Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 03 October 2014 and 07 August 2015.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
See below
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Reference substance name:
Reaction mass of trientine and trientine, mono- and di-propoxylated
EC Number:
942-835-1
Molecular formula:
(C6 H18 N4 . C3 H6 O)x
IUPAC Name:
Reaction mass of trientine and trientine, mono- and di-propoxylated
Test material form:
other: Liquid
Details on test material:
Identification: Triethylenetetramine, propoxylated (triethylene-tetramineadduct)
Physical State/Appearance: Very pale green liquid
Chemical Name: 1,2-Ethanediamine, N,N'-bis(2-aminoethyle)-polymer, with methyloxirane
Chemical Formula: C6H18N4 + C3H6O
Purity: 98%
Storage Conditions: Ambient temperature (10 to 30 ºC) and humidity, in dark
No correction for purity was made.

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
Animals and Animal Husbandry
A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, 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 ninety six animals (forty eight males and forty eight females) were accepted into the study. At the start of treatment the males weighed 306 to 353g, the females weighed 189 to 223g, and were approximately twelve weeks old.

Initially, all animals were housed in groups of three in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK) (see deviations from Study Plan). During the pairing phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.

The animals were allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK.) was used. 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) except for paired animals and mated females during late gestation and lactation. Mated females were also given softwood flakes, as bedding, throughout gestation and lactation. The diet, drinking water, bedding and environmental enrichment was 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 Harlan Laboratories Ltd., 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; there were no deviations from these targets.

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.


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.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
Test Item Preparation
For the purpose of this study the test item was prepared at the appropriate concentrations as a solution in distilled water. The formulations were visually assesed to be homogeneously prepared. In addition, stability of the test item formulations was determined by Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. Results show the formulations to be stable for at least twelve days when stored at +4 °C, in the dark. Initially, formulations were prepared and used on daily basis. With the availability of further stability data, formulations were prepared on an approximately weekly basis, aliquoted, and stored as above before use.

Samples of the test item formulation were taken and analyzed for concentration of Triethylenetetramine, propoxylated (triethylene-tetramineadduct) on weekly basis at Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. The results indicate that the prepared formulations were within 96% to 107% of the nominal concentration.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Introduction
The test item concentration in the test samples was determined by high performance liquid chromatography-mass spectrometry (HPLC-MS) using an external standard technique. The test item gave a chromatographic profile consisting of a profile of multiple peaks.

Test item
The test item described in the main part of the study was also used as the analytical standard.

Analytical procedure
Preparation of standard solutions
Stock solutions of test item in water were prepared for external standard calibration. An aliquot, approximately 0.01 g of test item was accurately weighed into a 200 mL volumetric flask and brought to volume with water to yield a solution with a concentration of 0.05 mg/mL. Aliquots of this stock standard solution were used to prepare working standard solutions in water with a concentration range of 0.0035 mg/mL to 0.007 mg/mL.
On each occasion standard solutions derived from two stock standard solutions were used for calculation.

Analysis of samples
The formulations received were diluted with water. An aliquot of test item formulation was accurately weighed into a volumetric flask and brought to volume with water this was then shaken to dissolve. Where necessary, sample solutions were further diluted with water to achieve the working concentration.

Preparation of accuracy samples
Samples of distilled water were accurately fortified with known amounts of test item equivalent to the lowest and highest anticipated dose concentrations. These samples were then prepared to analysis as the test samples.

Preparation of linearity standards
A range of standard solutions were prepared in water from a stock solution of 1.114 mg/L by serial dilution covering the concentration range 0 to 0.00780 mg/mL.

Instrument set up
HPLC system: Agilent Technologies 1200 MSD, incorporating autosampler and workstation
Mass selective detecto
Source: Electrospray
Fragmentation energy: 110 volts
Polarity: Positive
Mode: Single ion mode with 205.2, 263.2, 321.0 and 379.2 amu
Gas temperature: 350 °C
Drying gas: 12 L/min
Nebuliser pressure: 60 psi
Capillary voltage: 2000 v
Gain: 1
Column: X-terra phenyl, 3.5 µ (150 x 4.6 x 6 mm id)
Gradient elution
Eluent A: 0.1 % heptafluorobutyric acid in 1 lcms water
Eluent B: acetonitrile
Time (minutes) % A % B
0 95 5
10 20 80
12 20 80
Flow rate: 0.5 mL/min
Injection volume: 5 µL
Retention time: Approximately 9.5 minutes

Study samples and storage
Representative samples were dispatched to the analytical laboratories internally (under ambient conditions) and stored at room temperature until analysis.

Results
Validation of analytical method
Specificity
The control dose samples and an analysed solvent blank showed no significant interfering response at the retention time of the test item. The standard solutions contained a peak specific for the test item whose are changed accordingly with known concentration; hence the specificity of the method by retention time was confirmed.

Linearity
The data was found to have a quadratic correlation within the calibration range of 0 to 0.007 mg/mL. The R2 fit of the calibration curve to the data was in the range of 0.998 to 1.000 and was considered acceptable.

Accuracy
The fortified samples of distilled water were found to have a recovery value of ± 10 % of the fortification.

Test item formulation
The formulations investigated during the study were found to comprise test item in the range of 96 % to 107 % and thus the required content limit of ± 10 % with reference to the nominal content was met.
The test item was found to be stable in the formulations when kept for 12 days in the refrigerator (4 °C) due to results which met the variation limit of 10 % from the time-zero mean.
In conclusion, the results indicate the accurate use of the test item and distilled water as vehicle during this study. The formulations were visually assessed to be homogeneously prepared and sufficient formulation stability under storage conditions was proven.

Discussion
The detection system was found to have acceptable correlation to the fit of a quadratic equation. The analytical procedure had acceptable recoveries of test item in the vehicle. The method of analysis was validated and proven to be suitable for use.
Duration of treatment / exposure:
Males: Day 43 or 44.
Females: Up to 65 days.
Any female which did not produce a pregnancy was terminated on Day 26 post coitum.
Frequency of treatment:
Daily
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300 and 750 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
Twelve male and twelve female per dose
Control animals:
yes, concurrent vehicle
Details on study design:
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 5 mL/kg of distilled water.

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.

Chronological Sequence of Study
i. Groups of twelve male and twelve female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). The first day of dosing was designated as Day 1 of the study. The first six males in each dose group were dosed up to Day 42 whilst the remaining males were dosed up to Day 43.
ii. Prior to the start of treatment and once weekly thereafter, all animals were observed for signs of functional/behavioral toxicity (see deviations from Study Plan).
iii. On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a period of up to fourteen days. Female 14 (control) showed clinical signs of hunched posture and decreased respiratory rate after dosing on Day 15 and was therefore paired with Male 2 (control) on the following day.
iv. Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.
v. On completion of the pairing phase (during Week 6), five selected males per dose group were evaluated for functional/sensory responses to various stimuli (see deviations from Study Plan).
vi. Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Litter size, offspring weight and sex, surface righting and clinical signs were also recorded during this period.
vii. At Day 4 post partum, five selected females per dose group were evaluated for functional/sensory responses to various stimuli.
viii. Blood samples were taken from five males from each dose group for hematological and blood chemical assessments on Day 42 (see deviations from Study Plan). The male dose groups were killed and examined macroscopically on Day 43 or 44.
ix. Blood samples were taken from five randomly selected females from each dose group for hematological and blood chemical assessment on Day 4 post partum. At Day 5 post partum, all littering females and surviving offspring were killed and examined macroscopically. Any female which did not produce a pregnancy was also killed and examined macroscopically.
Positive control:
None

Examinations

Observations and examinations performed and frequency:
Clinical Observations
All animals were examined for overt signs of toxicity, ill-health and behavioral change immediately before dosing, soon after dosing, and one hour after dosing (except for females during parturition where applicable). All observations were recorded.


Functional Observations
Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioral toxicity. Functional performance tests were also performed on five selected males and females from each dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli (see deviations from Study Plan).


Behavioral Assessments
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.
Purpose-built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals were randomly allocated to the activity monitors. The tests were performed at approximately the same time on each occasion (at least two hours after dosing), under similar laboratory conditions. The evaluation period was thirty minutes for each animal. The percentage of time each animal was active and mobile was recorded for the overall thirty minute 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 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. The animal was pulled by the base of the tail until its grip was broken. 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 following parameters were observed:
Grasp response, Touch escape, Vocalization, Pupil reflex, Toe pinch, Blink reflex, Tail pinch, Startle reflex, Finger approach.


Body Weight
Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until pairing. During pairing phase females were weighed daily until mating was confirmed. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum. Body weights were also recorded at terminal kill.


Food Consumption
During the pre-pairing period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded on Days 1 and 4 post partum.

Food efficiency (the ratio of body weight change/dietary intake) was calculated retrospectively for males throughout the study period (with the exception of the mating phase) and for females during the pre-pairing phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated for females during gestation and lactation.



Water Consumption
Water intake was observed daily by visual inspection of water bottles for any overt changes.


Reproductive Performance
Mating
Animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of estrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages. Mated females were housed individually during the period of gestation and lactation.


Pregnancy and Parturition
Each pregnant female was observed at least three times a day (early morning, mid-day and as late as possible during the normal working day) around the period of expected parturition. Observations were carried out at approximately 08:30 AM and as late as possible at weekends. The following was recorded for each female:

i. Date of pairing
ii. Date of mating
iii. Date and time of observed start of parturition
iv. Date and time of observed completion of parturition


Litter Data
On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded. Offspring were individually identified within each litter by tattoo on Day 1 post partum.

For each litter the following was recorded:

i. Number of offspring born
ii. Number of offspring alive recorded daily and reported on Days 1 and 4 post partum
iii. Sex of offspring on Days 1 and 4 post partum
iv. Clinical condition of offspring from birth to Day 5 post partum
v. Individual offspring weights on Days 1 and 4 post partum (litter weights were calculated retrospectively from this data)


Physical Development
All live offspring were assessed for surface righting reflex on Day 1 post partum.


Laboratory Investigations
Hematological and blood chemical investigations were performed on five males and five females selected from each test and control group prior to termination (Day 42 for males and Day 4 post partum for females) (see deviations from Study Plan). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were taken by cardiac puncture at termination. Animals were not fasted prior to sampling.


Hematology
The following parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant:

Hemoglobin (Hb)
Erythrocyte count (RBC)
Hematocrit (Hct)
Erythrocyte indices
- mean corpuscular hemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular hemoglobin concentration (MCHC)
Total leukocyte count (WBC)
Differential leukocyte count
- neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count (PLT)
Reticulocyte count (Retic)
- Methylene blue stained slides were prepared but reticulocytes were not assessed

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


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

Urea, Calcium (Ca++), Glucose, Inorganic phosphorus (P), Total protein (Tot.Prot.), Aspartate aminotransferase (ASAT), Albumin, Alanine aminotransferase (ALAT), Albumin/Globulin (A/G) ratio (by calculation), Alkaline phosphatase (AP), Sodium (Na+), Creatinine (Creat), Potassium (K+), Total cholesterol (Chol), Chloride (Cl-), Total bilirubin (Bili), Bile acids.
Sacrifice and pathology:
Necropsy
Adult males were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 43 or 44. Adult females were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 5 post partum. Surviving offspring were terminated via intracardiac overdose of suitable barbiturate agent. Any females which failed to achieve pregnancy were killed on Day 26 post coitum.

For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced; as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution (Salewski 1964). The corpora lutea were also counted.

All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.


Organ Weights
The following organs were dissected free from fat and weighed before fixation from five selected males and five selected females from each dose group.

Adrenals, Prostate, Brain, Seminal vesicles, Epididymides, Spleen, Heart, Testes, Kidneys, Thymus, Liver, Thyroid (weighed post-fixation with Parathyroid), Ovaries, Uterus (weighed with Cervix), Pituitary (post fixation).

Tissues shown in below were weighed from all remaining animals (see deviations from Study Plan):
Prostate, Seminal vesicles, Epididymides, Testes, Ovaries, Pituitary (post fixation), Uterus (weighed with Cervix).


Histopathology
Samples of the following tissues were removed from five selected males and five selected females from each dose group and preserved in buffered 10% formalin, except where stated. Tissues shown in bold were preserved from all remaining animals:

Adrenals, Muscle (skeletal), Aorta (thoracic), Ovaries, Bone & bone marrow (femur including stifle joint), Pancreas, Bone & bone marrow (sternum), Pituitary, Brain (including cerebrum, cerebellum and pons), Prostate, Caecum, Rectum, Coagulating gland, Salivary glands (submaxillary), Colon, Sciatic nerve, Duodenum, Seminal vesicles, Epididymides•, Skin (hind limb), Esophagus, Spinal cord (cervical, mid-thoracic and lumbar), Eyes* , Gross lesions, Spleen, Heart, Stomach, Ileum (including peyer’s patches), Thyroid/parathyroid, Jejunum, Trachea, Kidneys, Testes•, Liver, Thymus, Lungs (with bronchi) #, Urinary bladder, Lymph nodes (mandibular and mesenteric), Uterus/Cervix, Mammary gland , Vagina.

Tissues shown in below were preserved from all remaining animals:
Ovaries, Pituitary, Prostate, Coagulating gland, Seminal vesicles, Epididymides•, Gross lesions, Testes• ,Uterus/Cervix, Mammary gland , Vagina.

• = preserved in Modified Davidson's fluid
* = eyes fixed in Davidson’s fluid
# = lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before
immersion in fixative

Tissues were dispatched to the Test Site (Huntingdon Life Sciences Ltd., Eye Research Centre, Eye, Suffolk, IP23 7PX) for processing (Principal Investigator: J Schofield). The tissues from five selected control and 750 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. The tissues shown in bold from the remaining control and 750 mg/kg bw/day animals were also processed; this included animals failing to achieve pregnancy whilst all females from the low and intermediate dose groups achieved pregnancy. In addition, sections of testes from all control and 750 mg/kg bw/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined.

Detailed qualitative examination of the testes was undertaken, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell-or stage-specificity of testicular findings was noted.

Since there were indications of treatment-related changes, examination was subsequently extended to include similarly prepared sections of the kidneys, stomach and lungs from animals in the low and intermediate groups.
Other examinations:
None specified
Statistics:
See below

Results and discussion

Results of examinations

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:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
See results
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
See results
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
See results
Histopathological findings: neoplastic:
no effects observed
Details on results:
Adult Responses
Mortality
There were no unscheduled deaths during the study.


Clinical Observations
There were no clinical observations considered to be related to the toxicity of the test item at any dose level.

At 750 mg/kg bw/day, sporadic instances of increased post-dose salivation were observed in 5/12 males between Days 30 to 38 of dosing and one female on Day 38. Such clinical observations are common in gavage studies and are often related to the taste of the formulation rather than an indication of the systemic toxicity of the test item.

Female 14 from the control group was observed with hunched posture and decreased respiratory rate following dosing on Day 15 of the study; these clinical signs were no longer apparent before dosing on the following day.
There were no clinical signs for any of the remaining animals on the study throughout the treatment period.


Functional Observations
Behavioral Assessments:
No treatment-related changes in the behavioral parameters were detected at any dose level.

Functional Performance Tests:
There were no intergroup differences considered to be related to treatment with the test item.

Motor activity monitoring during the final week of the treatment period revealed statistically significantly higher activity (last 20%) for females given the test item at 100 mg/kg bw/day in relation to controls (p<0.05). The corresponding values for the intermediate and high dose group females were similar to controls and as such this finding was deemed to be incidental. Grip strength testing also identified some statistically significant intergroup difference for animals of either sex when compared with the respective controls (p<0.05); there was generally no dose-dependence and any differences were considered to be incidental.


Sensory Reactivity Assessments:
There were no treatment-related intergroup differences in sensory reactivity scores across all dose groups including controls.


Body Weight
Throughout the study, there was no adverse effect of treatment with the test item on body weight development in animals of either sex.

At 750 mg/kg bw/day, group mean body weight gains for males over the first two weeks of dosing were slightly lower than controls, albeit without achieving statistical significance. Subsequent body weight gains for these animals were generally similar to controls with Week 5 being the exception when their group mean body weight gain was statistically significantly lower than controls (p<0.05); overall body weight gain for these males was approximately 24% lower than controls. Although this initial effect on body weight development may be treatment-related, in view of the overall body weight results it was considered unlikely to be of an adverse nature.

At the start of gestation, females treated with 750 mg/kg bw/day showed slightly lower group mean body weight gain in relation to controls, but without attaining statistical significance. Subsequent improvement was evident such that the mean body weight gain over Days 7 to 14 of gestation for these females was similar to controls and as such this finding was considered to be of no toxicological importance. Group mean body weight gains for these females during the pre-pairing and lactation phases of the study were similar to controls.

At 100 or 300 mg/kg bw/day, body weight development in animals of both sexes remained similar to controls throughout the study.


Food Consumption
There was no detrimental effect of treatment with the test item on dietary intake or food efficiency in male groups at any dose level.
There was no detrimental effect of treatment with the test item on food consumption or food efficiency in females during the pre-pairing phase of the study. During gestation and lactation, dietary intake in females treated with the test item at all dose levels remained similar to controls.


Water Consumption
Visual inspection of water bottles did not indicate any differences for the animals given the test item in relation to controls.


Reproductive Performance
Mating
There was no effect of treatment on mating performance with all animals mating within four days after pairing.


Fertility
Fertility as assessed by pregnancy index was unaffected by treatment with the test item at any dose level.

Two females from the control group (Females 19 and 23) and one female from the high dose group (Female 86) did not achieve pregnancy following positive evidence of mating. No histopathological correlates were evident in reproductive tissues from these animals and the males they were paired with which could have been considered to be the cause of their infertility.


Gestation Length
Gestation lengths were between 22 and 23½ days and the distribution of gestation lengths for treated females was similar to controls.


Litter Responses
In total ten, twelve, twelve and eleven females from control, 100, 300 or 750 mg/kg bw/day dose groups respectively gave birth to a live litter and successfully reared young to Day 5 of age. The following assessment of litter response is based on all litters reared to termination on Day 5 of lactation/age.

Offspring Litter Size, Sex Ratio and Viability
There was no detrimental effect of treatment with the test item on corpora lutea and implantation counts or pre- and post-implantation losses.

Of the litters born, litter size at birth and subsequently on Days 1 and 4 post partum from females treated with the test item at all dose levels were comparable with controls.
There were no intergroup differences in sex ratio (percentage male offspring) for litters from test item-treated groups when compared with controls.


Offspring Growth and Development
Offspring bodyweight gain and litter weights on Days 1 and 4 post partum were comparable with controls.

Surface righting reflex data did not reveal any intergroup differences when compared with controls.
Clinical signs for the offspring included small size, weak, no milk in stomach, physical injury, scab or mass on abdomen, found dead or missing and were considered to be low incidence findings observed in offspring in studies of this type and as such unrelated to the toxicity of the test item.

Laboratory Investigations
Hematology
No toxicologically significant effects were detected in animals of either sex receiving the test item at any dose level.

At the end of the treatment period, animals of both sexes treated with 750 mg/kg bw/day showed statistically significantly higher platelet counts when compared with controls (p<0.05). A dose-relationship was apparent in males, but most individual values were within the historical control data ranges. When compared with controls, other statistically significant haematology observations in males treated with 750 mg/kg bw/day included slightly lower red blood cell count (p<0.01) and slightly higher mean cell volume (p<0.05). Statistically significantly higher neutrophil counts were also observed in males given the test item at all dose levels in relation to controls (p<0.01 at 750 mg/kg bw/day and p<0.05 at 100 or 300 mg/kg bw/day). There were no clear dose-related trends for any of these parameters and individual values were within the background data ranges. In the absence of any histopathology correlates, these findings were considered to be of no toxicological relevance.


Blood Chemistry
At 750 mg/kg bw/day, group mean plasma concentration of total protein in males was statistically significantly lower than controls (p<0.05). Group mean creatinine concentration in these males was also statistically significantly lower than controls (p<0.05), but without any dose-dependence. Although individual values for these males were within the historical background data ranges, taken together with the microscopic findings in the kidneys from these animals (minimal or mild multifocal tubular basophilia with some of these males showing mild tubular dilation), the toxicological significance of these findings remains unclear.

At all dose levels, females receiving the test item showed statistically significantly lower aspartate aminotranferase activities when compared with controls (p<0.01). With the exception of one control female showing a value that was moderately above the background data ranges, individual values from the remaining control and treated animals were within these ranges. Group mean plasma concentration of cholesterol in females treated with 300 or 750 mg/kg bw/day was also statistically significantly higher than controls (p<0.05) albeit without any dose-dependence and all individual values from the high dose females were within the background data ranges. In the absence of any associated histopathology findings, these observations were considered to be of no toxicological relevance.

At 300 mg/kg bw/day, group mean plasma level of bile acid in females was statistically significantly higher than controls (p<0.05). As there was no dose-relationship and the corresponding value in the high dose females was similar to controls, this finding was considered unrelated to treatment with the test item.


Pathology
Necropsy
Offspring

Macroscopic necropsy findings for offspring on the study were typical for the age observed and neither the incidence nor the distribution of these observations indicated any adverse effect of maternal treatment on offspring development at 100, 300 or 750 mg/kg bw/day.

Adults
No changes were noted in any animals which could be related to treatment with the test item.

Organ Weights
At 750 mg/kg bw/day, group mean absolute and body weight-related kidney weights in males were moderately higher than controls (p<0.01). Although a dose-relationship was not evident, individual body weight-related kidney weights for 4/5 high dose males were above the background data range. Taken together with histopathological observations from kidneys, which included minimal or mild multifocal tubular basophilia with some males also showing mild tubular dilation, this finding was considered to be of toxicological significance.

At 750 mg/kg bw/day, group mean absolute and body weight-related liver weights in males showed a marginal increase when compared with controls (p<0.05). These males also showed statistically significantly lower mean absolute and body weight-related thyroid/parathyroid weights in comparison with controls (p<0.05). There were no dose-relationships and individual values for all high dose animals were within the background control data ranges. In the absence of any histopathology correlates, these observations were considered to be of no toxicological significance.

Other statistically significant differences included higher group mean absolute and body weight-related brain weights in males receiving 100 mg/kg bw/day when compared with controls (p<0.01). There was no dose-dependence and the corresponding values in the intermediate or high dose males were similar to controls. This finding was therefore considered to be incidental.


Histopathology
The following treatment-related microscopic abnormalities were detected.

Kidney: At 750 mg/kg bw/day, multifocal basophilic tubules was present in all males and 3/5 females at a minimal or mild level. Mild tubular dilation was also present in 2/5 of these males. This correlated with the absolute and body weight related kidney weight increase noted for these males at necropsy and is indicative of a toxic effect in the kidneys at the high dose level. No treatment related changes in the kidney were present in animals from the 100 or 300 mg/kg bw/day dose groups.

Stomach: At 750 mg/kg bw/day, inflammatory cell infiltrate in the submucosa of the glandular region, minimal or mild, was present in 3/5 males and females. These changes were considered to be indicative of an irritant effect of the test item at this dose level. Similar changes were not present in animals from the 100 or 300 mg/kg bw/day dose groups.

Lungs: At 300 or 750 mg/kg bw/day, there was an increase in severity of alveolitis (multifocal and mild) in females, and an increase in incidence in males treated with 300 mg/kg bw/day (focal and minimal) and in severity in males treated with750 mg/kg bw/day (multifocal and mild) of increased alveolar macrophages. One female receiving 300 mg/kg bw/day also had mild peribronchial inflammation. The type and distribution of the changes, such as peribronchial inflammation, is suggestive of irritation due to technical dosing issues rather than any systemic effect of the test item and indicative of an irritant effect. This is considered to be of limited toxicological significance as the gavage route of dosing is not relevant to human exposure.

Effect levels

Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Microscopic examination of the selected tissues revealed findings of toxicological significance in the kidneys from animals of either sex treated with 750 mg/kg bw/day.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Discussion

The oral administration of Triethylenetetramine, propoxylated (triethylene-tetramineadduct) to rats for a period of up to seven weeks (including two weeks of pre-pairing, gestation and early lactation for females) at dose levels of up to 750 mg/kg bw/day, was well tolerated. Sporadic instances of increased salivation towards the end of the treatment period were seen in some males and one female given the high dose; however, these were considered likely to be related to the taste of the test item formulation rather than an indication of its systemic toxicity. A slight reduction in group mean body weight gains for the high dose males during the pre-pairing phase and for the high dose females during the first week of gestation was not associated with any effect on dietary intake. Subsequent improvement in body weight was evident for these animals, and any slight effect on body weight development at high dose was considered to be of no toxicological significance. 

 

Blood chemistry investigations revealed slight decreases in plasma concentrations of total protein and creatinine in males given 750 mg/kg bw/day when compared with controls. Although these findings may correlate with microscopic observations in the kidneys from these males, individual total protein and creatinine values were within the historical data ranges and as such the toxicological significance of these intergroup differences was deemed not clear. Any other intergroup differences identified in hematological or blood chemistry assessments were considered to be of no toxicological significance.

 

Histopathology examination of the kidneys from animals of either sex identified the presence of multifocal tubular basophilia in animals of either sex receiving the test item at a dose level of 750 mg/kg bw/day, more notable in the males with some of these males also showing tubular dilation. These findings can be considered adverse as they are considered to be associated with degenerative change. This correlated with an increase in absolute and body weight-related kidney weights in these animals and was considered to be indicative of a toxic effect in the kidneys at this dose level. 

 

Inflammatory change in the glandular stomach was noted in males and females from the high dose group and this was considered to be indicative of an irritant effect of the test item at this dose level rather than direct systemic toxicity. Inflammatory changes in the lungs were apparent in animals across all groups but there was an apparent increase in animals from the intermediate and high dose groups above control animal limits. The type and distribution of the changes was suggestive of irritation due to technical dosing issues rather than any systemic effect of the test item and indicative of an irritant effect. These changes were considered to be of limited toxicological significance as the gavage route of dosing is not relevant to human exposure. 

 

Taking into view the above findings, it is considered that a dose level of 300 mg/kg bw/day could be assigned a No Observed Adverse Effect Level (NOAEL) for systemic toxicity.

 

Mating performance and fertility remained unaffected by treatment with the test item at all dose levels. In addition, there was no detrimental effect of treatment on pre- and post-implantation losses, viability indices, sex ratios and offspring growth and development. There were no treatment-related clinical signs or macroscopic findings for any of the pups. A dose level of 750 mg/kg bw/day was considered to be the No Observed Effect Level (NOEL) for reproductive toxicity.

Applicant's summary and conclusion

Conclusions:
The oral (gavage) administration of Triethylenetetramine, propoxylated (triethylene-tetramineadduct) to Wistar Han™:RccHan™:WIST strain rats, at dose levels of 100, 300 or 750 mg/kg bw/day was well tolerated. Microscopic examination of the selected tissues revealed findings of toxicological significance in the kidneys from animals of either sex treated with 750 mg/kg bw/day. Based on the available data, the ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was considered to be 300 mg/kg bw/day.

The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 750 mg/kg bw/day.
Executive summary:

Introduction

The study was designed to investigate the systemic toxicity and potential adverse effects of the test item on reproduction (including offspring development) and is designed to be compatible with the requirements of the OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test” (adopted 22 March 1996).

 

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 1907/2006 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 twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for up to seven weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 100, 300 and 750 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (distilled water).

 

Clinical signs, behavioral assessments, body weight change and food and water consumption were monitored during the study. 

 

Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation. Due to a decline in the clinical condition of Female 14 on Day 15 of the study following dosing, this female was paired with a male on the following day.

 

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex.

 

Extensive functional observations were performed on five selected males from each dose group after the completion of the pairing phase, and for five selected parental females from each dose group on Day 4post partum. Hematology and blood chemistry were evaluated prior to termination on five selected males and females from each dose group. 

 

Adult males were terminated on Day 43 or 44 of the study, followed by the termination of all littering females and offspring on Day 5 post partum. Any female which did not produce a pregnancy was terminated on Day 26 post coitum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

 

 

Results….

 

Adult Responses

Mortality

There were no unscheduled deaths during the study.

 

 

Clinical Observations

Throughout the study, there were no clinical signs considered to be related to the toxicity of the test item.

 

 

Behavioral Assessment

There were no treatment-related changes in the behavioral parameters measured.

 

 

Functional Performance Tests

There was no effect of treatment with the test item at any dose level on functional performance in animals of either sex.

 

 

Sensory Reactivity Assessments

Sensory reactivity scores across all treated groups were similar to controls.

 

 

Body Weight

There was no adverse effect of treatment with the test item on male body weight development during the study. There was also no adverse effect of treatment with the test item on body weight development in females during the pre-pairing, gestation or lactation phases of the study.

 

 

Food Consumption

Food intake and food efficiency across all of the test item-treated male groups remained similar to controls during the study. There was also no effect of treatment with the test item on food consumption and food efficiency in females during the pre-pairing phase of the study. During gestation and lactation, dietary intake in test item-treated females was comparable with controls.

 

 

Water Consumption

Visual inspection of water bottles did not indicate any differences for the animals given the test item in comparison with controls.

 

 

Reproductive Performance

Mating

There was no effect of treatment on mating performance. All animals mated within four days of pairing.

 

 

Fertility

There were no treatment-related effects in conception rates for test item-treated animals in relation to controls.

 

 

Gestation Lengths

There were no differences in gestation lengths in animals receiving the test item when compared with controls.

 

 

Litter Responses

Offspring Litter Size, Sex Ratio and Viability

There was no detrimental effect of treatment on corpora lutea count, pre-implantation loss, number of implantations, post-implantation loss, litter size, sex ratio and subsequent offspring survival to Day 5 of age at 100, 300 or 750 mg/kg bw/day.

 

 

Offspring Growth and Development

There was no detrimental effect of treatment indicated by offspring body weight or body weight gain and litter weights, surface righting ability on Day 1 or clinical signs to Day 5 of age at 100, 300 or 750 mg/kg bw/day.

 

 

Laboratory Investigations

Hematology

No toxicologically significant effects were detected in animals of either sex at any dose level.

 

 

Blood Chemistry

At 750 mg/kg bw/day, group mean plasma concentrations of total protein and creatinine in males were statistically significantly lower than controls (p<0.05). Although individual values for all high dose animals were within the historical control data ranges, taking into consideration the presence of multifocal tubular basophilia in the kidneys from these animals with some males also showing tubular dilation, the toxicological significance of these blood chemistry observations was unclear.

 

No other toxicologically significant differences were detected in animals of either sex at any dose level.

 

 

Pathology

Necropsy

Neither the type, incidence or distribution of macroscopic observations in adult animals or offspring indicated any treatment-related effect of treatment at 100, 300 or 750 mg/kg bw/day.

 

 

Organ Weights

At 750 mg/kg bw/day, group mean absolute and body weight-related kidney weights in males were moderately higher than controls (p<0.01). Taken together with histopathological observations from kidneys, which included minimal or mild multifocal tubular basophilia with some males also showing mild tubular dilation, this finding was considered to be of toxicological significance.

 

There were no other toxicologically significant intergroup differences.

 

 

Histopathology

The following treatment-related microscopic abnormalities were detected.

 

Kidney:At 750 mg/kg bw/day, minimal or mild multifocal basophilic tubules were present in all males and some females. Mild tubular dilation was also present in some of these males. These findings correlated with the absolute and body weight related kidney weight increase noted for these males and were considered to be indicative of a toxic effect in the kidneys at this dose level. 

 

Stomach:At 750 mg/kg bw/day, inflammatory cell infiltrate in the submucosa of the glandular region, at a minimal or mild level, was present in some males and females. These changes were considered to be indicative of an irritant effect of the test item at this dose level. 

 

Lungs:At 300 or 750 mg/kg bw/day, there was an increase in severity of alveolitis (multifocal and mild) in females and an increase in severity of increased alveolar macrophages (mulifocal and mild) in the high dose males. One female receiving 300 mg/kg bw/day also had mild peribronchial inflammation. The type and distribution of the changes, such as peribronchial inflammation, is suggestive of irritation due to technical dosing issues rather than any systemic effect of the test item and indicative of an irritant effect. This was considered to be of limited toxicological significance as the gavage route of dosing is not relevant to human exposure.

 

 

Conclusion

The oral (gavage) administration of Triethylenetetramine, propoxylated (triethylene-tetramineadduct) to Wistar Han™:RccHan™:WIST strain rats, at dose levels of 100, 300 or 750 mg/kg bw/day was well tolerated. Microscopic examination of the selected tissues revealed findings of toxicological significance in the kidneys from animals of either sex treated with 750 mg/kg bw/day. Based on the available data, the ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was considered to be 300 mg/kg bw/day.

 

The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 750 mg/kg bw/day.