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Diss Factsheets

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
1,2-Ethanediamine, N-(2-aminoethyl)-, reaction products with glycidyl tolyl ether
EC Number:
282-199-6
EC Name:
1,2-Ethanediamine, N-(2-aminoethyl)-, reaction products with glycidyl tolyl ether
Cas Number:
84144-79-6
Molecular formula:
C14H26N3O2
IUPAC Name:
1,2-Ethanediamine, N-(2-aminoethyl)-, reaction products with glycidyl tolyl ether
Test material form:
liquid: viscous
Details on test material:
Manufacturer Air Products and Chemicals
Batch # 8418406

Test animals

Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Envigo RMS (UK) Limited, Oxon, UK. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatized for seven days during which time their health status was assessed. A total of forty animals (twenty males and twenty females) were accepted into the study. At the start of treatment the males weighed 181 to 207g, the females weighed 131 to 156g, and were approximately six to eight weeks old.
The animals were housed in groups of five 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. A certificate of analysis of the batch of diet used is 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 the relative humidity 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.

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The test item was administered daily, for up to twenty-eight consecutive days, 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
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
28 days
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
30 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
200 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:
General Observations/Measurements
Clinical Observations
All animals were examined for overt signs of toxicity, ill-health or behavioral change
immediately before dosing, up to thirty minutes post dosing and one hour after dosing. All
observations were recorded.
Body Weight
Individual body weights were recorded on Day 1 and at weekly intervals thereafter. Body
weights were also performed prior to terminal kill.
Food Consumption
Food consumption was recorded for each cage group at weekly intervals throughout the
study. Food conversion efficiency was calculated retrospectively.
Water Consumption
Water intake was observed daily, for each cage group, by visual inspection of the water
bottles for any overt changes except during Week 3 where water intake was measured
gravimetrically.
Special Evaluations
Functional Observations
Prior to the start of treatment and on Days 7, 14, 21 and 27, all animals were observed for
signs of functional/behavioral toxicity. Functional performance tests were also performed on
all animals during Week 4, together with an assessment of sensory reactivity to different
stimuli. Observations were carried out from approximately two hours after dosing on each
occasion.
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

In-Life Sampling and Analysis
Hematological and blood chemical investigations were performed on all animals from each
test and control group at the end of the study (Day 28). Blood samples were obtained from
the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior
to necropsy on Day 29. Animals were not fasted prior to sampling.

Hematology
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)
Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time
(APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11
mol/L).

Blood Chemistry
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+) Triglycerides (Trigs)
Potassium (K+) Total cholesterol (Chol)
Chloride (Cl-) Total bilirubin (Bili)
Calcium (Ca++) Bile acids
Sacrifice and pathology:
Necropsy
On completion of the dosing period all animals were killed by intravenous overdose of
sodium pentobarbitone followed by exsanguination.
All animals were subjected to a full external and internal examination, and any macroscopic
abnormalities were recorded.

Thyroid Hormone Assessment
At termination, blood samples were taken from the exsanguination procedure, allowed to
clot, centrifuged and the serum from each animal was stored frozen at lower than -60 °C. No
treatment-related effects on the pituitary-thyroid axis were identified, therefore these samples
were discarded.

Organ Weights
The following organs, removed from animals that were killed either at the end of the dosing
period or at the end of the treatment-free period, were dissected free from fat and weighed
before fixation:
Adrenals Liver
Brain Ovaries
Epididymides Spleen
Heart Testes
Kidneys Thymus
Pituitary (post-fixation) Thyroid/Parathyroid (post fixation)
Prostate and Seminal Vesicles Uterus with Cervix
(with coagulating glands and fluids)

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 Rectum
pons) Salivary glands (submaxillary)
Caecum Sciatic nerve
Colon Seminal vesicles (with coagulating glands and fluids)
Duodenum
Epididymides Skin (hind limb)
Esophagus Spinal cord (cervical, mid thoracic
Eyes and lumbar)
Gross lesions Spleen
Heart Stomach
Ileum Testes
Jejunum Thymus
Kidneys Thyroid/Parathyroid
Liver Trachea
Lungs (with bronchi)# Urinary bladder
Lymph nodes (mandibular and mesenteric) Uterus & Cervix
Mammary gland Vagina

All tissues were dispatched to the histology processing Test Site (Envigo CRS Limited, Eye,
Suffolk, IP23 7PX) for processing (Principal Investigator: D Roberts). The tissues shown in
bold from all control and 200 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. In addition, sections of testes from all Control and 200 mg/kg bw/day males were
stained with Periodic Acid-Schiff (PAS) stain and examined.
Since there were indications of treatment-related changes, examination was subsequently
extended to include similarly prepared sections of mesenteric lymph nodes from animals in
the low and intermediate groups.

Pathology
Microscopic examination was conducted by the Study Pathologist Wendy Henderson. A peer
review of the findings observed was conducted by Vasanthi Mowat at Envigo CRS Limited,
Woolley Road, Alconbury, Huntingdon, Cambridgeshire, PE28 4HS. A complete
histopathology phase report is presented in Annex 1 and represents the consensus view of
both pathologists.

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)

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Animals of either sex treated with 100 or 200 mg/kg bw/day showed sporadic instances of
increased post-dose salivation between Days 2 to 28 of dosing. Intermittent instances of
noisy respiration were noted in all males and three female animals from the 200 mg/kg
bw/day dose group between Days 5 to 28, in three male animals from the 100 mg/kg bw/day
treatment group between Days 14 to 26 of dosing and in one female animal from the
30 mg/kg bw/day treatment group on Day 14 of dosing. Such observations are fairly
common in this type of study and may reflect an irritant nature of the test item and/or
formulation; these observations were therefore considered to be of no toxicological
significance.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Female animals from the 200 mg/kg bw/day treatment group showed a reduced body weight
gain in comparison to control animals during Week 3 but this did not attain statistical
significance. Female animals from the 30 mg/kg bw/day and 100 mg/kg bw/day treatment
groups showed reduced body weight gains during the first two weeks of treatment, reduced
body weight gains attained statistical significance (p<0.05) during Week 1 for the lower of
these treatment groups. These animals subsequently showed recovery over the remainder of
the treatment period such that overall body weight gain was similar to controls for female
animals in all treatment groups.
Male animals from all treatment groups showed similar weight gain to controls throughout
the treatment period.
Overall, it could be concluded that there did not appear to be any detrimental effect of
treatment with the test item at any dose level on body weight performance in animals of
either sex.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Male animals from all treatment groups showed a slight reduction in food consumption when
compared to controls throughout the treatment period (with the exception of Group 2 animals
during Week 2) but without any dose relationship. There was no effect of treatment up to a
dose level of 200 mg/kg bw/day on food consumption for female animals.
Overall, there did not appear to be any detrimental effect of treatment with the test item at
any dose level on food consumption in animals of either sex.
Food efficiency:
no effects observed
Description (incidence and severity):
At all dose levels, males showed similar food conversion efficiency when compared with
controls. In contrast, the corresponding values in females were sporadically lower that the
respective controls, reflecting intergroup differences in body weight gains for these animals.
Water consumption and compound intake (if drinking water study):
effects observed, non-treatment-related
Description (incidence and severity):
Gravimetric measurement of water consumption during Week 3 of the treatment period
identified sporadic instances of slightly lower water intake for males receiving 30 mg/kg
bw/day and slightly higher water intake for females receiving 100 mg/kg bw/day, resulting in
slightly lower and slightly higher overall water consumption for each of these dose groups
respectively. As all other dose groups were similar to controls this could be considered to be
due to biological variation and is considered not to be treatment related.
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
One male animal from the 200 mg/kg bw/day treatment group exhibited corneal opacity on Day 16.
Haematological findings:
no effects observed
Description (incidence and severity):
No toxicologically significant effects were detected at any dose level in animals of either sex.
At the end of the dosing period, group mean reticulocytes in all treated animals were higher
than controls (with a dose relationship being noted) statistical significance was attained in
both sexes treated with 200 mg/kg bw/day (p<0.05 and p<0.01 for males and females
respectively) and in female animals from the other two treatment groups (p<0.05 and p<0.01
for 30 mg/kg bw/day and 100 mg/kg bw/day respectively). Females from all dose groups
also exhibited a slight shortening in activated partial thromboplastin time with a dose
relationship being apparent, however, statistical significance was not achieved.
With the exception of two male animals (one from the 100 mg/kg bw/day and one from the
200 mg/kg bw/day treatment groups) which exhibited Reticulocyte counts slightly above the
control ranges, all individual values (including activated partial thromboplastin time) for the
corresponding animals generally remained within the historical control data ranges.
These findings were, therefore, deemed to be of no toxicological significance.
Clinical biochemistry findings:
not examined
Description (incidence and severity):
No toxicologically significant effects were detected at any dose level in animals of either sex.
When compared with controls, males treated with 30 mg/kg bw/day showed statistically
significantly higher Bilirubin levels (p<0.05). Female animals treated with 200 mg/kg
bw/day showed statistically significantly higher Phosphorous levels (p<0.05).
All individual values from affected treated and control groups were within the historical
control data ranges and as such these findings were considered to be of no toxicological
significance.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Behavioral Assessments
There were no treatment-related intergroup differences at any dose level.
Functional Performance Tests
There were no treatment-related changes in functional performance at any dose level.
Sensory Reactivity Assessments
There were no treatment-related changes detected at any dose level.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No toxicologically significant effects were detected at any dose level in animals of either sex.
A statistically significant increase (p<0.05) in both absolute and body weight-relative Kidney
weights were noted in female animals from the 200 mg/kg bw/day treatment group.
Two control female animals showed Absolute Kidney weights that were slightly lower than
the control data ranges, however, all values were within the control ranges for the affected
treated female group and as such the lower values from the control group are considered to
have influenced the overall apparent effect in the high dose females. Four females from the
200 mg/kg bw/day treatment group exhibited relative Kidney weights that were slightly
higher than the control data ranges with one control animal also showing a value that was
higher than the normal control ranges.
A statistically significant decrease (p<0.05) in both absolute and body weight-related
Thyroid/Parathyroid weights were noted in female animals from the 200 mg/kg bw/day and
100 mg/kg bw/day treatment groups.
Two control female animals exhibited relative Thyroid/Parathyroid weights that were slightly
higher than the control data ranges. All affected treated animals exhibited values that were
within the normal control ranges.
Taking into account the above it is considered that the findings were of no toxicological
significance.
Gross pathological findings:
no effects observed
Description (incidence and severity):
At necropsy, macroscopic abnormalities were limited to an enlarged spleen in one male from the 200 mg/kg bw/day dose group and an increased pelvic space in the left kidney of one female from the 30 mg/kg bw/day dose group.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The following microscopic change was evident:
Mesenteric Lymph Node
Histiocytosis (histiocyte aggregates) minimal or mild, was present in 1/5 males and 3/5
females in Group 4. This was not apparent in animals in Groups 2 and 3.
This change is known to occur in response to the oral administration of some test items,
likely representing accumulation of material and indicating delayed clearance of the test item
or its metabolites. At the severities seen in this study and in the absence of necrosis in the
lymph node, there is no evidence of functional impact thus in the confines of this study it is
considered likely to be non-adverse.
Other effects:
not examined

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
behaviour (functional findings)
body weight and weight gain
clinical biochemistry
clinical signs
food consumption and compound intake
food efficiency
gross pathology
haematology
histopathology: non-neoplastic
mortality
ophthalmological examination
organ weights and organ / body weight ratios
water consumption and compound intake
Dose descriptor:
NOEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
behaviour (functional findings)
body weight and weight gain
clinical biochemistry
clinical signs
food consumption and compound intake
food efficiency
gross pathology
haematology
histopathology: non-neoplastic
mortality
ophthalmological examination
organ weights and organ / body weight ratios
water consumption and compound intake

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
The oral (gavage) administration of 1,2-Ethanediamine, N-(2-aminoethyl)-, reaction products with glycidyl tolyl ether to male and female Wistar Han™:RccHan™:WIST strain rats at a dose level of 200 mg/kg bw/day resulted in microscopic changes in the mesenteric lymph node which were considered to be non-adverse. As such, 200 mg/kg bw/day is considered to be the ‘No Observed Adverse Effect Level’ (NOAEL) and 100 mg/kg bw/day is considered
to be the No Observed Effect Level’ (NOEL) for systemic toxicity within the confines of this type of study.
Executive summary:

The oral (gavage) administration of 1,2-Ethanediamine, N-(2-aminoethyl)-, reaction products with glycidyl tolyl ether to male and female Wistar Han™:RccHan™:WIST strain rats at a dose level of 200 mg/kg bw/day resulted in microscopic changes in the mesenteric lymph node which were considered to be non-adverse. As such, 200 mg/kg bw/day is considered to be the ‘No Observed Adverse Effect Level’ (NOAEL) and 100 mg/kg bw/day is considered to be the No Observed Effect Level’ (NOEL) for systemic toxicity within the confines of this type of study.