Registration Dossier

Administrative data

Description of key information

The oral administration of the test substance to rats by gavage (OECD 408), at dose levels of 10, 100 and 350 mg/kg bw/day, resulted in findings associated with the irritant nature of the test item at 350 and 100 mg/kg bw/day with histopathological changes in the stomach, duodenum and kidneys. Therefore based on these results on this study the ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was considered to be 350 mg/kg bw/day for both males and females.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental Starting date: 24 May 2016 and Experimental Completion date: 02 February 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
Adopted 21 September 1998
Deviations:
yes
Remarks:
Please see below in Materials and Methods field.
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
Updated 21 August 2001
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Justification
The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.

Male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Envigo RMS (UK) Limited, Oxon, UK.
Sex:
male/female
Details on test animals and environmental conditions:
On receipt the animals were examined for signs of ill-health or injury.
The animals were acclimatized for at least nine days during which time their health status was assessed. A total of eighty animals (forty males and forty females) were accepted into the study. At the start of treatment the males weighed 228 to 268g, the females weighed 153 to 180g, and were approximately six to eight weeks old.

Animal Care and Husbandry
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 (Datesand Ltd., Cheshire, UK).
The animals were allowed free access to food and water. A pelleted diet (Rodent 2014C Teklad Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK.) was used. Certificates of analysis of the batches of diet used are given in Annex 5. Mains drinking water was supplied from polycarbonate bottles attached to the cage. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK). The diet, drinking water bedding and environmental enrichment were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

The animals were housed in a single air-conditioned room within the Envigo Research Limited, Shardlow, UK Barrier Maintained Rodent Facility. 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. Environmental conditions were continuously monitored by a computerized system, and print-outs of hourly temperatures and humidities are included in the study records. The Study Plan target ranges for temperature and relative humidity were 22 ± 3 °C and 50 ± 20% respectively. Short term deviations from these targets were considered not to have affected the purpose or integrity of the study; see deviations from Study Plan.

The animals were randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomized. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.
Route of administration:
oral: gavage
Details on route of administration:
The test item was administered daily by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 mL/kg of Polyethylene glycol 400.
Vehicle:
polyethylene glycol
Remarks:
PEG 400
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test item was prepared at the appropriate concentrations as a solution in Polyethylene glycol 400. The stability and homogeneity of the test item formulations were determined by Envigo Research Limited, Shardlow, UK, Analytical Services. Results showed the formulations to be stable for at least 12 days at 2.5 mg/mL and 21 days at 250 mg/mL. Formulations were therefore prepared weekly and stored at approximately 4 °C in the dark.


VEHICLE
- Justification for use and choice of vehicle: The test item is stable in PEG 400 (The results indicate that that the prepared formulations were within acceptable ranges for the purpose of this study).
- Concentration in vehicle:
- Amount of vehicle (if gavage): 4 mL/kg of Polyethylene glycol 400.
Analytical verification of doses or concentrations:
yes
Remarks:
Homogeneity and stability determined with respect to the concentration of Test Item in PEG 400 formulations at nominal concentrations of 2.5 mg/L and 250 mg/L.
Details on analytical verification of doses or concentrations:
The test item concentration in the test samples was determined by high performance liquid chromatography-mass spectometry (HPLC-MS) using an external standard technique. The test item gave a chromatographic profile consisting of a single peak.

Apparatus: Analytical and top-pan balances and general laboratory apparatus and glassware.

Reagents:
- Control vehicle: PEG 400
- Acatonitrile: Gradient HPLC grade
- Wster: LC-MS
- Formic acid: Analytical reagent
- Dilution solvent: Acetonitrile

PREPARATION OF CALIBRATION STANDARDS
Stock solutions of test item in dilution solvent were prepared for external standard calibration. An aliquot, approximately 0.1 gr of test item was exactly weighed into a 100 mL volumetric flask and brought to volume with dilution solvent to yield a solution with a concentration of 1 mg/mL. Aliquots of this stock standard solution were used to prepare working standard solutions in dilution solvent with a concentration range of 0.001 mg/mL to 0.025 mg/mL.
On each occasion standard solutions derived from two stock standard solution were used for calculation.

Calibration solutions were injected onto instrument, at the beginning and end of each sample analysis sequence as a minimum, using the conditions detailed in the instrument.
To assess the calibration range of the method, a range of standard solutions were prepared in dilution solvent from a stock solution of 1.156 mg/mL by serial dilution covering the concentration range 0.001156 mg/mL to 0.02665 mg/mL.

PREPARATION OF TEST SAMPLES:
The formulations received were diluted with dilution solvent. An aliquot of the test item formulation was accurately weighed into a volumetric flask and brought to volume with dilution solvent this was then shaken to dissolve. Where necessary, sample solutions were further diluted with dilution solvent to achieve to working concentration.

PREPARATION OF ACCURACY AND PRECISISON SAMPLES.
Sample of PEG400 were accurately fortified with known amounts of test item equivalent to the lowest and highest anticipated dose concentrations. These sample were then prepared for analysis as the test samples.
The concentration of the test item in the final solution was quantified by HPLC using MS detection as detailed in the instrument parameters section.

INSTRUMENTATION PARAMETERS:
HPLC system: Agilent technologies 1200 MSD, incorporating autosample and workstation.
Mass selective detector
- Source: Electrospray
- Fragmentation energy: 110 volts
- Polarity: Positive
- Mode: Single ion mode with 302.1 amu
- Gas temperature: 350°C
- Drying gas: 9 liter/minute
- Nebuliser pressure: 40 psi
- Capillary voltage: 1500 volts
- Gain: 1
- Column: symmetry, 3.5 micro, (50x3 mm id)
- Column Temperature: 30°C
- Gradient elution: Eluent A: 10 mM ammonium formate with 0.1% formic acid in LC-MS water
Eluent B: 0.1% formic acid in methanol

Time (minutes) %A %B
0 95 5
5 5 95
10 5 95

- Flow rate: 0.4 mL/min.
- Injection volume: 5 microliter
- Retention time: approximately 6.8 minutes


DATA EVALUATION AND CALCULATIONS:
The peak area for Araldite CY 184 in each calibration standard chromatogramm was measured. Calibration curves were constructed by non-linear regression of calibration standard response versus calibration standard concentration. The area response of the peak observed at the characteristic retention time for test item in sample and procedural recovery chromtograms was measured.

The recovery rate (R) of the spiked recovery sample was calculated using the following equation:

R= (C / C fort) x100

Where:
R= Recovery rate %
C= Determined concentration of the test item in the spiked recovery sample (mg/mL)
C fort= Fortified concentration of the test item in the spiked recovery sample (mg/mL)

VALIDATION OF THE ANALYTICAL PROCEDURE:
The analytical procedure was validated by determining the following parameters:
- The specificity of the chromatographic analysis in control sample chromatograms.
- The linearity of detector response over the calibration standard concentration range.
- The method accuracy (recovery) and precision, by analyzing five recovery samples at nominal concantrations of 2.5 mg/mL and 250 mg/mL
- The limit of quantification (LOQ) was determined as the lowest standard concentration used during the study.

HOMOGENEITY AND STABILITY IN VEHICLE FORMULATIONS
The homogeneity and satbility of the test item in PEG 400 formulations was assessed at nominal concentrations of 2.5 mg/mL and 250 mg/mL after refrigerated storage.

REFRIGERATED STORAGE (nominally + 4°C)
The formulation were analysed and refrigerated on receipt. On day 12 (low dose) and day 21 (high dose); the formulations were removed from storage and equilibrated to ambient temperature. The formulations were removed from storage and equilibrated to ambient temperature. The formulations were mixed as stated in the mixing procedure (described in the main part of this study) and single samples were removed for analysis from the top, middle and bottom of the mixed formulation.


CONCENTRATION OF DOSE FORMULATIONS:
For each analysis occasion, freshly prepared test formulations were analyzed. Duplicate samples were analyzed in accordance with the analytical procedure. Samples were disposed of once satisfactory result were achieved

MAJOR COMPUTERIZED SYSTEMS:
The computerized systems that have been used in this project are:
Chemstation and Delta BMS


RESULTS:
METHOD VALIDATION:
The analytical procedure was successfully validated for test item in PEG 400 with respect to the specificity of chromatographic analysis, the linearity of detector response, method accuracy and precision
The specificity of the analytical method was demonstrated by the basence of a peak at the characteristic retention time for test item in the control sample chromatogram.

The data was found to have a quadratic correlation within the calibration range of 0.001047 mg/mL to 0.02583 mg/mL. The R2 fit of the calibration curve to the data was considered to be acceptable.

A mean recovery value of 125% (CV=2.80%, n=5) was obtained for 2.5 mg/mL and 108% (CV= 1.68%, n=5) was obtained for 250 mg/mL.

The limit of quantification (LOQ) was determined as the lowest standard concentration used during the study.


HOMOGENEITY AND STABILITY OF DOSE FORMULATIONS:
The homogeneity and stability of test item in PEG 400 formulations was assessed with respect to the level of concentration at nominal concentrations of 2.5 mg/mL and 250 mg/mL.
Homogeneity was confirmed at the initial stability time point. The mean analyzed concentration for the nine samples remained within 10% of the initial time zero value and the variation was less than 10%.

CONCENTRATION OF DOSE FORMULATIONS:
The mean concentrations were within applied limits +/- 10%, confirming accurate formulation.

CONCLUSION:
The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, linearity of detector response, method accuracy and precision.

The homegneity and satbilty was confirmed for test item in PEG 400 formulations at nominal concentrations of 2.5 mg/mL and 250 mg/mL when refrigerated for 12 days (low dose) and 21 days high dose)

The mean concentrations of test item in test formulations analyzed for the study were within +/- 10% of nominal concentrations, confirming accurate formulations.
Duration of treatment / exposure:
90 consecutive days.
Frequency of treatment:
Daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Treatment volume: 4 mL/kg
Control treatment group
The volume of test and control item administered to each animal was based on the most
recent scheduled body weight and was adjusted at weekly intervals.
Dose / conc.:
10 mg/kg bw/day (nominal)
Remarks:
Treatment volume: 4 mL/kg
Low treatment group
The volume of test and control item administered to each animal was based on the most
recent scheduled body weight and was adjusted at weekly intervals.
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Treatment volume: 4 mL/kg
Intermediate treatment group
The volume of test and control item administered to each animal was based on the most
recent scheduled body weight and was adjusted at weekly intervals.
Dose / conc.:
350 mg/kg bw/day (nominal)
Remarks:
Treatment volume: 4 mL/kg
High treatment group
The volume of test and control item administered to each animal was based on the most
recent scheduled body weight and was adjusted at weekly intervals.
No. of animals per sex per dose:
10 male and 10 female per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
- Rationale for animal assignment (if not random):
- Rationale for selecting satellite groups:
- Post-exposure recovery period in satellite groups:
- Section schedule rationale (if not random): - Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: no satellite group used
- Post-exposure recovery period in satellite groups: no post exposure recovery period in satellite groups as no satellite groups used
Positive control:
Not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: No

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: 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: Yes
- Time schedule for examinations: 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.

FOOD EFFICIENCY: No

WATER CONSUMPTION : Yes
- Time schedule for examinations: Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes.

OPHTHALMOSCOPIC EXAMINATION: Yes
The eyes of all animals from Groups 1 to 4 were examined pre-treatment. During Week 12, the eyes of all control and high dose animals (Groups 1 and 4, respectively) were examined.
Examinations included observation of the anterior structures of the eye and following pupil dilation with 0.5% Tropicamide solution (Mydriacyl® 0.5%, Alcon Laboratories (UK) Ltd., Pentagon Park, Boundary Way, Hemel Hampstead, Hertfordshire), detailed examination of the internal structure of the eye using an ophthalmoscope was performed.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Hematological and blood chemical investigations were performed on all surviving animals from each test and control group at the end of the study (Day 90). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Day 91. Animals were not fasted prior to sampling.
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).


CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Specialist Evaluations
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 performances 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

This test was developed from the methods used by Irwin (1968) and Moser et al (1988). The scoring system used is outlined in The Key to Scoring System and Explanation for Behavioral Assessments and Sensory Reactivity Tests.


Functional Performance Tests
Motor Activity.
Twenty purpose built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals of one sex were tested at each occasion and were randomly allocated to the activity monitors. The tests were performed at approximately the same time each occasion (at least two hours after dosing), under similar laboratory conditions. The evaluation period was one hour for each animal. The time in seconds each animal was active and mobile was recorded for the overall one hour period and also during the final 20% of the period (considered to be the asymptotic period, Reiter and Macphail 1979).

Forelimb/Hindlimb Grip Strength.
An automated grip strength meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal. The assessment was developed from the method employed by Meyer et al (1979).

Sensory Reactivity
Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. This assessment was developed from the methods employed by Irwin (1968) and Moser et al (1988). The scoring system used is outlined in The Key to Scoring System and Explanation for Behavioral Assessments and Sensory Reactivity Tests. The following parameters were observed:

Grasp response
Touch escape
Vocalization
Pupil reflex
Toe pinch
Blink reflex
Tail pinch
Startle reflex
Finger approach


Blood Chemistry
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:
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++)

Terminal Investigations
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
The following organs, removed from animals that were killed at the end of the study, were dissected free from fat and weighed before fixation:
Adrenals
Ovaries
Brain
Spleen
Epididymides
Testes
Heart
Thymus
Kidneys
Uterus
Liver


Histopathology
Samples of the following tissues were removed from all animals and preserved in buffered 10% formalin, except where stated:
Adrenals
Ovaries
Aorta (thoracic)
Pancreas
Bone & bone marrow (femur including stifle joint)•
Pituitary
Bone & bone marrow (sternum)
Prostate
Brain (including cerebrum, cerebellum and pons)
Rectum
Caecum
Salivary glands (submaxillary)
Colon
Sciatic nerve
Duodenum
Seminal vesicles
Epididymides ♦
Skin
Esophagus
Spinal cord (cervical, mid-thoracic and lumbar)
Eyes*
Gross lesions
Spleen
Heart
Stomach
Ileum (including Peyer’s patches)
Testes ♦
Jejunum
Thymus
Kidneys
Thyroid/Parathyroid
Liver
Tongue•
Lungs (with bronchi) #
Trachea
Lymph nodes (mandibular and mesenteric)
Urinary bladder
Mammary glands
Uterus (with cervix)
Muscle (skeletal)•
Vagina

• Retained only and not processed
* Eyes fixed in Davidson’s fluid
♦ preserved in Modified Davidson’s fluid
# Lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative
• Retained only and not processed

All tissues were dispatched to the Test Site (Envigo CRS Limited, Eye, Suffolk, IP23 7PX) for processing (Principal Investigator: D Roberts). All selected tissues from control and 350 mg/kg bw/day dose group animals were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with Hematoxylin and Eosin for subsequent microscopic examination. Any macroscopically observed lesions were also processed.

Since there were indications of treatment-related changes, examination was subsequently extended to include similarly prepared sections of the stomach, duodenum and kidneys to include animals of both sexes from the low and intermediate groups.
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.

Pathology
Microscopic examination was conducted by the Study Pathologist (W Henderson). A review of the histopathology results for the study was conducted by an appointed Responsible Scientist (V Mowat) working at a designated Test Site.
Statistics:
Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. 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.

Data were analyzed using the decision tree from the ProvantisTM Tables and Statistics Module as detailed as follows:
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 nonhomogeneity 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):
There were no clinical observations to indicate systemic toxicity.

At 350 mg/kg bw/day animals showed incidences of increased salivation: males from Day 10 and females from Day 3 of treatment. At 100 mg/kg bw/day there were incidences of increased salivation during the latter stage of the treatment period, for which this observation showed a dose related response. This observation is commonly associated with test items of irritant nature and therefore considered not to represent systemic toxicity.

One male treated with 350 mg/kg bw/day was observed to have staining around the snout and one male treated with 100 mg/kg bw/day showed noisy respiration.

One female treated with 350 mg/kg bw/day showed generalised fur loss during Week 12.
These were isolated observations and considered incidental and to be of no toxicological significance.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One control female (No.14) was found dead on Day 4 of treatment. There were no clinical signs prior to death, and macroscopic examination showed the thoracic cavity was filled with a clear fluid. The histopathological examination confirmed no specific microscopic changes.
Macroscopic changes suggest dosing trauma may have contributed to the animal’s death.
One high dose female (no.77) was found dead on Day 53 of treatment. Clinical signs observed prior to death included hunched posture, lethargy, pilo-erection and staining of the snout. The animal had also lost 18g from the previous scheduled body weight on Day 50.
Macroscopic findings included reddened lungs, reddened jejunum filled with red fluid and dark and distended stomach, filled with red fluid. The stomach showed raised limiting ridge and sloughing of the glandular region. The histopathological examination of female 77 showed general signs of malaise with lymphoid depletion/atrophy and had notable ulceration in the non-glandular stomach which was the likely cause of death and was considered to be related to the administration of the test item.
There were no further unscheduled deaths.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There was no adverse effect on body weight development and overall body weight gains for animals of either sex receiving the test item were similar to controls.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There was no adverse effect on food consumption or food conversion efficiency as values were similar to controls.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
The gravimetric measuring of the daily water consumption at the beginning of treatment for two weeks and two weeks at a later stage of the study showed fluctuations in water intake without a dose relationship, therefore, there was no adverse effect detected.
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related ocular effects detected
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no toxicologically significant effects on hematology parameters.
At 350 mg/kg bw/day males showed statistically significantly lower (p<0.05) hemoglobin, mean corpuscular hemoglobin concentration and activated partial thromboplastin time when compared to controls. Females treated with 350 or 100 mg/kg bw/day showed a statistically significant reduction (p>0.01) in mean corpuscular hemoglobin concentration when compared to controls. Most individual values for the latter parameter were slightly below the background control ranges, but in the absence of any histopathological correlates, these findings were deemed to be of no toxicological significance.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no toxicologically significant effects on blood chemical parameters.
All male treatment groups showed statistically significant reductions in alkaline phosphatase and cholesterol when compared to controls, with the corresponding females showing a nondose related increase in glucose. At 350mg/kg bw/day, males also showed a statistically significant increase in albumin/globulin ratio and phosphorus when compared to controls.
The majority of individual values for these parameters were within the background control ranges and in the absence of any associated microscopic findings these findings were considered to be of no toxicological relevance.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Behavioral Assessemnt:
There were no treatment-related changes in behavioural parameters measured.
One male treated with 10 mg/kg bw/day showed pilo-erection during the final assessment. This was an isolated incident and considered to be unrelated to treatment.

Functional Performance Tests
There were no treatment-related changes in functional performance.
Males treated with 350 mg/kg bw/day showed a statistically significant reduction in the third forelimb grip strength test when compared to controls. This finding was considered to be incidental.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Kidneys: At 350 mg/kg bw/day, four males showed enlarged kidneys with the left kidney from one of these also showing increased pelvic space and being fluid-filled. One female also showed increased pelvic space. One control male and one 350 mg/kg bw/day female also showed increased pelvic space. This correlated with the histopathological findings.

Stomach: At 350 mg/kg bw/day all males showed raised limiting ridge in the stomach with one male also showing raised white patches on the non-glandular region of the stomach. The corresponding females also showed raised limiting ridge with one female also showing thickening of the glandular region of the stomach and red patches on it. These observations correlated with the histopathological findings but were considered to be specific to the anatomy of the rat and therefore not relevant to humans
At 100 mg/kg bw/day two males and two females showed raised limiting ridge in the stomach.
At 10 mg/kg bw/day one female showed sloughing of the glandular region of the stomach.
As there were no histopathological correlates at this dose level, this finding was considered to be incidental.
Spleen: One female treated with 100 mg/kg bw/day was shown to have a dark spleen. Due to no histopathological correlates this finding was considered to be incidental.
Lungs: A small number of animals including controls showed red discoloration of the lungs.
There was no dose-dependence and this was considered to be incidental.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Early decedents
One control female found dead on Day 4 of treatment showed a fluid-filled thoracic cavity.
One female treated with 350 mg/kg bw/day found dead on Day 53 of dosing showed reddened lungs, reddened jejunum filled with red fluid, dark and distended stomach filled with red fluid and raised limiting ridge and sloughing of the glandular region of the stomach.


Kidneys: At 350 mg/kg bw/day, four males showed enlarged kidneys with the left kidney from one of these also showing increased pelvic space and being fluid-filled. One female also showed increased pelvic space. One control male and one 350 mg/kg bw/day female also showed increased pelvic space. This correlated with the histopathological findings.
Stomach: At 350 mg/kg bw/day all males showed raised limiting ridge in the stomach with one male also showing raised white patches on the non-glandular region of the stomach. The corresponding females also showed raised limiting ridge with one female also showing thickening of the glandular region of the stomach and red patches on it. These observations correlated with the histopathological findings.
At 100 mg/kg bw/day two males and two females showed raised limiting ridge in the stomach.
At 10 mg/kg bw/day one female showed sloughing of the glandular region of the stomach.
As there were no histopathological correlates at this dose level, this finding was considered to be incidental.
Spleen: One female treated with 100 mg/kg bw/day was shown to have a dark spleen. Due to no histopathological correlates this finding was considered to be incidental.
Lungs: A small number of animals including controls showed red discoloration of the lungs. There was no dose-dependence and this was considered to be incidental.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Sensory Reactivity Assessments
There were no treatment-related changes in functional performance.
Males treated with 350 mg/kg bw/day showed a statistically significant reduction in the third forelimb grip strength test when compared to controls (p<0.05). This finding was considered to be incidental as the values in the remaining 2/3 tests were similar to controls and there were no clinical signs of neurotoxicity evident throughout the treatment period.
Details on results:
Histopathology.

Early decedents
One control female (No.14) showed general signs of malaise with lymphoid depletion/ atrophy, but no specific histopathological changes were identified to explain its death. Misdosing may have occurred, given the fluid-filled cavity noted at necropsy. One female (No.77) treated with 350 mg/kg bw/day showed general signs of malaise with lymphoid depletion/atrophy and had notable ulceration in the non-glandular stomach, which is the likely cause of death in this case and is considered to be related to the administration of the test item.

Scheduled necropsy
Stomach: Hyperplasia of the non-glandular stomach, of minimal or mild severity, was noted in 8/10 males and all females treated with 350 mg/kg bw/day. Ulceration of the nonglandular region was noted in one 350 mg/kg bw/day male with inflammatory cell infiltrate and noted in 2/9 females. These findings were not present in any 10 or 100 mg/kg bw/day animals. This correlated with the necropsy findings noted for the 350 mg/kg bw/day dose group.

Duodenum: Hypertrophy of the mucosa of the duodenum, minimal or mild severity, was noted in 9/10 males and 5/9 females at 350 mg/kg bw/day. These findings were also present in 3/10 males and one female from the 100 mg/kg bw/day dose group. It was not present in any of the 10 mg/kg bw/day animals.

Kidneys: Microscopic findings of minimal diffuse tubular hypertrophy was noted in 8/10 males and 6/9 females from the 350 mg/kg bw/day dose group. Cortical tubules appeared larger in diameter with pale staining and sometimes slightly dilated lumen. This appearance was not present in any 10 or 100 mg/kg bw/day animals. This change correlated with the enlarged kidneys noted at necropsy and the increase in organ weight for 350 mg/kg bw/day males.

Conclusion of histopathology report:
Administration of Araldite CY 184 at 350 mg/kg bw/day resulted in changes in the stomach, duodenum and kidneys.
Administration of Araldite CY 184 at 100 mg/kg bw/day resulted in changes in the duodenum.
Administration of Araldite CY 184 at 10 mg/kg bw/day is considered to be the No Observed Effect Level (NOEL)
Key result
Dose descriptor:
NOAEL
Effect level:
350 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
water consumption and compound intake
ophthalmological examination
haematology
clinical biochemistry
behaviour (functional findings)
organ weights and organ / body weight ratios
gross pathology
Key result
Dose descriptor:
NOEL
Effect level:
10 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: N/A
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (nominal)
System:
other: Please refer to organ field.
Organ:
stomach
duodenum
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

DISCUSSION:

The oral administration of the test substance to rats by gavage, at dose levels of 10, 100 and 350 mg/kg bw/day, resulted in one premature death at 350 mg/kg bw/day on Day 53 which was considered to be related to the administration of the test item, due to the microscopic findings in the stomach. The premature death of one control female on Day 4 was considered to be due to trauma during the dosing procedure.

The increased salivation observed in animals treated with 350 or 100 mg/kg bw/day can be attributed to the irritant nature of the test item, evident from the microscopic findings observed in the stomach. These clinical observations are therefore considered not to represent systemic toxicity.

There were no adverse effects on body weight development, food or water intake for animals of either sex treated with 10, 100 and 350 mg/kg bw/day.

There were statistically significant reductions in the level of alkaline phosphatase and cholesterol in males from all dose groups which may be associated with the increased liver weights. However, there were no microscopic findings of the liver and therefore these findings were considered to be of no toxicological significance. The hematological assessment revealed treated animals showed a reduction in activated partial thromboplastin time, with 350 mg/kg bw/day males attaining statistical significance (p<0.05). However, there was no dose relationship present for animals of either sex and no microscopic correlates. Therefore, the hematological and blood chemistry assessment in animals of either sex did not reveal any definitive findings of toxicological significance and there were no effects on the organ weights of toxicological importance.

The microscopic changes in the stomach at 350 mg/kg bw/day are likely to reflect an irritant effect of the test item on the non-glandular mucosa where there is prolonged exposure due to the unique structure of the rodent stomach. The hyperplasia and ulceration correlated with necropsy findings. Whilst this is an adverse change in the animals affected it is not generally considered to be significant to man as the corresponding anatomical area is not present.

There were no microscopic changes present in any of the animals treated with 100 or 10 mg/kg bw/day. Therefore, the macroscopic findings at necropsy were considered to be of no toxicological significance.

The diffuse tubular hypertrophy noted was a minimal change and correlates with the necropsy finding of enlarged kidneys and the statistically significant increase in organ weight. This is considered likely to be a functional, adaptive change due to increased demand which may be related to excretion of the test item or metabolites (Frazier K. S. et al 2012). This was only present in animals treated with 350 mg/kg bw/day.

The hypertrophy of the mucosa in the duodenum is seen occasionally related to the administration of some test items (Nolte T. et al 2016). The significance is unclear however it may be a response to changes in ingesta (pH for example) or a minor local irritant effect. Given the lack of any inflammatory or degenerative change it was considered likely to be adaptive. These microscopic changes were not present in any of the animals treated with 10 mg/kg bw/day.

Therefore taking into consideration the overall results from this study, it is considered that a dose level of 350 mg/kg bw/day could be considered as the No Observed Adverse Effect Level (NOAEL) for systemic toxicity in both males and females within the confines of this study.

CONCLUSION

The oral administration of the test substance to rats by gavage (OECD 408), at dose levels of 10, 100 and 350 mg/kg bw/day, resulted in findings associated with the irritant nature of the test item at 350 and 100 mg/kg bw/day with histopathological changes in the stomach, duodenum and kidneys. Therefore based on these results on this study the ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was considered to be 350 mg/kg bw/day for both males and females.

Conclusions:
The oral administration of the test substance to rats by gavage, at dose levels of 10, 100 and 350 mg/kg bw/day, resulted in findings associated with the irritant nature of the test item at 350 and 100 mg/kg bw/day with histopathological changes in the stomach, duodenum and kidneys. Therefore based on these results on this study the ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was considered to be 350 mg/kg bw/day for both males and females.
Executive summary:

The study was designed to investigate the systemic toxicity of the test item and is compatible with the following regulatory guidelines:

The OECD Guidelines for Testing of Chemicals No. 408 "Subchronic Oral Toxicity - Rodent: 90 Day Study” (Adopted 21 September 1998).

The EU Annex B Method B26 - “Subchronic Oral Toxicity Test - Repeated Dose 90- Day Oral Toxicity Study in Rodents” - updated 21 August 2001.

The United States Environmental Protection Agency (EPA), Health Effects Test Guidelines, OPPTS 870.3100 - 90 Day Oral Toxicity in Rodents.

This study was also designed to be compatible with Commission Regulation (EC) No 440/2008 of 30 May 2008, laying down test methods pursuant to Regulation (EC) No 907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Methods

The test item was administered by gavage to three groups, each of ten male and ten female Wistar Han™:RccHan™:WIST strain rats, for up to ninety consecutive days, at dose levels of 10, 100 and 350 mg/kg bw/day A control group of ten males and ten females was dosed with vehicle alone (Polyethylene glycol 400).

Clinical signs, functional observations, body weight change, dietary intake and water consumption were monitored during the study. Hematology and blood chemistry were evaluated for all animals at the end of the study. Ophthalmoscopic examination was also performed on all animals for Groups 1 to 4 before treatment and only animals from Groups 1 and 4 were examined during Week 12 of treatment.

All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues was performed.

Results

Mortality

One control female (No.14) was found dead on Day 4 of treatment following dosing trauma.

There were no previous clinical signs prior to death or effects on body weight. Macroscopic examination showed the thoracic cavity was filled with a clear fluid. The histopathological examination confirmed no specific changes. Macroscopic post mortem evaluation suggests dosing trauma may have contributed to the animals death due to finding the thoracic cavity fluid filled.

One high dose female (no.77) was found dead on Day 53 of treatment. The clinical signs prior to death included hunched posture, lethargy, pilo-erection and staining of the snout. The animal had shown actual body weight loss. Macroscopic post mortem findings included reddened lungs and reddened jejunum filled with red fluid. The stomach was dark, distended and filled with red fluid. In addition there was a raised limiting ridge and sloughing of the glandular region. The microscopic examination showed general signs of malaise with lymphoid depletion/atrophy and had notable ulceration in the non-glandular stomach which is

the likely the cause of death in this case and is considered to be related to the administration of the test item.

There were no further unscheduled deaths.

Clinical Observations

There were no clinical observations to indicate systemic toxicity.

Behavioral Assessment

There were no treatment-related changes in behavioural parameters measured.

Functional Performance Tests

There were no treatment-related changes in functional performance.

Sensory Reactivity Assessments

There were no treatment-related changes in sensory reactivity.

Body Weight

There was no adverse effect on body weight development in animals of either sex.

Food Consumption

There was no adverse effect on food consumption or food conversion efficiency.

Water Consumption

The gravimetric measuring of the daily water consumption at the beginning of treatment for two weeks and two weeks at later stage of the study showed fluctuations in water intake, without a dose relationship. Therefore there was no adverse effect on water consumption.

Ophthalmoscopy

There were no treatment related ocular effects detected.

Hematology

There were no changes on hematology parameters of toxicological significance.

Blood Chemistry

There were no changes on blood chemical parameters of toxicological significance.

Necropsy

Stomach: At 350 mg/kg bw/day all males showed raised limiting ridge in the stomach with one male also showing raised white patches on the non-glandular region of the stomach. The corresponding females also showed raised limiting ridge with one female also showing thickening with red patches of the glandular region of the stomach. These observations correlated with the subsequent histopathological findings.

At 100 mg/kg bw/day two males and two females showed raised limiting ridge in the stomach.

At 10 mg/kg bw/day one female showed sloughing of the glandular region of the stomach but with no histopathological correlates.

Kidneys: At 350 mg/kg bw/day four males showed enlarged kidneys; including one with increased pelvic space and one fluid filled. One control male and one female treated with 350 mg/kg bw/day also showed increased pelvic space.

Organ Weights

At 350 mg/kg bw/day animals of either sex showed a statistically significant increase in kidney weights; both absolute and relative to terminal body weight. Males treated with 100 mg/kg bw/day also showed a statistically significant increase in kidney weights; both absolute and relative to terminal body weight. The histopathological findings correlated with the increased kidney weights at 350 mg/kg bw/day.

Males at 350 mg/kg bw/day also showed a statistically significant increase in liver weights; both absolute and relative to terminal body weight. There were no histopathological correlates and therefore this increase was considered to be of no toxicological significance.

No such effects were noted for females treated with 100 mg/kg bw/day or animals of either sex at 10 mg/kg bw/day.

Histopathology

The following treatment related microscopic abnormalities were detected:

Stomach

Hyperplasia of the non-glandular stomach, of minimal or mild severity, was noted in 8/10 males and all females from the high dose group. Ulceration of the non-glandular region was noted in one male and inflammatory cell infiltrate noted in 2/9 females treated with 350 mg/kg bw/day. This finding was not present in the lower dose groups. These findings correlated with the necropsy findings.

Duodenum

Hypertrophy of the mucosa of the duodenum, minimal or mild severity, was noted in 9/10 males and 5/9 females treated with 350 mg/kg bw/day. At 100 mg/kg bw/day this finding was also present in 3/10 males and one female and not present in animals treated with 10 mg/kg bw/day.

Kidneys

Minimal, diffuse tubular hypertrophy was noted in 8/10 males and 6/9 females treated with 350 mg/kg bw/day. Cortical tubules appeared larger in diameter with pale staining and sometimes slightly dilated lumen. These changes correlated with the enlarged kidneys noted at necropsy and the increased organ weights. These findings were not seen in animals treated with 10 mg/kg bw/day.

Conclusion

The oral administration of the test substance to rats by gavage, at dose levels of 10, 100 and 350 mg/kg bw/day, resulted in findings associated with the irritant nature of the test item at 350 and 100 mg/kg bw/day with histopathological changes in the stomach, duodenum and kidneys. Therefore based on these results on this study the ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was considered to be 350 mg/kg bw/day for both males and females.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
350 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Conducted in accordance with OECD guidelines and GLP
System:
gastrointestinal tract
Organ:
stomach
intestine

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no 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

Additional information

Justification for classification or non-classification

According to Annex IX, the extended one-generation reproductive toxicity study (EOGRTS) is required if the 28- or 90-day study or the screening study indicates adverse effects on reproductive organs or tissues. In case there are no triggers for the EOGRTS, a screening study (or a study providing equivalent information) is enough at Annex IX.

The results of the OECD 408 and OECD 414 did not identify any adverse effects on reproductive organs or tissues, as a consequence there is no trigger for the EOGRTs and classification is not required.