Reaction products of 12-hydroxyoctadecanoic acid with ethane-1,2-diamine and hexane-1,6-diamine and 1,3-phenylenedimethanamine

Endpoint summary

Description of key information

 In a Dose Range-finding toxicity study performed by oral route, rats treated for 14 days showed no remarkable effects up to a dose-level of 1000 mg/kg bw/day.  
In a  subacute inhalation toxicity study performed in accordance with OECD guideline No 412 and in compliance with GLP, no systemic effects were observed in rats exposed up to 219 mg/m3 air  but histopathological evaluation reported local pulmonary effects. The test substance was responsible for a mild pulmonary inflammation. The changes in haematology correlated with the microscopic findings. Based on these findings, the NOAEC was set to 8.11 mg/m3.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From2013/06/11 to 2013/07/11

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study with acceptable restrictions

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)

GLP compliance:

no

Remarks:

Range -finding toxicity study

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Italia, Calco, Italy.
- Age at study initiation: approximately 10 weeks old on the day of treatment
- Mean body weight at study initiation: for the males, 355 g (range: 324 g to 397 g) and the females 258 g (range: 240 g to 284 g).
- Fasting period before study: no
- Housing: in polycarbonate cages with stainless steel lids
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: a period of 6 days before the beginning of the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12

IN-LIFE DATES: 11 June 2013 to 01 July 2013

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION
The test item was administered as a suspension in the vehicle. The test item was ground to a fine powder, using a mortar and pestle, and then mixed with the required quantity of vehicle.
The test item dose formulations were prepared daily and were delivered to the study room at room temperature and protected from light.
This preparation process was validated after this Dose-Range Finding study and prior to the subsequent reproduction/developmental toxicity screening test performed in rats with this test item(CiToxLAB France/Study No. 40216 RSR). During the homogeneity and stability study (CiToxLAB
France/Study No. 40218 AHS), the analytical sequences of the samples prepared according to the preparation procedure described in the section “Dose formulation preparation” failed due to standard variability. Nevertheless and considering the obtained profile of the dose formulation samples, it was decided to modifiy the preparation process for the lowest dose formulations. Thus, only the dose formulation of the test item E99434 at 1000 mg/kg bw/day (i.e. 200 mg/mL) could be considered as homogeneous. This dose-level did not show any adverse or clinical and macroscopic toxic effect, it was considered that the lack of homogeneity data at the other dose-levels did not compromise the validity and integrity of the results of
this study.

VEHICLE
- Justification for use and choice of vehicle (if other than water): homogeneous 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

Duration of treatment / exposure:

14 days

Frequency of treatment:

7 days/week

Remarks:

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

No. of animals per sex per dose:

5 males and 5 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale:
The dose-levels used in this study (0, 100, 300 and 1000 mg/kg bw/day) were selected on the basis of the results of an acute oral toxicity study. In this study, two groups of three Sprague-Dawley females were treated once with the test item at 2000 mg/kg bw and one group of three animals were treated once with the test item at 300 mg/kg bw in corn oil and then observed for fourteen days. Piloerection was observed in three out six females given 2000 mg/kg bw 4 hours after treatment on day 1 and on day 2. No clinical signs were observed in other animals.
Based on these observations, a dose-level of 1000 mg/kg bw/day was selected for the high dose. The dose levels for the mid and low doses (300 and 100 mg/kg bw/day respectively) were selected to explore any possible dose relationship.

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a day.

BODY WEIGHT: Yes
The body weight of each animal was recorded once before the beginning of the treatment period, on the first day of treatment, and then on days 4, 8, 11 and 14 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 quantity of food consumed by the animals in each cage was measured twice weekly (days 1-4, 4-8, 8-11 and 11-14) during the treatment period.
Food consumption was calculated per animal and per day.When one of the animals in the same cage died, the number of days for which that animal had been present in the cage was taken into consideration for the calculation of food consumption.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER: ORGAN WEIGHTS
The body weight of each animal was recorded before sacrifice at the end of the treatment period. Epididymides, kidneys, liver , ovaries with oviducts, spleen and testes were weighed wet as soon as possible after dissection.
The ratio of organ weight to body weight was calculated.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
A complete macroscopic post-mortem examination was performed on all study animals. This included
examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain, the
thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its
associated organs and tissues.

HISTOPATHOLOGY: No
No microscopic examination was performed in first instance.

Other examinations:

None

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, Dunnett 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 normally 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 Dunn test.

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):

not examined

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:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Details on results:

MORTALITY:
One control female was prematurely sacrificed on day 4. Prior to death, this female showed piloerection, dyspnea, hunched posture, increase in size of the thorax (right lateral region) and weight loss from days 1 to 4 (-14 g). These clinical signs were suspected to be related to a trauma induced by the technical gavage procedure, and were not considered to be test item-related.
They were no other mortalities observed during the study.

CLINICAL SIGNS:
No clinical signs were observed in surviving animals at any dose-levels.

BODY WEIGHT:
There were no test item-related changes in body weight and body weight gain during the study at any dose level.
Between days 1 and 4, statistical differences were observed between control and treated animals. These variations were due to body weight loss in control animals, particularly for the preterminally euthanized female.

FOOD CONSUMPTION:
There were no test item-related effects on food consumption during the study.

ORGAN WEIGHTS:
There were no changes in the mean organ weights which suggested a test item-related effect.
The mean relative kidney weights were statistically significantly lower (10%, p<0.05) in females given 100 or 300 mg/kg/day. In the absence of a similar trend at 1000 mg/kg/day, these differences were considered to be fortuitous.
Other changes in the mean organ weights were considered to be incidental as they were small in amplitude, seen in one sex only or not dose-related.

GROSS PATHOLOGY:
There were no changes at necropsy which indicated a test item-related effect.
The red discoloration seen in the thymus of a male given the test item at 100 mg/kg/day and the focal yellow discoloration of the median lobe of the liver in a female given 1000 mg/kg/day were considered to be part of the normal background of changes commonly seen in rats and therefore without any relationship with the test item.
The necropsy of the female sacrificed on day 4 showed a pouch with a white thick content in the subcutaneous tissue. This observation was consistent with a trauma induced by the technical gavage procedures.

Dose descriptor:

other: maximum tolerated dose

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Critical effects observed:

not specified

Conclusions:

The administration of the test item daily by the oral route to male and female Sprague-Dawley rats at dose-levels of 100, 300 or 1000 mg/kg bw/day for two weeks was clinically well tolerated.
Consequently, the same dose-levels were chosen for the subsequent reproduction/developmental toxicity screening test in this species.

Executive summary:

The objective of this dose-range finding study was to evaluate the potential toxicity of the test item following daily oral administration by gavage to rats for 14 days in order to select the dose-levels for a further OECD 421 reproductive/developmental toxicity screening in this species.

Three groups of five male and five female Sprague-Dawley rats received the test item daily, by gavage at dose-levels of 100, 300 or 1000 mg/kg bw/day for 14 days. A group of five males and five females received the vehicle alone,corn oil under the same experimental conditions and acted as a control group. The dose formulations were administered under a constant dosage-volume of 5 mL/kg/day.

The animals were checked daily for mortality and clinical signs. Body weights were recorded once during the pre-treatment period, on the first day of treatment and then on days 4, 8, 11 and 14 before necropsy. Food consumption was recorded twice weekly during the treatment period.

On completion of the treatment period, animals were sacrificed. The epididymides, kidneys, liver, ovaries, spleen and testes were weighed and a full macroscopic post‑mortem examination was performed.

  No unscheduled deaths related to the test item treatment occurred during the study. No clinical signs were observed in animals.

There were no test item-related changes in body weight and body weight gain during the study at any dose‑level, and no relevant effects were recorded on food consumption.

No test item-related changes were observed in the mean organ weights or at the macroscopic post-mortem examination.

The administration of the test item daily by the oral route to male and female Sprague‑Dawley rats at dose‑levels of 100, 300 or 1000 mg/kg/day for 2 weeks was clinically well tolerated.

Consequently, the same dose-levels were chosen for a further reproduction/developmental toxicity screening test in this species.

 

 

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Dose Range-Finding study with a limited number of parameters assessed

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:

From 2014-03-07 to 2015-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.8 (Subacute Inhalation Toxicity: 28-Day Study)

Deviations:

no

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: Harlan (UK) Ltd.
- Age at study initiation:60 to 66 days
- Weight at study initiation: 217 to 261 g (males) and 153 to 188 g (females)
- Fasting period before study:no
- Housing: The animals were housed five of one sex per cage
- Diet: Ad libitum, standard rodent diet (Rat and Mouse n°1 Maintenance Diet). This diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
- Water: Ad libitum, potable water taken from the public supply.
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19 to 23°C
- Humidity: 40 to 70%
- Air changes (per hr):Each animal room was supplied with 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:

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 1.3-1.6 µm with Geometric Standard Deviation (GSD) of 2.52-2.75.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: flow through nose only chamber. This system was an aluminium alloy construction comprising a base unit, three animal exposure sections and a top section.
- Method of holding animals in test chamber: Rats were held in polycarbonate tubes with their snouts protruding from the end of the tubes into the exposure chamber.
- Source and rate of air: From in-house compressed air system(breathing quality), Generator flow: 19L/minute - Supplementary flow: 10-60 L/minute
- System of generating particulates/aerosols: Wright Dust Feed mechanism designed to produce and maintain atmospheres of dust by suspending material scraped from the surface of a compressed powder in a stream of dry air. The concentrations of dust were altered by changing the gear ratio (and therefore the speed of rotation of the compressed powder towards the scraper blade) of the mechanism.
- Temperature, humidity, pressure in air chamber:
Temperature was measured using an electronic thermometer inserted into the inhalation chamber. The mean chamber temperatures were all within expected ranges (22.2; 22.2; 22.4 and 22.0 °C for controls, low, mid and high-dosed groups).
Humidity and pressure in air chamber were not reported.
- Air flow rate: Airflow to Wright dust Feed was 19 L/minute for all groups. Supplementary airflows were 10, 60, 10 and 10 L/minute for the control, low, mid and high-dose groups respectively.
- Method of particle size determination: Particle size analysis of generated atmospheres was performed using a 6-stage cascade impactor (Marple 296). Samples were collected at least once during each week of exposure for each concentration tested. Samples were also 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, was 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 < 0.42, 0.76, 1.27, 2.86, 4.9 and 8.0 µm (aerodynamic diameter) was calculated. From this data, the Mass Median Aerodynamic Diameter (MMAD), and Geometric Standard Deviation (GSD) were calculated.
- Treatment of exhaust air: Extract airflow was drawn by in-house vacuum system at a flow rate of 30, 80, 30 and 30 L/minute for the control, low, mid and high-dose groups respectively. the airflow was filtered locally.

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 a glass fibre filters. Sampling was performed at least three times during each exposure and for each dose-group.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.
In addition, the aerosol concentrations measured by gravimetric analysis were checked by a chemical analysis once every week. High Performance Liquid Chromatography with UV detection analytical method was used.

- 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 during each exposure by gravimetrical analysis of the test item deposited on a sampling filter. Once every week, the gravimetric analysis was coupled to analytical analysis by HPLC with UV detection to check the accuracy of the gravimetric method.
The mean achieved concentrations were 8.11; 39.6 and 219 µg/L and corresponded to 113; 110 and 122% of the target concentrations respectively. The mean exposure concentrations measured by chemical and gravimetric analysis correlated well.

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

5 days / week

Remarks:

Doses / Concentrations:
7.2, 36, 180 µg/L
Basis:
other: target

Remarks:

Doses / Concentrations:
8.11, 39.6, 219 µg/L
Basis:
analytical conc.

No. of animals per sex per dose:

3 groups, each comprising 5 male and 5 female rats received the test substance at target exposure levels of 7.2, 36 or 180 µg/L. A similarly constituted Control group received air only, at the same operating conditions as the high dose group.

Control animals:

yes, sham-exposed

Details on study design:

- Dose selection rationale:
In a previously conducted two-week repeated dose inhalation toxicity study, rats were exposed 5 days/week, 6 hours/day to 0.0691, 0.298 or 1.55 mg/L (69.1, 298 or 1550 μg/L ) of the test substance. Histopathological treatment related findings were evident in the lungs and tracheobronchial lymph nodes at all exposure levels. These findings were of minimal severity and were not considered to be adverse.
In another study with a similar compound, adverse respiratory tract histopathological findings were apparent at 21.2 and 101 µg/L following 13 weeks of exposures; the NOAEL in that study was 3.30 µg/L.
Considering the results from the previous studies and the likely progression of lung changes following exposure to the test substance over a longer duration, a high exposure level targeted at 180 μg/L was selected and was anticipated to induce treatment related changes similar to those previously seen but was expected to be tolerated for 4 weeks. Target exposure levels of 36 and 7.2 μgL were selected for the intermediate and low groups respectively to identify a no-observed adverse effect level and to explore any possible dose relationship.

Positive control:

none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Cages were inspected daily for evidence of ill-health amongst the occupants.
Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.
During the acclimatisation period, observations of the animals and their cages were recorded at least once per day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily during Weeks 1 to 4 of treatment, detailed observations were recorded at the following times in relation to dose administration:
* Pre-exposure observation
* As each animal is returned to its home cage
* As late as possible in the working day

BODY WEIGHT: Yes
- Time schedule for examinations:The weight of each animal was recorded one week before treatment commenced (Day -7), on the day that treatment commenced (Day 1), twice weekly throughout the study and before necropsy.

FOOD CONSUMPTION: Yes
-The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded for the week before treatment started (Week -1), and each week throughout the treatment. From these records the mean weekly consumption per animal (g/animal/week) was calculated for each cage.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: During Week 4 of treatment and before dosing for all study animals.
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: Yes, overnight withdrawal of food.
- How many animals: 40.
- Parameters examined: Haematocrit (Hct), Haemoglobin concentration (Hb), Erythrocyte count (RBC), Reticulocyte count (RETA), Mean cell haemoglobin (MCH), Mean cell haemoglobin concentration (MCHC), Mean cell volume (MCV), Total white cell count (WBC), Differential WBC count including Neutrophils (N), Lymphocytes (L), Eosinophils (E), Basophils (B), Monocytes (M), Large unstained cells (LUC), Platelet count (Plt), Prothrombin time (PTP) and Activated partial thromboplastin time (APTT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: During Week 4 of treatment and before dosing for all study animals.
- Animals fasted: Yes, overnight withdrawal of food.
- How many animals: 40
- Parameters examined: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Total bilirubin (Bili), Urea, Creatinine (Creat), Glucose (Gluc), Total cholesterol (Chol), Triglycerides (Trig), Sodium (Na), Potassium (K), Chloride (Cl), Calcium (Ca), Inorganic phosphorus (Phos), Total protein (Total Prot) and Albumin (Alb).

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

All study animals were sacrificed following 4 weeks of exposures.
GROSS PATHOLOGY: Yes
All animals were subject to a detailed necropsy. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded.
HISTOPATHOLOGY: Yes
The following tissues were examined for all study animals of Groups 1 (Control) and 4 (219 µg/L) sacrificed on completion of the 4-week treatment period:
Adrenals - cortex and medulla
Brain - cerebellum, cerebrum and midbrain
Heart - included auricular and ventricular regions
Kidneys - included cortex, medulla and papilla regions
Larynx - 5 sections
Liver - section from two main lobes
Lungs - section from all major lobes, to include bronchi
Nasal turbinates -4 levels including nasal cavity, paranasal sinuses, nasopharynx and nasopharynx duct and nasal associated lymphoid tissue
Oesophagus
Ovaries
Seminal vesicles
Spinal cord - transverse and longitudinal section at the cervical, lumbar and thoracic levels
Spleen
Stomach - included keratinised, glandular and antrum in sections
Testes
Thymus
Thyroid - included parathyroids in section where possible
Trachea - including bifurcation
Uterus - uterine body with cervix section
In addition:
- all mediastinal lymph nodes showing macroscopic abnormality were examined,
- Lungs, larynx, trachea (including bifurcation), nasal turbinates and tracheobronchial lymph nodes were examined for all study animals of groups 2 (8.11 µg/L) and 3 (39.6 µg/L).

Other examinations:

- HAEMATOLOGY, BONE MARROW:
Bone marrow smears were prepared immediately following death on completion of the scheduled treatment period. The smears from all animals of Groups 1 (Control) and 4 (219 µg/L) were examined to assess the cellularity, distribution and morphology of the marrow. The smears from animals of the intermediate and low dose groups were not examined.

- ORGAN WEIGHTS:
The following organs, taken from each animal killed after 4 weeks of treatment were weighted: Adrenals, Brain, Heart, Kidneys, Liver, Lungs with mainstem bronchi, Spleen, Testes and Thymus.
Bilateral organs were weighed together. Organ weights were also adjusted for terminal bodyweight, using the weight recorded before necropsy.

Statistics:

All analyses were carried out using the individual animal as the basic experimental unit.
# The following sequence of statistical tests was used for bodyweight, organ weight and clinical pathology:
A parametric analysis was performed if Bartlett's test for variance homogeneity was not significant at the 1% level. The F1 approximate test was applied. If the F1 approximate test was not significant at the 1% level, Williams' test was applied. If the F1 approximate test was significant, suggesting that the dose response was not monotone, Dunnett's test was performed instead.
A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. The H1 approximate test was applied. If the H1 approximate test was not significant at the 1% level, Shirley's test was applied. If the H1 approximate test was significant, suggesting that the dose-response was not monotone, Steel's test was performed instead.
For pathology if 75% of the data (across all groups) were the same value, for example c, Fisher’s Exact tests were performed. Treatment groups were compared using pairwise comparisons of each dose group against the control both for i) values c, as applicable.
# For organ weight data, analysis of covariance was performed using terminal bodyweight as covariate, unless non-parametric methods were applied.

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):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no mortalities and no test article-related clinical signs during the study.

BODY WEIGHT AND WEIGHT GAIN
There were no treatment related effects on body weight and weight gain.
A weight loss occurred on Day 25 for all groups, including control and was due to overnight food withdrawal before the blood sampling procedures for haematology and blood chemistry on Day 24.

FOOD CONSUMPTION
There were no treatment related changes on food consumption.

HAEMATOLOGY
Higher group mean neutrophil counts were evident for females exposed to 39.6 µg/L and both sexes exposed to 219 µg/L, up to 1.52X and 2.5X control for males and females, respectively. The neutrophil count was dose-dependently increased in females.
Lower group mean lymphocyte counts were evident in males exposed to 219 µg/L compared with control (0.77X control). In treated females, there was a decrease without exposure relationship in mean lymphocyte counts when compared with control (0.75X, 0.66X and 0.79X for animals exposed to 8.11, 39.6 or 219 µg/L, respectively).
All other differences between the control and treated groups, were minor or were confined to one sex and were therefore attributed to normal biological variation.

CLINICAL CHEMISTRY
Mean phosphorus concentrations were lower in males exposed to 39.6 or 219 µg/L (as low as 0.82X ) compared with control. There was no dose relationship or similar effect in the females, this was therefore considered not to be an effect of treatment.
Compared with control, higher creatinine concentrations, not exposure related were observed in males exposed to 39.6 or 219 µg/L (1.13X control) and all treated female groups (1.24X, 1.15X and 1.12X for animals exposed to 8.11, 39.6 or 219 µg/L, respectively). The higher creatinine concentrations were not associated with any histopathological changes and, were not dose-related. They were considered to be due to normal biological variation and not a reaction to treatment with E99434.
Lower mean triglycerides were evident in males exposed to 219 µg/L, however the individual values were not consistently out of the control range, a low value for one animal lowered the group mean, and this was therefore considered not to be test article-related.
All other differences between the control and treated groups, including those that attained a degree of statistical significance were minor, lacked exposure relationship or were confined to one sex and were therefore attributed to normal biological variation.

ORGAN WEIGHTS
There was a statistical significant increase in mean lung and bronchi weights (adjusted for terminal bodyweight) in both sexes from 39.6 µg/L, (up to 1.7X and 1.9X, for males and females respectively).
All other differences between the control and treated groups, including those that attained statistical significance were minor, lacked exposure-relationship, or were confined to one sex and were therefore attributed to normal biological variation.

GROSS PATHOLOGY
Enlargement and pale appearance of the tracheobronchial lymph nodes were noted in nearly all animals receiving 39.6 or 219 µg/L. Enlargement and pale appearance of the mediastinal lymph nodes were also noted in half or more of the animals receiving 39.6 or 219 µg/L (See 7.5.2/1 Macropathology results in any other information on results incl.tables).

HISTOPATHOLOGY: NON-NEOPLASTIC
Histopathological findings related to treatment were seen in the lungs, larynx, tracheobronchial and mediastinal lymph nodes:

A variety of findings indicative of mild irritation and clearance were seen in the lungs of both sexes exposed to 39.6 or 219 µg/L. These findings included bronchiolo-alveolar hyperplasia in the terminal bronchioles and alveoli, diffuse foamy alveolar macrophages, foamy alveolar macrophages around terminal bronchioles, and eosinophilic material in the alveoli and in one female (exposed to 219 µg/L), in the bronchioles. Findings in the lung of rats given 8.11 μg/L were confined to a minimal macrophage response around the terminal bronchioles in two males and one female.

In the tracheobronchial and mediastinal lymph nodes, increased cellularity and macrophage aggregates were observed in the majority of treated rats exposed to 39.6 or 219 µg/L. These findings correlated with enlargement and pallor observed during necropsy (See 7.5.2/2 Histopathology results in any other information on results incl.tables).

OTHER FINDINGS
Bone marrow analysis:
The cellularity, distribution and morphology of the marrow was unaffected by the treatment.

Dose descriptor:

NOEC

Effect level:

8.11 other: µg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: At 39.6 and 219 µg/L: -Microscopic findings observed in lungs suggestive of hyperplasia, - Changes in Haematology indicative of inflammatory reaction and consistent with the findings recorded microscopically.

Critical effects observed:

not specified

7.5.2 /1 Macropathology results

Tracheobronchial and Mediastinal lymph nodes

Enlargement and/or abnormal colour (pale) of these lymph nodes was seen in the majority of animals exposed to 39.6 or 219 µg/L.

Summary of findings in the tracheobronchial and mediastinal lymph nodes for animals killed after 4 weeks of treatment
Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Exposure level (µg/L)	0	8.11	39.6	219	0	8.11	39.6	219
Tracheobronchial LN enlarged	0	0	4	5	0	0	5	4
Tracheobronchial LN abnormal colour (pale)	0	0	5	4	0	0	4	3
Number of animals examined	5	5	5	5	5	5	5	5
Mediastinal LN enlarged	0	0	3	3	0	0	4	3
Mediastinal LN abnormal colour (pale)	0	0	4	3	0	0	4	3
Number of animals examined	5	5	5	5	5	5	5	5

 

7.5.2 /2 Histopathology results

Lungs

Summary of treatment related findings in the lungs for animals killed after 4 weeks of treatment
Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Exposure level (µg/L)	0	8.11	39.6	219	0	8.11	39.6	219
Hyperplasia, bronchiolo-alveolar, terminal bronchioles
Minimal	0	0	0	2	0	0	0	3
Total	0	0	0	2	0	0	0	3
Hyperplasia, bronchiolo-alveolar, alveoli
Minimal	0	0	4	2	0	0	2	0
Slight	0	0	0	2	0	0	2	5
Total	0	0	4	4	0	0	4	5
Alveolar macrophages, foamy, diffuse
Minimal	0	0	2	2	0	0	4	0
Slight	0	0	3	3	0	0	1	5
Total	0	0	5	5	0	0	5	5
Macrophages, foamy, terminal bronchioles
Minimal	0	2	2	0	0	1	3	0
Slight	0	0	1	0	0	0	0	0
Total	0	2	3	0	0	1	3	0
Eosinophilic material, alveoli
Minimal	0	0	0	0	0	0	3	0
Slight	0	0	0	2	0	0	1	0
Moderate	0	0	0	3	0	0	0	5
Total	0	0	0	5	0	0	4	5
Eosinophilic material, terminal bronchioles
Minimal	0	0	0	0	0	0	0	1
Total	0	0	0	0	0	0	0	1
Number of tissues examined	5	5	5	5	5	5	5	5

Tracheobronchial lymph nodes

Summary of treatment related findings in the tracheobronchial lymph nodes for animals killed after   4 weeks of treatment
Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Exposure level (µg/L)	0	8.11	39.6	219	0	8.11	39.6	219
Cellularity increased, Generalised
Minimal	0	0	0	0	0	0	0	2
Slight	0	0	3	4	0	0	4	1
Total	0	0	3	4	0	0	4	3
Aggregates, macrophages
Minimal	0	0	5	4	0	0	4	3
Total	0	0	5	4	0	0	4	3
Number of tissues examined	5	5	5	5	5	5	5	4

Mediastinal lymph nodes

Summary of treatment related findings in the tracheobronchial lymph nodes for animals killed after 4 weeks of treatment
Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Exposure level (µg/L)	0	8.11	39.6	219	0	8.11	39.6	219
Cellularity increased, Generalised
Minimal	0	0	0	0	0	0	0	1
Slight	0	0	3	2	0	0	3	0
Moderate	0	0	0	1	0	0	0	0
Total	0	0	3	3	0	0	3	1
Aggregates, macrophages
Minimal	0	0	4	4	0	0	3	3
Total	0	0	4	4	0	0	3	3
Number of tissues examined	0	0	4	5	0	0	4	3

Conclusions:

The test article was administered by snout-only inhalation administration, for 6 hours a day, 5 days a week, for 4 weeks at achieved aerosol concentrations of 8.11, 39.6 or 219 µg/L.
In the lungs, higher concentrations of the test article produced bronchioloalveolar hyperplasia in the terminal bronchioles and alveoli which are a common site for hyperplastic and inflammatory findings in rat inhalation studies. There were also increased macrophages and eosinophilic material in the alveoli and terminal bronchioles. These findings were indicative of mild irritation. They correlated with the elevation of plasma neutrophils in the lack of any abnormalities in bone marrow and with the dose-dependent increase in lungs and bronchi weight. Higher concentrations of the test article also resulted in increased cellularity and macrophage aggregates in the tracheobronchial and mediastinal lymph nodes. These findings correlated with enlargement and pallor observed at necropsy .They were considered a likely secondary response to the increased influx of macrophages clearing the test material from the terminal airways and alveolar spaces and trafficking to the local draining lymph nodes.
At 8.11 μg/L, a minimal macrophage response around the terminal bronchioles was noted in two males and one female. These findings were considered to be the result of the normal physiological response following inhalation of particulate matter, and given the low incidence and severity, and their focal nature , they were considered to be non-adverse.
On the basis of these findings, the exposure level of 8.11 µg/L was therefore considered to represent the no observed adverse effect level (NOAEL) for this study.

Executive summary:

The cumulative toxicity of the test item was assessed when administered to Wistar rats by snout-only inhalation administration for 6 hours per day, 5 days per week, over a period of 4 weeks. The study was designated to provide a rational basis for the assessment of the toxicological risk to man.

Three groups, each comprising five male and five female rats, received the test item at target exposure levels of 7.2, 36 or 180 µg/L. A similarly constituted control group received air at the same operating conditions as the 180 µg/L group. During the study, clinical condition, body weight, food consumption, haematology (peripheral blood), haematology (bone marrow), blood chemistry,organ weight, macropathology and histopathology investigations were undertaken.

The achieved gravimetric aerosol concentrations were 8.11, 39.6 and 219 µg/L (113, 110 and 122% of the target concentrations). The Mass Median Aerodynamic Diameters for all treated groups were within the ideal range (1-3µm) for a repeat dose inhalation study.

Test article-related effects were observed in haematology at 39.6 and 219μg/L for both sexes during the treatment. Higher neutrophil counts were observed for females exposed to 39.6 µg/L and both sexes exposed to 219 µg/L. Lower group mean lymphocyte counts were evident in males exposed to 219 µg/L. In treated females, a decrease in mean lymphocyte counts without exposure relationship was observed when compared with control. No abnormalities were observed in the bone marrow thus confirming that changes in the haematology parameters were secondary to the local inflammatory effects observed in the lungs.

Lower phosphorus concentrations in males exposed to 39.6 or 219 µg/L and higher creatinine concentrations in both sexes were observed at all exposure levels. They were not associated with any histopathological changes and not dose-related and were therefore considered not to be of toxicological importance.

A statistical significant increase in mean lung and bronchi weights was observed in both sexes from 39.6 µg/L. Macroscopically enlargement and/or abnormal colour (pale) of the tracheobronchial and mediastinal lymph nodes was also seen in animals exposed to 39.6 or 219 µg/L.

Microscopically changes related to treatment with E99434 were seen in the lungs, tracheobronchial and mediastinal lymph nodes. A variety of findings indicative of mild irritation (hyperplasia) and clearance (macrophage) were seen in the lungs of both sexes exposed to 39.6 or 219 µg/L. These findings included bronchiolo-alveolar hyperplasia in the terminal bronchioles and alveoli, diffuse foamy alveolar macrophages, foamy alveolar macrophages around terminal bronchioles, and eosinophilic material in the alveoli and in one female exposed to 219 µg/L, in the bronchioles. In the tracheobronchial and mediastinal lymph nodes generalised increased cellularity and macrophage aggregates were observed in animals exposed to 39.6 or 219µg/L. These findings correlated with enlargement and pallor observed at necropsy . They were considered a likely secondary response to the increased influx of macrophages clearing the test material from the terminal airways and alveolar spaces and trafficking to the local draining lymph nodes.

At 8.11μg/L , the findings in the lungs were confined to a minimal macrophage response around the terminal bronchioles in two males and one female. These findings were considered to be a normal physiological response following inhalation of particle matter and were not adverse. The increase in macrophages correlated to the lung load with the test item and were considered to be related to pulmonary clearance.

On the basis of these findings, the exposure level of 8.11 µg/L was therefore considered to represent the no observed adverse effect level (NOAEL) for this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

219 mg/m³

Study duration:

subacute

Species:

rat

Quality of whole database:

28-day oral toxicity study complete and sufficient to fulfill the REACh annex VIII requirements

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:

From 2014-03-07 to 2015-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.8 (Subacute Inhalation Toxicity: 28-Day Study)

Deviations:

no

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: Harlan (UK) Ltd.
- Age at study initiation:60 to 66 days
- Weight at study initiation: 217 to 261 g (males) and 153 to 188 g (females)
- Fasting period before study:no
- Housing: The animals were housed five of one sex per cage
- Diet: Ad libitum, standard rodent diet (Rat and Mouse n°1 Maintenance Diet). This diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
- Water: Ad libitum, potable water taken from the public supply.
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19 to 23°C
- Humidity: 40 to 70%
- Air changes (per hr):Each animal room was supplied with 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:

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 1.3-1.6 µm with Geometric Standard Deviation (GSD) of 2.52-2.75.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: flow through nose only chamber. This system was an aluminium alloy construction comprising a base unit, three animal exposure sections and a top section.
- Method of holding animals in test chamber: Rats were held in polycarbonate tubes with their snouts protruding from the end of the tubes into the exposure chamber.
- Source and rate of air: From in-house compressed air system(breathing quality), Generator flow: 19L/minute - Supplementary flow: 10-60 L/minute
- System of generating particulates/aerosols: Wright Dust Feed mechanism designed to produce and maintain atmospheres of dust by suspending material scraped from the surface of a compressed powder in a stream of dry air. The concentrations of dust were altered by changing the gear ratio (and therefore the speed of rotation of the compressed powder towards the scraper blade) of the mechanism.
- Temperature, humidity, pressure in air chamber:
Temperature was measured using an electronic thermometer inserted into the inhalation chamber. The mean chamber temperatures were all within expected ranges (22.2; 22.2; 22.4 and 22.0 °C for controls, low, mid and high-dosed groups).
Humidity and pressure in air chamber were not reported.
- Air flow rate: Airflow to Wright dust Feed was 19 L/minute for all groups. Supplementary airflows were 10, 60, 10 and 10 L/minute for the control, low, mid and high-dose groups respectively.
- Method of particle size determination: Particle size analysis of generated atmospheres was performed using a 6-stage cascade impactor (Marple 296). Samples were collected at least once during each week of exposure for each concentration tested. Samples were also 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, was 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 < 0.42, 0.76, 1.27, 2.86, 4.9 and 8.0 µm (aerodynamic diameter) was calculated. From this data, the Mass Median Aerodynamic Diameter (MMAD), and Geometric Standard Deviation (GSD) were calculated.
- Treatment of exhaust air: Extract airflow was drawn by in-house vacuum system at a flow rate of 30, 80, 30 and 30 L/minute for the control, low, mid and high-dose groups respectively. the airflow was filtered locally.

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 a glass fibre filters. Sampling was performed at least three times during each exposure and for each dose-group.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.
In addition, the aerosol concentrations measured by gravimetric analysis were checked by a chemical analysis once every week. High Performance Liquid Chromatography with UV detection analytical method was used.

- 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 during each exposure by gravimetrical analysis of the test item deposited on a sampling filter. Once every week, the gravimetric analysis was coupled to analytical analysis by HPLC with UV detection to check the accuracy of the gravimetric method.
The mean achieved concentrations were 8.11; 39.6 and 219 µg/L and corresponded to 113; 110 and 122% of the target concentrations respectively. The mean exposure concentrations measured by chemical and gravimetric analysis correlated well.

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

5 days / week

Remarks:

Doses / Concentrations:
7.2, 36, 180 µg/L
Basis:
other: target

Remarks:

Doses / Concentrations:
8.11, 39.6, 219 µg/L
Basis:
analytical conc.

No. of animals per sex per dose:

3 groups, each comprising 5 male and 5 female rats received the test substance at target exposure levels of 7.2, 36 or 180 µg/L. A similarly constituted Control group received air only, at the same operating conditions as the high dose group.

Control animals:

yes, sham-exposed

Details on study design:

- Dose selection rationale:
In a previously conducted two-week repeated dose inhalation toxicity study, rats were exposed 5 days/week, 6 hours/day to 0.0691, 0.298 or 1.55 mg/L (69.1, 298 or 1550 μg/L ) of the test substance. Histopathological treatment related findings were evident in the lungs and tracheobronchial lymph nodes at all exposure levels. These findings were of minimal severity and were not considered to be adverse.
In another study with a similar compound, adverse respiratory tract histopathological findings were apparent at 21.2 and 101 µg/L following 13 weeks of exposures; the NOAEL in that study was 3.30 µg/L.
Considering the results from the previous studies and the likely progression of lung changes following exposure to the test substance over a longer duration, a high exposure level targeted at 180 μg/L was selected and was anticipated to induce treatment related changes similar to those previously seen but was expected to be tolerated for 4 weeks. Target exposure levels of 36 and 7.2 μgL were selected for the intermediate and low groups respectively to identify a no-observed adverse effect level and to explore any possible dose relationship.

Positive control:

none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Cages were inspected daily for evidence of ill-health amongst the occupants.
Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.
During the acclimatisation period, observations of the animals and their cages were recorded at least once per day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily during Weeks 1 to 4 of treatment, detailed observations were recorded at the following times in relation to dose administration:
* Pre-exposure observation
* As each animal is returned to its home cage
* As late as possible in the working day

BODY WEIGHT: Yes
- Time schedule for examinations:The weight of each animal was recorded one week before treatment commenced (Day -7), on the day that treatment commenced (Day 1), twice weekly throughout the study and before necropsy.

FOOD CONSUMPTION: Yes
-The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded for the week before treatment started (Week -1), and each week throughout the treatment. From these records the mean weekly consumption per animal (g/animal/week) was calculated for each cage.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: During Week 4 of treatment and before dosing for all study animals.
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: Yes, overnight withdrawal of food.
- How many animals: 40.
- Parameters examined: Haematocrit (Hct), Haemoglobin concentration (Hb), Erythrocyte count (RBC), Reticulocyte count (RETA), Mean cell haemoglobin (MCH), Mean cell haemoglobin concentration (MCHC), Mean cell volume (MCV), Total white cell count (WBC), Differential WBC count including Neutrophils (N), Lymphocytes (L), Eosinophils (E), Basophils (B), Monocytes (M), Large unstained cells (LUC), Platelet count (Plt), Prothrombin time (PTP) and Activated partial thromboplastin time (APTT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: During Week 4 of treatment and before dosing for all study animals.
- Animals fasted: Yes, overnight withdrawal of food.
- How many animals: 40
- Parameters examined: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Total bilirubin (Bili), Urea, Creatinine (Creat), Glucose (Gluc), Total cholesterol (Chol), Triglycerides (Trig), Sodium (Na), Potassium (K), Chloride (Cl), Calcium (Ca), Inorganic phosphorus (Phos), Total protein (Total Prot) and Albumin (Alb).

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

All study animals were sacrificed following 4 weeks of exposures.
GROSS PATHOLOGY: Yes
All animals were subject to a detailed necropsy. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded.
HISTOPATHOLOGY: Yes
The following tissues were examined for all study animals of Groups 1 (Control) and 4 (219 µg/L) sacrificed on completion of the 4-week treatment period:
Adrenals - cortex and medulla
Brain - cerebellum, cerebrum and midbrain
Heart - included auricular and ventricular regions
Kidneys - included cortex, medulla and papilla regions
Larynx - 5 sections
Liver - section from two main lobes
Lungs - section from all major lobes, to include bronchi
Nasal turbinates -4 levels including nasal cavity, paranasal sinuses, nasopharynx and nasopharynx duct and nasal associated lymphoid tissue
Oesophagus
Ovaries
Seminal vesicles
Spinal cord - transverse and longitudinal section at the cervical, lumbar and thoracic levels
Spleen
Stomach - included keratinised, glandular and antrum in sections
Testes
Thymus
Thyroid - included parathyroids in section where possible
Trachea - including bifurcation
Uterus - uterine body with cervix section
In addition:
- all mediastinal lymph nodes showing macroscopic abnormality were examined,
- Lungs, larynx, trachea (including bifurcation), nasal turbinates and tracheobronchial lymph nodes were examined for all study animals of groups 2 (8.11 µg/L) and 3 (39.6 µg/L).

Other examinations:

- HAEMATOLOGY, BONE MARROW:
Bone marrow smears were prepared immediately following death on completion of the scheduled treatment period. The smears from all animals of Groups 1 (Control) and 4 (219 µg/L) were examined to assess the cellularity, distribution and morphology of the marrow. The smears from animals of the intermediate and low dose groups were not examined.

- ORGAN WEIGHTS:
The following organs, taken from each animal killed after 4 weeks of treatment were weighted: Adrenals, Brain, Heart, Kidneys, Liver, Lungs with mainstem bronchi, Spleen, Testes and Thymus.
Bilateral organs were weighed together. Organ weights were also adjusted for terminal bodyweight, using the weight recorded before necropsy.

Statistics:

All analyses were carried out using the individual animal as the basic experimental unit.
# The following sequence of statistical tests was used for bodyweight, organ weight and clinical pathology:
A parametric analysis was performed if Bartlett's test for variance homogeneity was not significant at the 1% level. The F1 approximate test was applied. If the F1 approximate test was not significant at the 1% level, Williams' test was applied. If the F1 approximate test was significant, suggesting that the dose response was not monotone, Dunnett's test was performed instead.
A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. The H1 approximate test was applied. If the H1 approximate test was not significant at the 1% level, Shirley's test was applied. If the H1 approximate test was significant, suggesting that the dose-response was not monotone, Steel's test was performed instead.
For pathology if 75% of the data (across all groups) were the same value, for example c, Fisher’s Exact tests were performed. Treatment groups were compared using pairwise comparisons of each dose group against the control both for i) values c, as applicable.
# For organ weight data, analysis of covariance was performed using terminal bodyweight as covariate, unless non-parametric methods were applied.

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):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no mortalities and no test article-related clinical signs during the study.

BODY WEIGHT AND WEIGHT GAIN
There were no treatment related effects on body weight and weight gain.
A weight loss occurred on Day 25 for all groups, including control and was due to overnight food withdrawal before the blood sampling procedures for haematology and blood chemistry on Day 24.

FOOD CONSUMPTION
There were no treatment related changes on food consumption.

HAEMATOLOGY
Higher group mean neutrophil counts were evident for females exposed to 39.6 µg/L and both sexes exposed to 219 µg/L, up to 1.52X and 2.5X control for males and females, respectively. The neutrophil count was dose-dependently increased in females.
Lower group mean lymphocyte counts were evident in males exposed to 219 µg/L compared with control (0.77X control). In treated females, there was a decrease without exposure relationship in mean lymphocyte counts when compared with control (0.75X, 0.66X and 0.79X for animals exposed to 8.11, 39.6 or 219 µg/L, respectively).
All other differences between the control and treated groups, were minor or were confined to one sex and were therefore attributed to normal biological variation.

CLINICAL CHEMISTRY
Mean phosphorus concentrations were lower in males exposed to 39.6 or 219 µg/L (as low as 0.82X ) compared with control. There was no dose relationship or similar effect in the females, this was therefore considered not to be an effect of treatment.
Compared with control, higher creatinine concentrations, not exposure related were observed in males exposed to 39.6 or 219 µg/L (1.13X control) and all treated female groups (1.24X, 1.15X and 1.12X for animals exposed to 8.11, 39.6 or 219 µg/L, respectively). The higher creatinine concentrations were not associated with any histopathological changes and, were not dose-related. They were considered to be due to normal biological variation and not a reaction to treatment with E99434.
Lower mean triglycerides were evident in males exposed to 219 µg/L, however the individual values were not consistently out of the control range, a low value for one animal lowered the group mean, and this was therefore considered not to be test article-related.
All other differences between the control and treated groups, including those that attained a degree of statistical significance were minor, lacked exposure relationship or were confined to one sex and were therefore attributed to normal biological variation.

ORGAN WEIGHTS
There was a statistical significant increase in mean lung and bronchi weights (adjusted for terminal bodyweight) in both sexes from 39.6 µg/L, (up to 1.7X and 1.9X, for males and females respectively).
All other differences between the control and treated groups, including those that attained statistical significance were minor, lacked exposure-relationship, or were confined to one sex and were therefore attributed to normal biological variation.

GROSS PATHOLOGY
Enlargement and pale appearance of the tracheobronchial lymph nodes were noted in nearly all animals receiving 39.6 or 219 µg/L. Enlargement and pale appearance of the mediastinal lymph nodes were also noted in half or more of the animals receiving 39.6 or 219 µg/L (See 7.5.2/1 Macropathology results in any other information on results incl.tables).

HISTOPATHOLOGY: NON-NEOPLASTIC
Histopathological findings related to treatment were seen in the lungs, larynx, tracheobronchial and mediastinal lymph nodes:

A variety of findings indicative of mild irritation and clearance were seen in the lungs of both sexes exposed to 39.6 or 219 µg/L. These findings included bronchiolo-alveolar hyperplasia in the terminal bronchioles and alveoli, diffuse foamy alveolar macrophages, foamy alveolar macrophages around terminal bronchioles, and eosinophilic material in the alveoli and in one female (exposed to 219 µg/L), in the bronchioles. Findings in the lung of rats given 8.11 μg/L were confined to a minimal macrophage response around the terminal bronchioles in two males and one female.

In the tracheobronchial and mediastinal lymph nodes, increased cellularity and macrophage aggregates were observed in the majority of treated rats exposed to 39.6 or 219 µg/L. These findings correlated with enlargement and pallor observed during necropsy (See 7.5.2/2 Histopathology results in any other information on results incl.tables).

OTHER FINDINGS
Bone marrow analysis:
The cellularity, distribution and morphology of the marrow was unaffected by the treatment.

Dose descriptor:

NOEC

Effect level:

8.11 other: µg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: At 39.6 and 219 µg/L: -Microscopic findings observed in lungs suggestive of hyperplasia, - Changes in Haematology indicative of inflammatory reaction and consistent with the findings recorded microscopically.

Critical effects observed:

not specified

7.5.2 /1 Macropathology results

Tracheobronchial and Mediastinal lymph nodes

Enlargement and/or abnormal colour (pale) of these lymph nodes was seen in the majority of animals exposed to 39.6 or 219 µg/L.

Summary of findings in the tracheobronchial and mediastinal lymph nodes for animals killed after 4 weeks of treatment
Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Exposure level (µg/L)	0	8.11	39.6	219	0	8.11	39.6	219
Tracheobronchial LN enlarged	0	0	4	5	0	0	5	4
Tracheobronchial LN abnormal colour (pale)	0	0	5	4	0	0	4	3
Number of animals examined	5	5	5	5	5	5	5	5
Mediastinal LN enlarged	0	0	3	3	0	0	4	3
Mediastinal LN abnormal colour (pale)	0	0	4	3	0	0	4	3
Number of animals examined	5	5	5	5	5	5	5	5

 

7.5.2 /2 Histopathology results

Lungs

Summary of treatment related findings in the lungs for animals killed after 4 weeks of treatment
Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Exposure level (µg/L)	0	8.11	39.6	219	0	8.11	39.6	219
Hyperplasia, bronchiolo-alveolar, terminal bronchioles
Minimal	0	0	0	2	0	0	0	3
Total	0	0	0	2	0	0	0	3
Hyperplasia, bronchiolo-alveolar, alveoli
Minimal	0	0	4	2	0	0	2	0
Slight	0	0	0	2	0	0	2	5
Total	0	0	4	4	0	0	4	5
Alveolar macrophages, foamy, diffuse
Minimal	0	0	2	2	0	0	4	0
Slight	0	0	3	3	0	0	1	5
Total	0	0	5	5	0	0	5	5
Macrophages, foamy, terminal bronchioles
Minimal	0	2	2	0	0	1	3	0
Slight	0	0	1	0	0	0	0	0
Total	0	2	3	0	0	1	3	0
Eosinophilic material, alveoli
Minimal	0	0	0	0	0	0	3	0
Slight	0	0	0	2	0	0	1	0
Moderate	0	0	0	3	0	0	0	5
Total	0	0	0	5	0	0	4	5
Eosinophilic material, terminal bronchioles
Minimal	0	0	0	0	0	0	0	1
Total	0	0	0	0	0	0	0	1
Number of tissues examined	5	5	5	5	5	5	5	5

Tracheobronchial lymph nodes

Summary of treatment related findings in the tracheobronchial lymph nodes for animals killed after   4 weeks of treatment
Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Exposure level (µg/L)	0	8.11	39.6	219	0	8.11	39.6	219
Cellularity increased, Generalised
Minimal	0	0	0	0	0	0	0	2
Slight	0	0	3	4	0	0	4	1
Total	0	0	3	4	0	0	4	3
Aggregates, macrophages
Minimal	0	0	5	4	0	0	4	3
Total	0	0	5	4	0	0	4	3
Number of tissues examined	5	5	5	5	5	5	5	4

Mediastinal lymph nodes

Summary of treatment related findings in the tracheobronchial lymph nodes for animals killed after 4 weeks of treatment
Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Exposure level (µg/L)	0	8.11	39.6	219	0	8.11	39.6	219
Cellularity increased, Generalised
Minimal	0	0	0	0	0	0	0	1
Slight	0	0	3	2	0	0	3	0
Moderate	0	0	0	1	0	0	0	0
Total	0	0	3	3	0	0	3	1
Aggregates, macrophages
Minimal	0	0	4	4	0	0	3	3
Total	0	0	4	4	0	0	3	3
Number of tissues examined	0	0	4	5	0	0	4	3

Conclusions:

The test article was administered by snout-only inhalation administration, for 6 hours a day, 5 days a week, for 4 weeks at achieved aerosol concentrations of 8.11, 39.6 or 219 µg/L.
In the lungs, higher concentrations of the test article produced bronchioloalveolar hyperplasia in the terminal bronchioles and alveoli which are a common site for hyperplastic and inflammatory findings in rat inhalation studies. There were also increased macrophages and eosinophilic material in the alveoli and terminal bronchioles. These findings were indicative of mild irritation. They correlated with the elevation of plasma neutrophils in the lack of any abnormalities in bone marrow and with the dose-dependent increase in lungs and bronchi weight. Higher concentrations of the test article also resulted in increased cellularity and macrophage aggregates in the tracheobronchial and mediastinal lymph nodes. These findings correlated with enlargement and pallor observed at necropsy .They were considered a likely secondary response to the increased influx of macrophages clearing the test material from the terminal airways and alveolar spaces and trafficking to the local draining lymph nodes.
At 8.11 μg/L, a minimal macrophage response around the terminal bronchioles was noted in two males and one female. These findings were considered to be the result of the normal physiological response following inhalation of particulate matter, and given the low incidence and severity, and their focal nature , they were considered to be non-adverse.
On the basis of these findings, the exposure level of 8.11 µg/L was therefore considered to represent the no observed adverse effect level (NOAEL) for this study.

Executive summary:

The cumulative toxicity of the test item was assessed when administered to Wistar rats by snout-only inhalation administration for 6 hours per day, 5 days per week, over a period of 4 weeks. The study was designated to provide a rational basis for the assessment of the toxicological risk to man.

Three groups, each comprising five male and five female rats, received the test item at target exposure levels of 7.2, 36 or 180 µg/L. A similarly constituted control group received air at the same operating conditions as the 180 µg/L group. During the study, clinical condition, body weight, food consumption, haematology (peripheral blood), haematology (bone marrow), blood chemistry,organ weight, macropathology and histopathology investigations were undertaken.

The achieved gravimetric aerosol concentrations were 8.11, 39.6 and 219 µg/L (113, 110 and 122% of the target concentrations). The Mass Median Aerodynamic Diameters for all treated groups were within the ideal range (1-3µm) for a repeat dose inhalation study.

Test article-related effects were observed in haematology at 39.6 and 219μg/L for both sexes during the treatment. Higher neutrophil counts were observed for females exposed to 39.6 µg/L and both sexes exposed to 219 µg/L. Lower group mean lymphocyte counts were evident in males exposed to 219 µg/L. In treated females, a decrease in mean lymphocyte counts without exposure relationship was observed when compared with control. No abnormalities were observed in the bone marrow thus confirming that changes in the haematology parameters were secondary to the local inflammatory effects observed in the lungs.

Lower phosphorus concentrations in males exposed to 39.6 or 219 µg/L and higher creatinine concentrations in both sexes were observed at all exposure levels. They were not associated with any histopathological changes and not dose-related and were therefore considered not to be of toxicological importance.

A statistical significant increase in mean lung and bronchi weights was observed in both sexes from 39.6 µg/L. Macroscopically enlargement and/or abnormal colour (pale) of the tracheobronchial and mediastinal lymph nodes was also seen in animals exposed to 39.6 or 219 µg/L.

Microscopically changes related to treatment with E99434 were seen in the lungs, tracheobronchial and mediastinal lymph nodes. A variety of findings indicative of mild irritation (hyperplasia) and clearance (macrophage) were seen in the lungs of both sexes exposed to 39.6 or 219 µg/L. These findings included bronchiolo-alveolar hyperplasia in the terminal bronchioles and alveoli, diffuse foamy alveolar macrophages, foamy alveolar macrophages around terminal bronchioles, and eosinophilic material in the alveoli and in one female exposed to 219 µg/L, in the bronchioles. In the tracheobronchial and mediastinal lymph nodes generalised increased cellularity and macrophage aggregates were observed in animals exposed to 39.6 or 219µg/L. These findings correlated with enlargement and pallor observed at necropsy . They were considered a likely secondary response to the increased influx of macrophages clearing the test material from the terminal airways and alveolar spaces and trafficking to the local draining lymph nodes.

At 8.11μg/L , the findings in the lungs were confined to a minimal macrophage response around the terminal bronchioles in two males and one female. These findings were considered to be a normal physiological response following inhalation of particle matter and were not adverse. The increase in macrophages correlated to the lung load with the test item and were considered to be related to pulmonary clearance.

On the basis of these findings, the exposure level of 8.11 µg/L was therefore considered to represent the no observed adverse effect level (NOAEL) for this study.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

8.11 mg/m³

Study duration:

subacute

Species:

rat

Quality of whole database:

28-day oral toxicity study complete and sufficient to fulfill the REACh annex VIII requirements

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The substance was tested in two repeated-dose toxicity studies performed by the oral and inhalation routes:

Oral route:

The objective of this dose-range finding study was to select the dose-levels for a further OECD 421 reproductive/developmental toxicity screening.

Three groups of five male and five female Sprague-Dawley rats received the test item daily, by gavage at dose-levels of 100, 300 or 1000 mg/kg bw/day for 14 days. A group of five males and five females received the vehicle alone,corn oil under the same experimental conditions and acted as a control group.

The animals were checked daily for mortality and clinical signs. Body weights were recorded once during the pre-treatment period, on the first day of treatment and then on days 4, 8, 11 and 14 before necropsy. Food consumption was recorded twice weekly during the treatment period.

On completion of the treatment period, animals were sacrificed. The epididymides, kidneys, liver, ovaries, spleen and testes were weighed and a full macroscopic post-mortem examination was performed.

 No unscheduled deaths related to the test item treatment occurred during the study. No clinical signs were observed in animals.

There were no test item-related changes in body weight and body weight gain during the study at any dose‑level, and no relevant effects were recorded on food consumption.

No test item-related changes were observed in the mean organ weights or at the macroscopic post-mortem examination.

No adverse effects were observed in rats treated up to a dose-level of 1000 mg/kg/day for 14 days with the test item (Leclère, 2013b).

 

Inhalation:

The cumulative toxicity of the test item was assessed in a 4 -week inhalation toxicity study in rat. The study was performed in accordance with OECD guideline 412 and in compliance with Good Laboratory Practices.

The test item was administered by snout-only inhalation administration, for 6 hours a day, 5 days a week, for 4 weeks at achieved aerosol concentrations of 8.11, 39.6 or 219 µg/L. A similarly constituted control group received air at the same operating conditions as the 219 µg/L group.

There were no test item-related deaths or effects on bodyweight or food consumption.

Test item-related effects were observed in haematology at 39.6 and 219μg/L for both sexes during the treatment. Higher neutrophil counts were observed for females exposed to 39.6 µg/L and both sexes exposed to 219 µg/L. Lower group mean lymphocyte counts were evident in males exposed to 219 µg/L. In treated females, a decrease in mean lymphocyte counts without exposure relationship was observed when compared with control. No abnormalities were observed in the bone marrow thus confirming that changes in the haematology parameters were secondary to the local inflammatory effects observed in the lungs.

Lower phosphorus concentrations in males exposed to 39.6 or 219 µg/L and higher creatinine concentrations in both sexes were observed at all exposure levels. They were not associated with any histopathological changes and not dose-related and were therefore considered not to be of toxicological importance.

A statistical significant increase in mean lung and bronchi weights was observed in both sexes from 39.6 µg/L. Macroscopically enlargement and/or abnormal colour (pale) of the tracheobronchial and mediastinal lymph nodes was also seen in animals exposed to 39.6 or 219 µg/L.

Microscopically changes related to treatment with the test item were seen in the lungs, tracheobronchial and mediastinal lymph nodes.

A variety of findings indicative of mild irritation (hyperplasia) and clearance (macrophage) were seen in the lungs of both sexes exposed to 39.6 or 219 µg/L. These findings included bronchiolo-alveolar hyperplasia in the terminal bronchioles and alveoli, diffuse foamy alveolar macrophages, foamy alveolar macrophages around terminal bronchioles, and eosinophilic material in the alveoli and in one female exposed to 219 µg/L, in the bronchioles.In the tracheobronchial and mediastinal lymph nodes generalised increased cellularity and macrophage aggregates were observed in animals exposed to 39.6 or 219µg/L. These findings correlated with enlargement and pallor observed at necropsy .They were considered a likely secondary response to the increased influx of macrophages clearing the test material from the terminal airways and alveolar spaces and trafficking to the local draining lymph nodes.

At 8.11μg/L , the findings in the lungs were confined to a minimal macrophage response around the terminal bronchioles in two males and one female.These findings were considered to be a normal physiological response following inhalation of particle matter and not adverse. The increase in macrophages correlated to the lung load with the test item and were considered to be related to pulmonary clearance.

On the basis of these findings, the exposure level of 8.11 µg/L was therefore considered to represent the no observed adverse effect level (NOAEL) for this study (Beebe, 2015b).

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Study well conducted, meets acceptable scientific principles.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
Study performed according to the OECD guideline 412 and GLP compliant.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
Study performed according to the OECD guideline 412 and GLP compliant.

Justification for classification or non-classification

No classification for repeated dose toxicity is warranted according to regulation (EC) No. 1272/2008 and its subsequent amendments on classification, labeling and packaging (CLP) of substances and mixtures.