (animals breathing zone). The difference in the pre- and post-sampling weights, divided by the volume of atmosphere sampled, was equal to the actual achieved test atmosphere concentration.

- Samples taken from breathing zone: yes, samples were collected from a vacant animal exposure port.

VEHICLE (if applicable)
- none

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 deposited on a sampling filter. Sometimes, marked fluctuations in sample concentration occurred. These fluctuations can be explained by the short duration of the sampling, however the average concentration was equal to the target concentrations. The nominal concentration (mass of the test item dispersed into the exposure system in total air flow used for exposure) deviates significantly from mean achieved concentration due to loss of particles in pre-separation devices used for particle size optimization.
The mean achieved concentrations were 0.10; 0.50 and 2.02 mg/L and corresponded to 100; 100 and 101% of the target concentrations respectively.

Duration of treatment / exposure:

14 days

Frequency of treatment:

5 days/ week

Remarks:

Doses / Concentrations:
0.1; 0.5 and 2.0 mg/L
Basis:
other: target concentrations

Remarks:

Doses / Concentrations:
0.10 ; 0.50 and 2,02 mg/L
Basis:
analytical conc.

No. of animals per sex per dose:

5

Control animals:

yes, sham-exposed

Details on study design:

- Dose selection rationale: The target concentration levels were 0.10, 0.50 and 2.0 mg/L of test item. They were set on the basis of available data and information from previous experimental work, including the results of an acute inhalation toxicity study (see section 7.2.2). In this 4–hour exposure study, no death occurred in group of six male and female rats exposed to a mean achieved atmosphere of 5.05 mg/l.

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Clinical observations were performed prior to exposure on the working days, and at least twice during the exposure whilst the animals were still restrained. Following exposure clinical observation was performed at least twice (as soon as practicable after removal from restraint, and approximately one hour after completion of the exposure. During the weekend (no exposure) the clinical signs were recorded once per day only.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were made on all animals outside the home cage weekly, on Day 1, 7, 14.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight of each animal was recorded with precision of 1 g at randomization, then on Day 1 and at least on Days 4, 8, 11, 14 and 15 (prior to necropsy, fasted before the scheduled euthanasia).

FOOD CONSUMPTION: Yes
- Food was recorded on Day 1 and at least on Days 4, 8, 11 and 14. The remaining, non-consumed food given was weighed with a precision of 1g for each cage. The mean individual daily food consumption was calculated per animal.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 15
- Anaesthetic used for blood collection: Yes (pentobarbital)
- Animals fasted: Yes
- How many animals: all animals of all groups
- Parameters checked in table 7.5.2/1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 15
- Animals fasted: Yes
- How many animals: all animals of all groups
- Parameters checked in table 7.5.2/2 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Gross necropsy and macroscopic examination were performed on all animals. After exsanguination, the external appearance was examined, cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed macroscopically. Any abnormality were recorded with details of the location, colour, shape and size, as appropriate.

HISTOPATHOLOGY: Yes
Initial processing was limited to larynx, trachea, lungs, tracheobronchial lymph nodes, nasal cavity and any gross abnormalities for all animals of all groups.

Other examinations:

Yes
The following organs were weighed : Brain, heart, kidneys, liver, lungs, spleen, testes, thymus, adrenals and ovaries.

Statistics:

The heterogeneity of variance between groups were checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance was carried out. If the obtained result was positive, Duncan’s Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorov-Smirnov test. If the data was not normal distributed, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was used. If there was a positive result, the inter-group comparisons was performed using Mann-Whitney U-test.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In the animals exposed to the concentrations of 2.02 mg/L, the following clinical signs were observed:
Slight increase in respiratory rate was observed in 4/5 females on Day 1 and in 2/5 males on Days 3-4,
Slight and in few occasion moderate laboured respiration was noted throughout the study in females. In males, a slight laboured respiration was observed on Day 2 in 3/5 animals,
Slight noisy respiration turning to moderate in two occasions, was observed from Day 2 to Day 10 in 1 to 2 females,
Hunched posture was noted from Day 4 to Day 12 in 1 to 3 females and on Days 10-11 in 1 male,
Sneezing was noted for 2 females on Days 10-11.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In the animals exposed to the concentrations of 2.02 mg/L,, a very slight mean bodyweight loss was recorded during the first days of treatment in both sexes. In comparison with controls, the decrease in mean bodyweight during days 1 to 4 was -2.2% and -0.43% in males and females respectively. In addition, females showed a statistically significant decrease in mean bodyweight gain on the overall period of treatment (-36% compared with controls).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In the animals exposed to the concentrations of 2.02 mg/L, there was a significant decrease in food consumption in both males and females. The lower food consumption was in accordance with the effect on body weight.

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):

An increase in White Blood Count (WBC) was observed in both sexes at all doses but without dose-dependency. In parallel, increase of relative Neutrophils and concomitant decrease of relative Lymphocytes were observed during the hematology analysis. Slight decreased of Red Blood Count and slight increase of Mean Cell Haemoglobin were observed in males. However the changes were slight in magnitude, and were within the physiological range. Therefore these changes were not considered toxicologically significant (see table 7.5.2/1).

Clinical biochemistry findings:

no effects observed

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):

The mean absolute and relative lungs weights, adjusted for both brain and terminal body weight, were significantly higher in all treated groups. They increased in a dose dependent manner up to 2 times in the 2.02 mg/L Group (see table 7.5.2/2).

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

There was evidence of test item-related macroscopic changes in the lungs at the dose levels of 0.50 and 2.02 mg/L.
Enlargement/pale discoloration were observed in 5/5 males and 5/5 females in the 2.02 mg/L group. Pale foci were seen in 2/5 females of the 0.50 mg/L group.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Substance-related findings were noted in the lungs and the tracheobronchial lymph nodes at all dose levels. There was clear relationship of these microscopic findings to the dose.
The alterations in the lungs were characterized as presence of white/pink foreign material in the alveoli, granular in consistency. The presence of foreign alveolar material was generally minimal at low dose and moderate at high dose.
The inflammatory alterations in the lungs ranged from minimal to moderate severity, with the presence of mixed cell population (prominent feature of lymphocytes, macrophages and neutrophils) in the bronchiolar and alveolar compartments. The incidence and severity of the inflammatory responses were generally increased consistently to dose. Additionally, the formation of microgranulomas was appeared by light microscopy in the alveoli of animals treated at 2.02 mg/L. A similar pattern of dose relationship was recorded for the accumulation of foamy macrophages in the tracheobronchial lymph nodes. The low severity (minimal) at0.1 mg/L, increased (mild) at 0.50 mg/L and was moderateat 2.02 mg/L (see table 7.5.2/3).

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
There was no mortality observed during the study.
In the animals exposed to the concentrations of 2.02 mg/L, the following clinical signs were observed:
• Slight increase in respiratory rate was observed in 4/5 females on Day 1 and in 2/5 males on Days 3-4,
• Slight and in few occasion moderate laboured respiration was noted throughout the study in females. In males, a slight laboured respiration was observed on Day 2 in 3/5 animals,
• Slight noisy respiration turning to moderate in two occasions, was observed from Day 2 to Day 10 in 1 to 2 females,
• Hunched posture was noted from Day 4 to Day 12 in 1 to 3 females and on Days 10-11 in 1 male,
• Sneezing was noted for 2 females on Days 10-11.

In the animals exposed to the concentrations of 0.50 mg/L, no significant clinical signs were noted except for 1 male where initially slight subcutaneous mass in the left axillary was recorded from Day 5 which continuously grew until the end of the study.

In the animals exposed to the concentrations of 0.10 mg/L, slightly laboured respiration was noted in 1 female on day 5. No significant clinical signs were noted for the other animals in this group during the exposure period.

No clinical signs were recorded for the air control animals.


BODY WEIGHT AND WEIGHT GAIN
In the animals exposed to the concentrations of 2.02 mg/L,, a very slight mean bodyweight loss was recorded during the first days of treatment in both sexes. In comparison with controls, the decrease in mean bodyweight during days 1 to 4 was -2.2% and -0.43% in males and females respectively. In addition, females showed a statistically significant decrease in mean bodyweight gain on the overall period of treatment (-36% compared with controls).

No effects on bodyweight and bodyweight gain were noted in the animals exposed to 0.50 or 0.10 mg/L when compared with controls.

FOOD CONSUMPTION
In the animals exposed to the concentrations of 2.02 mg/L, there was a significant decrease in food consumption in both males and females. The lower food consumption was in accordance with the effect on body weight.

No effects on food consumption were noted in the animals exposed to 0.50 or 0.10 mg/L when compared with controls.

HAEMATOLOGY
An increase in White Blood Count (WBC) was observed in both sexes at all doses but without dose-dependency. In parallel, increase of relative Neutrophils and concomitant decrease of relative Lymphocytes were observed during the hematology analysis. Slight decreased of Red Blood Count and slight increase of Mean Cell Haemoglobin were observed in males. However the changes were slight in magnitude, and were within the physiological range. Therefore these changes were not considered toxicologically significant (see table 7.5.2/1).

CLINICAL CHEMISTRY

There was no effect of treatment on clinical chemistry.

ORGAN WEIGHTS
The mean absolute and relative lungs weights, adjusted for both brain and terminal body weight, were significantly higher in all treated groups. They increased in a dose dependent manner up to 2 times in the 2.02 mg/L Group (see table 7.5.2/2).

GROSS PATHOLOGY
There was evidence of test item-related macroscopic changes in the lungs at the dose levels of 0.50 and 2.02 mg/L.
Enlargement/pale discoloration were observed in 5/5 males and 5/5 females in the 2.02 mg/L group. Pale foci were seen in 2/5 females of the 0.50 mg/L group.

HISTOPATHOLOGY: NON-NEOPLASTIC
Test item-related findings were noted in the lungs and the tracheobronchial lymph nodes at all dose levels. There was clear relationship of these microscopic findings to the dose.
The alterations in the lungs were characterized as presence of white/pink foreign material in the alveoli, granular in consistency. The presence of foreign alveolar material was generally minimal at low dose and moderate at high dose.
The inflammatory alterations in the lungs ranged from minimal to moderate severity, with the presence of mixed cell population (prominent feature of lymphocytes, macrophages and neutrophils) in the bronchiolar and alveolar compartments. The incidence and severity of the inflammatory responses were generally increased consistently to dose. Additionally, the formation of microgranulomas was appeared by light microscopy in the alveoli of animals treated at 2.02 mg/L. A similar pattern of dose relationship was recorded for the accumulation of foamy macrophages in the tracheobronchial lymph nodes. The low severity (minimal) at0.1 mg/L, increased (mild) at 0.50 mg/L and was moderateat 2.02 mg/L (see table 7.5.2/3).

Key result

Dose descriptor:

LOAEC

Effect level:

0.1 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: At 0.1 mg/L, slight increase in neutrophils and slight concomitant decrease in lymphocytes were observed during the Hematology analysis. These effects were associated with a slight inflammation process in the respiratory tract of the animals

Key result

Critical effects observed:

no

Table 7.5.2/1 Mean values in affected haematologic parameters

GROUP	1 (Control)	2 (Low)	3 (Mid)	4 (High)	Remarks
Concentration (mg/L)	0	0.1	0.5	2.02
Males
White Blood Count – WBC (K/mL)	2.776	3.244	3.664	3.314	NS
Red Blood Count – RBC (M/mL)	8.726	8.384	8.272	8.096*	DN
Neutrophils –NEU, Relative (%)	25.160	21.480	28.320	34.720**	DN
Lymphocytes – LYMPH, Relative (%)	69.620	74.080	66.780	59.040**	DN
Mean Cell Haemoglobin – MCH, pg	18.18	19.00**	19.12**	19.06**	DN
Females
White Blood Count – WBC (K/mL)	1.216	2.394*	2.130*	1.948*	U
Red Blood Count – RBC (M/mL)	7.932	8.108	7.986	8.034	NS
Neutrophils –NEU, Relative (%)	14.660	20.920*	20.420*	26.360**	DN
Lymphocytes – LYMPH, Relative (%)	80.460	73.920*	74.480*	67.900**	DN
Mean Cell Haemoglobin – MCH, pg	18.94	19.32	19.18	19.62	NS

NS = Not Significant

DN =Duncan's Multiple Range Test

U = Mann- Whitney U-Test

*= p<0.05

** = p<0.01

Table 7.5.2/2 Mean values of lungs weights following the 14-day exposure period

GROUP	1 (Control)	2 (Low)	3 (Mid)	4 (High)	Remarks
Concentration (mg/L)	0	0.1	0.5	2.02
Males
Lungs Weight (g)Absolute	1.318	1.620**	2.092**	2.884**	U
Relative to Brain Weight (%)	69.52	83.78**	109.68**	145.41**	U
Relative to Body Weight (%)	0.44	0.54**	0.68**	0.99**	DN
Females
Lungs Weight (g)Absolute	1.014	1.286**	1.804**	2.256**	DN
Relative to Brain Weight (%)	55.01	73.04**	98.92**	124.25**	DN
Relative to Body Weight (%)	0.51	0.64**	0.88**	1.17**	DN

DN =Duncan's Multiple Range Test

U = Mann- Whitney U-Test

*= p<0.05

** = p<0.01

Table 7.5.3/3 Summary of histopatology data for lungs and tracheobronchial lymph nodes

Sex	male	male	male	male	female	female	female	female
Concentration (mg/l)	0	0.10	0.50	2.02	0	0.10	0.50	2.02
Number of Animals on Study	5	5	5	5	5	5	5	5
LUNGS;
Examined	(5)	(5)	(5)	(5)	(5)	(5)	(5)	(5)
WithinLimits	5	0	0	0	5	0	0	0
Foreign Material; alveolar; all lobes, Glanular, white/pink	(0)	(5)	(5)	(5)	(0)	(5)	(5)	(5)
minimal	0	4	1	0	0	5	0	0
mild	0	1	4	0	0	0	3	0
Foreign Material; alveolar; all lobes, Glanular, white/pink; correlated with necropsy.	(0)	(5)	(5)	(5)	(0)	(5)	(5)	(5)
mild	0	0	0	1	0	0	2	2
moderate	0	0	0	4	0	0	0	3
Inflammation, Bronchioloalveolar; mixed; all lobes	(0)	(0)	(5)	(5)	(0)	(0)	(5)	(5)
minimal	0	0	5	0	0	0	5	0
mild	0	0	0	4	0	0	0	1
moderate	0	0	0	1	0	0	0	1
Inflammation, Bronchioloalveolar; mixed; all lobes correlated with necropsy	(0)	(0)	(5)	(5)	(0)	(0)	(5)	(5)
mild	0	0	0	4	0	0	0	3
Microgranuloma; alveolar; multifocal	(0)	(0)	(0)	(3)	(0)	(0)	(0)	(3)
minimal	0	0	0	1	0	0	0	2
mild	0	0	0	2	0	0	0	1
Cell Infiltrate, Mixed Cellular; bronchiole; alveolus, all lobes	(0)	(5)	(0)	(0)	(0)	(5)	(0)	(0)
minimal	0	3	0	0	0	5	0	0
mild	0	2	0	0	0	0	0	0
LYMPH NODE, LUNG-ASSOCIATED;
Examined	(5)	(5)	(5)	(5)	(4)	(5)	(5)	(5)
WithinLimits	5	0	0	0	4	2	0	0
Not Examined: NOT PRESENT	0	0	0	0	1	0	0	0
Accumulation, Foamy Macrophage; medulla; paracortex	(0)	(5)	(5)	(5)	(0)	(3)	(5)	(5)
minimal	0	2	1	0	0	3	2	0
mild	0	3	4	0	0	0	3	1
moderate	0	0	0	5	0	0	0	4

Conclusions:

Exposure to the test substance at gravimetrically determined dose levels of 0.10, 0.50 and 2.02 mg/L air resulted in findings in the lungs and tracheobronchial lymph nodes. In the lungs, they consisted of inflammatory reactions ranging from minimal to moderate severity, with the presence of mixed cell population. Also, foreign material was seen in the alveoli. The incidence and severity of the inflammatory responses and presence of foreign alveolar material were generally increased consistently to dose. A similar pattern of dose relationship was recorded for the accumulation of macrophages in the tracheobronchial lymph nodes. The low severity (minimal) in Low dose, increased (mild) in Mid dose and was moderate in High dose animals.

Executive summary:

In a pilot study performed in compliance with Good Laboratory Practices, the test substance was administered by nose-only inhalation exposure for 6 hours per day (5 days per week) to Wistar rats.

The test item was administered as a dry aerosol. The animals were exposed on 10 occasions during a 14-day period. Three groups, each comprising five male and five female rats received the test substance at target exposure levels of of 0.1, 0.5 and 2.00 mg/L. A similarly constituted Control group received air at the same operating conditions as the other groups.

The mortality observations were performed twice daily. The general clinical observations were recorded five times for the exposed animals on the days of exposure. Detailed clinical examination was performed on Days 1, 7 and 14 prior to the 6-hour exposure. Body weight and food consumption were measured on Days 1, 4, 8, 11 and 14. Blood samples were collected for clinical pathology at necropsy. Gross macroscopic examination was performed at necropsy one day after the last treatment. Selected organs were weighed. Histopathology investigation was performed on selected tissues.

The achieved aerosol concentrations were 0.10, 0.50 and 2.02 mg/L (100, 100 and101% of target). The Mass Median Aerodynamic Diameters for all treated groups were within the ideal range (1-3 µm) for a repeat dose inhalation study. 

There was no mortality during the study. Clinical signs related to treatment were noted only in animals given 2.02 mg/L. All but one female and only 2 males had a transient slight increase in respiratory rate. In females, slight and in few occasions moderate laboured and/ or noisy respiration was observed throughout the study. In males, slight laboured respiration was noted only on day 2 in 3 animals. Hunched posture was observed from day 4 to day 10 in 1 to 3 females and on days 10-11 in 1 male. In addition, sneezing was noted for two females on Day 10 and 11. 

Changes in bodyweight and bodyweight gain were noted only in animals that received 2.02 mg/L . Temporary mean bodyweight loss was observed at the beginning of the study in both sexes on (Days 1-4) ( -2.2% and -0.43% in males and females respectively). This bodyweight loss correlated with the significant decrease in food consumption noted during the same treatment-period. In addition, females, showed a statistically significant decrease in mean bodyweight gain on the overall period of treatment (-36% compared with controls).

Following 2 weeks of treatment, an increase in White Blood Count was observed in both sexes at all doses but without dose-dependency. In addition, statistically significant increase of relative Neutrophils and concomitant decrease of relative Lymphocytes were observed in males given 2.02 mg/l and in all treated females. These changes in haematology might be contributed to changes in the lungs. Slight decreased of Red Blood Count and slight increase of Mean Cell Haemoglobin were also observed in males. However the changes were slight in magnitude, and were within the physiological range. Therefore these changes were not considered toxicologically significant. There was no effect of treatment noted during the evaluation of the clinical chemistry parameters.

Macroscopic examination revealed enlargement and/or pale discoloration of the lungs in animals given 0.50 or 2.02 mg/L. Treatment related histopathological changes were seen in lungs and tracheobronchial lymph nodes at all doses levels.There was clear relationship of these microscopic findings to the dose. The alterations in the lungs were characterized as presence of white/pink foreign material in the alveoli. The presence of foreign alveolar material was generally minimal at low dose and moderate at high dose. The inflammatory reactions in the lungs ranged from minimal to moderate severity, with the presence of mixed cell population in the bronchiolar and alveolar compartments. The incidence and severity of the inflammatory responses were generally increased consistently with dose. The formation of microgranulomas was observed microscopically in the alveoli of animals treated at a dose level of 2.02 mg/L. A similar pattern of dose relationship was recorded for the accumulation of foamy macrophages in the tracheobronchial lymph nodes with low severity (minimal) in Low dose, increased (mild) in Mid dose and moderate in the High dose group. The mean absolute and relative lungs weights adjusted for both brain and terminal body weight were significantly higher in males and females at dose levels of 0.1, 0.5 and 2.02 mg/L compared to controls.

Based on these findings, it was anticipated that a target exposure level of 100 µg/L at maximum would be tolerated for a 90 day exposure in the same species.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEC

100 mg/m³

Study duration:

subacute

Species:

rat

Quality of whole database:

One study available.

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available study data, classification for repeated dose toxicity is not justified.