Triglycerides, C16-18 (even numbered), reaction products with diethylenetriamine

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The purpose of this study was to assess the potential systemic toxicity in rats, including a screen for reproductive/developmental effects and assessment of endocrine disruptor relevant endpoints, with administration of Amidoamine (UVCB) by oral gavage administration for at least five weeks.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

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)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Triglycerides, C16-18 (even numbered), reaction products with diethylenetriamine

Molecular formula:

not applicable (UVCB)

IUPAC Name:

Triglycerides, C16-18 (even numbered), reaction products with diethylenetriamine

Test material form:

solid: compact

Details on test material:

- Name of test material (as cited in study report): Amidoamine (UVCB)
- Substance type: Amidoamine
- Chemical name: Glycerides, C14-20, reaction products with diethylenetriamine (preliminary naming)/Amides, from diethylenetriamine and hydrogenated palm oil
- CAS 85409-11-6/1618093-67-6
- Physical state: pale yellowish solid at 20 °C
- Batch No.: K8 4309 L481
- Expiry date of batch: 09 March 2017
- Purity: 100 % (UVCB)
- Storage condition of test material: Room temperature, protected from light
- Stability: stable under test conditions

Specific details on test material used for the study:

Test item without emulsifier was investigated.

Amidoamine (UVCB)
Pulcra ID: DE07_2014_012_BEL66 (amidoamine without emulsifier)
Physical state: pale yellowish solid at 20 °C
Batch No.: K8 4309 L481
Expiry date of batch: 09 March 2018
Purity: 100 % (UVCB)
Stability: stable under test conditions
Storage condition of test material: Room temperature, protected from light

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Strain/Species: RccHan; WIST rat.
Supplier: Envigo (RMS) B.V.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test animals

The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The RccHan;WIST (Han Wistar) strain was used because of the historical control data available at this laboratory.

Strain/Species: RccHan™;WIST rat.
Supplier: Envigo (RMS) B.V.
Number of animals ordered: 44 males and 48 females.
Duration of acclimatization Males: five days prior to the commencement of treatment.
Duration of acclimatization Females: 19 days prior to the commencement of treatment.
Age of the animals at the start of treatment Males: nominally 12 weeks old.
Age of the animals at the start of treatment Females: nominally 14 weeks old.
Weight range of the animals at the start of treatment Males: 325 to 356 g.
Weight range of the animals at the start of treatment Females: 202 to 252 g.

Environmental conditions

Rodent facility: Limited access - to minimize entry of external biological and chemical agents and to minimize the transference of such agents between rooms.
Air supply: Filtered fresh air which was passed to atmosphere and not recirculated. The air change rate per hour was 18.6 to 35.5 (assessed May 2017).
Temperature and relative humidity: Monitored and maintained within the range of 20-24ºC and 40-70%. There were no deviations from these ranges.
Lighting: Artificial lighting, 12 hours light : 12 hours dark (See Section 4).
Electricity supply: Public supply with automatic stand-by generators.
Cages: Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals. Solid (polycarbonate) bottom cages were used during the acclimatization, pre-pairing, treatment, gestation, littering and lactation periods. Grid bottomed polypropylene cages were used during pairing. These were suspended above absorbent paper which was changed daily during pairing.
Cage distribution: The cages were distributed on the racking to equalize, as far as possible, environmental influences amongst the groups.
Bedding: Solid bottom cages contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 1% methylcellulose in 0.1% Tween 80

Details on oral exposure:

Administration volume was 10 mL/kg bw/day.
The test item was dissolved in the vehicle to concentrations of 10, 33 and 100 mg test item/mL vehicle. The test item formulations were freshly prepared and adjusted to the animal's current body weight on each administration day.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

An analytical methods was developed to quantify Amidoamine (UVCB) or Amidoamine 2 (UVCB, low nitrogen content) in liquid vehicle at concentrations from 1 – 200 mg/mL (approximately). The homogeneity and stability of Amidoamine (UVCB) formulations during storage were determined as part of another study, Envigo Study Number TD87XR. Stability was confirmed for 15 days when stored refrigerated (2 to 8 °C) and for one day when stored at ambient temperature (15 to 25 °C) between the concentration range 5 to 200 mg/mL.

Duration of treatment / exposure:

Males: Two weeks before pairing up to necropsy after a minimum of five weeks of treatment.
Females: Two weeks before pairing, then throughout pairing and gestation until Day 13 of lactation. Two females were also treated on Day 14 of lactation

Frequency of treatment:

Once daily at approximately the same time each day.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

10 animals of each sex per dose.

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale:
The dose levels have been selected in agreement with the Sponsor based on the results of a 14-day dose-range-finding study in rats dosed at 100, 300 and 1000 mg (active ingredient)/kg bw by oral gavage (Envigo Study No. YT38XB).
No animals died prematurely. Oral treatment with 1000 mg/kg bw/day caused no signs of systemic toxicity.
In that study Amidoamine (UVCB) or Amidoamine 2 (UVCB, low nitrogen content) were administered to rats at doses of 100 or 1000 mg/kg/day, there were no clear effects of treatment from either test item on body weight, food consumption, water consumption, organ weights or tissue appearance at necropsy. Therefore the test material of Amidoamine (UVCB) was selected for this study. The high dose of 1000 mg/kg/day is the limit dose for the OECD 422 study guideline and the intermediate and low doses of 330 and 100 mg/kg/day were selected to investigate any dose relationship should effects be observed in the longer duration study.

Positive control:

no positive control included

Examinations

Observations and examinations performed and frequency:

Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate. During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

Detailed observations were performed to establish and confirm a pattern of signs in association with dosing according to the following schedule:
F0 males
Week 1 - daily
Week 2 onwards - once each week

F0 females
Week 1 - daily
Week 2 - once
Gestation phase - Days 0, 7, 14 and 20
Lactation phase - Days 1, 6 and 12

Detailed observations were recorded at the following times in relation to dose administration:
Pre-dose observation
One to two hours after completion of dosing
As late as possible in the working day

Before treatment commenced and during each week of treatment and on Days 0, 7, 14 and 20 after mating and Days 1, 6 and 12 of lactation, detailed physical examination and arena observations were performed on each animal. On each occasion, the examinations were performed at approximately the same time of day (before dosing during the treatment period), by an observer unaware of the experimental group identities.

After removal from the home cage, animals were assessed for physical condition and behavior during handling and after being placed in a standard arena. Any deviation from normal was recorded with respect to the nature and, where appropriate, degree of severity. Particular attention was paid to possible signs of neurotoxicity, such as convulsions, tremor and abnormalities of gait or behavior. Findings were either reported as "present" or assigned a severity grade - slight, moderate or marked.

Sensory reactivity and grip strength

Sensory reactivity and grip strength assessments were performed (before dosing) on the five lowest numbered surviving males in each group during Week 5 of treatment and on the first five lactating females in each group at Day 7-9 of lactation.

Motor activity

During Week 5 of treatment for males and at Day 7-9 of lactation for females, the motor activity of the five lowest numbered surviving males and the first five lactating females in each group was measured (before dosing) using a Rodent Activity Monitoring System (Version 2.0.6), with hardware supplied by Pearson Technical Services and software developed and maintained by Envigo. Animals were tested individually in clear polycarbonate cages and motor activity was measured by counting infra-red beam breaks over ten 6-minute intervals (one hour total). Ten beams were set at two height levels (five low and five high) to detect cage floor and rearing activity respectively.

Body weight

The weight of animals was recorded as follows:

F0 males Weekly during acclimatization. Before dosing on the day that treatment commenced (Week 0) and weekly thereafter.On the day of necropsy.

F0 females Weekly during acclimatization. Before dosing on the day that treatment commenced (Week 0) and weekly before pairing. Days 0, 7, 14 and 20 after mating.
Day 1, 4, 7 and 13 of lactation. On the day of necropsy.

Food consumption

The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded as follows:
F0 animals Weekly, from the day that treatment commenced. Food consumption was not recorded for males and females during the period when paired for mating (Week 3), but recommenced for males in Week 4. For females after mating food consumption was performed to match the body weight recording:
Days 0-6, 7-13 and 14-19 after mating
Days 1-3 , 4-6 and 7-12 of lactation.

Estrous Cycles

Dry and wet smears were taken as follows:

Dry smears
For 15 days before pairing using cotton swabs.

Wet smears
Using pipette lavage during the following phases:
For 14 days before treatment (all females including spares); animals that failed to exhibit 4-5 day cycles were not allocated to study.
After pairing until mating. For four days before scheduled termination (nominally Days 11-14 of lactation).

Hematology, Peripheral Blood

Blood samples were collected after overnight withdrawal of food at the following occasion:
Occasion: At termination
Animals: The five lowest numbered surviving males and the first five lactating females with a surviving litter, in each dose group.

Blood sampling was performed on the morning after collection of urine. Animals were, therefore, deprived of food and water overnight but were allowed access to water for a minimum period of one hour prior to the commencement of blood sampling procedures. Animals were held under light general anesthesia induced by isoflurane. Blood samples (nominally 0.5 mL) were withdrawn from the sublingual vein, collected into tubes containing EDTA anticoagulant and examined for the following characteristics using a Bayer Advia 120 analyzer:

Hematocrit (Hct)*
Hemoglobin concentration (Hb)
Erythrocyte count (RBC)
Absolute reticulocyte count (Retic)
Mean cell hemoglobin (MCH)*
Mean cell hemoglobin concentration (MCHC)*
Mean cell volume (MCV)
Red cell distribution width (RDW)
Total leucocyte count (WBC)
Differential leucocyte count:
Neutrophils (N)
Lymphocytes (L)
Eosinophils (E)
Basophils (B)
Monocytes (M)
Large unstained cells (LUC)
Platelet count (Plt)

Blood Chemistry

Occasion: At termination
Animals: The five lowest numbered surviving males and the first five lactating females with a surviving litter, in each dose group.

Blood sampling was performed on the morning after collection of urine. Animals were, therefore, deprived of food and water overnight but were allowed access to water for a minimum period of one hour prior to the commencement of blood sampling procedures. Animals were held under light general anesthesia induced by isoflurane. Blood samples (nominally 0.7 mL) were withdrawn from the sublingual vein and collected into tubes containing lithium heparin as anticoagulant. After separation, the plasma was examined using a Roche P Modular Analyzer in respect of:

Alkaline phosphatase (ALP)
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Total bilirubin (Bili)
Bile acids (Bi Ac)
Urea
Creatinine (Creat)
Glucose (Gluc)
Total cholesterol (Chol)
Triglycerides (Trig)
Sodium (Na)
Potassium (K)
Chloride (Cl)
Calcium (Ca)
Inorganic phosphorus (Phos)
Total protein (Total Prot)
Albumin (Alb)

Albumin/globulin ratio (A/G Ratio) was calculated from total protein concentration and analyzed albumin concentration.


Urinalysis

Animals were placed in an individual metabolism cage, without food or water. Urine samples were collected overnight at the following occasion:
Occasion: At termination
Animals: The five lowest numbered surviving males and the first five lactating females with a surviving litter, in each dose group.

Using manual methods:
Clarity and Color (App) - by visual assessment
Volume (Vol) - using a measuring cylinder
pH - using a pH meter
Specific gravity (SG) - by direct refractometry using a SG meter

Using Multistix reagent strips interpreted using the Clinitek®500 instrument:
Ketones (Keto)
Bile pigments (Bili)
Urobilinogen (Urob)
Blood pigments (UBld)

Using a Roche P Modular Analyzer:
Protein (T-Prot and Prot)
Glucose (T-Gluc and U-Gluc)
Creatinine (T-Creat and U-Creat)
Sodium (T-Na)
Potassium (T-K)
Chloride (T-Cl)

A microscopic examination of the urine sediment was performed. An aliquot of the urine sample was centrifuged, stained with Kova stain and the resulting deposit spread on a microscope slide. The number of elements seen in nine high or low power fields (HPF or LPF) was recorded in the raw data and entered onto the database and the number seen /HPF or /LPF was derived from these data as described below.
Epithelial cells (Epi)
Leucocytes (WBC)
Erythrocytes (RBC)
Casts
Other abnormal components (A)

The slide was also examined for abnormalities in spermatozoa and crystals.

Thyroid Hormone Analysis

Blood samples were collected as follows:
Occasion: At termination

Animals: All F0 adult males and females.
Day 4 of age F1 offspring, two females per litter (where possible).
No pups were allocated to these procedures if the resultant live litter size would fall below eight pups.
- one for T4 (serum)#
- one for TSH (plasma)
# priority given to serum sample
Day 13 of age F1 offspring, two males and two females per litter (where possible).
- two for T4 (serum): where possible one male and one female#
- two for TSH (plasma): where possible one male and one female)
# priority given to serum sample

Sequence of blood sampling on each occasion

In order to minimize any potential confounding effect of the time of day of blood sampling, the order of blood sampling was controlled to allow satisfactory inter-group comparisons.

Conditions

F0 animals: Following overnight deprivation of food

F1offspring: No overnight deprivation of food

Anesthetic F0 animals: Isoflurane

F1 offspring Day 4 and Day 13 of age: None

Blood sample site

F0 adults: Sublingual vein

F1 offspring Day 4 and Day 13 of age: Decapitation

Anticoagulant Plasma samples: K2EDTA. Microtainers used for collection of samples did not contain separator gel.

Serum samples: None (Greiner Minicollect tubes with clotting activators)

Blood volume

F0 animals: 2 x 0.5 mL

F1 offspring: maximum possible

Processing

Plasma samples: Samples were kept on wet ice prior to centrifugation and commenced within 30 minutes of sampling.

Serum samples: Samples were kept at ambient temperature for a minimum of 30 minutes prior to centrifugation.

Centrifugation conditions: At 2000g for ten minutes at 4°C.

Number of aliquots per sample: All available plasma/serum was transferred to appropriately labelled polypropylene “cryo” tubes using micropipettes.

Final storage conditions: Deep frozen (approximately -60 to -90ºC).

Fate of samples: Dispatched to the Department of Biomarkers, Bioanalysis and Clinical Sciences, Envigo.


Necropsy

All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

Sacrifice and pathology:

Method of sacrifice
All adult animals: Carbon dioxide asphyxiation (no exposure to carbon dioxide took place until after the completion of blood sampling for thyroid hormone assays).
Offspring - selected for thyroid hormone sampling on Day 4 and 13 of age: Decapitation.
Offspring - all other: Intraperitoneal injection of sodium pentobarbitone
Sequence: To allow satisfactory inter-group comparison.

Statistics:

Statistical analyses were performed on the majority of data presented and results of these tests, whether significant or non-significant. For some parameters, including estrous cycles before treatment, pre-coital interval, mating performance, gestation index and stage of estrous cycle at termination the similarity of the data was such that analyses were not considered to be necessary. All statistical analyses were carried out separately for males and females using the individual animal as the basic experimental unit. For litter/fetal findings the litter was taken as the treated unit and the basis for statistical analysis and biological significance was assessed with relevance to the severity of the anomaly and the incidence of the finding within the background control population.
The following data types were analyzed at each timepoint separately:
Grip strength and motor activity
Body weight, using absolute weights and gains over appropriate study periods
Food consumption, over appropriate study periods during gestation and lactation
Hematology, blood chemistry and urinalysis
Estrous cycles during treatment
Gestation length
Ano-genital distance, adjusted for pup body weight
Litter (implantations, litter size, sex ratio - percentage male, post implantation survival index, live birth index and viability index), for before blood sampling study periods
Organ weights, both absolute and adjusted for terminal body weight

A sequence of statistical tests was used for inter alia grip strength, motor activity, body weight, food consumption, implantations, litter size, sex ratio - percentage male, post implantation survival index, ano-genital distance, organ weight and clinical pathology data-
A parametric analysis was performed if Bartlett's test for variance homogeneity (Bartlett 1937) was not significant at the 1% level.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

The clinical condition of the animals, their behavior in the arena, sensory reactivity, grip strength, motor activity, body weight gain and food intake were all unaffected by treatment and no treatment-related abnormalities were identified histopathologically.

Mortality:

no mortality observed

Description (incidence):

No premature deaths occurred during the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Overall group mean body weight gain was considered unaffected by treatment. In females before pairing, body weight gain was high, when compared with the controls at all doses levels (50, 48 and 99% higher than control, respectively) however, during gestation and lactation body weight gain was similar to controls. It was therefore, considered that this was fortuitous and unrelated to treatment.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was no effect of treatment upon food intake.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The hematological examination of peripheral blood performed at termination revealed, when compared with controls, a slight increase in neutrophil, lymphocyte and monocyte counts (with a consequential increase of total leucocyte count) among females receiving 100, 330 or 1000 mg/kg/day, generally attaining statistical significance at all dose levels for lymphocyte, monocyte and total leucocyte counts. A similar effect was seen in males (neutrophils and monocyte counts only). In the absence of a clear dose-response and a high individual value in a single male (No.14) a relationship to treatment is unclear.
All inter-group differences from control, including those attaining statistical significance, were minor, lacked dose-relationship or were confined to one sex and were therefore attributed to normal biological variation. Such differences included the low group mean prothrombin time at all doses in females, but this was a consequence of the high control group mean value that was partly due to a particularly high value for one animal (Animal No. 47; 30.1 seconds).

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

The biochemical examination of plasma performed at termination did not reveal any changes that were clearly related to treatment with Amidoamine (UVCB).
All inter-group differences from control, including those attaining statistical significance, were minor, lacked dose-relationship or were confined to one sex and were therefore attributed to normal biological variation. Such differences included the slightly low plasma urea concentrations in males given 1000 mg/kg/day, but the individual differences from control were slight, and there was no similar effect observed in the females. Plasma glucose concentrations were marginally, but statistically significantly low, in males at all dose levels, but there was no dose-relationship, and the majority of individual values were similar to the controls. There were a few statistically significant differences from control among the plasma electrolytes (sodium and potassium in males and chloride in females) but there was no dose-relationship in any of the affected parameters and the urinalysis investigations did not reveal any effect on the urinary output of electrolytes. Plasma bile acid concentrations were statistically significantly low in males at all dose levels, but there no clear dose-relationship and the individual values were highly variable.

Endocrine findings:

no effects observed

Description (incidence and severity):

Thyroid hormone analysis was performed. No effects of treatment upon circulating levels of thyroxine (T4) were identified in adult males or offspring on Day 13 of age.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

The urinalysis investigations performed at termination did not identify any treatment-related changes. All inter-group differences from control, including those attaining statistical significance, were minor, lacked dose-relationship or were confined to one sex and were therefore attributed to normal biological variation. Such differences included the marginally, but statistically significantly, low pH in males receiving 1000 mg/kg/day, where the difference form control was minor and the individual values were similar to the controls.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There we no signs seen at the routine examination that were considered to be related to treatment with Amidoamine and no premature deaths occurred during the study.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There was no histopathological correlate for the slightly low heart weights in males given 330 or 1000 mg/kg/day or the low liver weights in males given 1000 mg/kg/day, and no similar changes were observed in the females, therefore these changes were considered to be fortuitous and of no consequence.

Gross pathological findings:

no effects observed

Description (incidence and severity):

The macroscopic examination revealed no test item related lesions. The incidence and distribution of all findings were considered to be unrelated to treatment.

Neuropathological findings:

no effects observed

Description (incidence and severity):

There we no signs seen at the routine examination that were considered to be related to treatment with Amidoamine and no premature deaths occurred during the study.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No changes related to treatment with Amidoamine (UVCB) were seen.
There were no test item-related microscopic findings in the testes, following the qualitative examination of the stages of spermatogenesis (no test item-related abnormalities were identified in the integrity of the various cell types present within the different stages of the sperm cycle) or following the evaluation of the uterus or of the follicles and corpora lutea in the ovaries. The incidence and distribution of all findings were considered to be unrelated to treatment.
Pathology procedures for the five lowest numbered surviving males and females with a surviving litter per group at scheduled termination.

- The following organs or parts of organs of all adult animals were fixed in 10% Neutral Buffered Formalin; testes and epididymides were fixed in modified Davidson's fluid/Davidson's fluid:
Abnormalities, cowper's gland, epididymis (2, caput, corpus and cauda), glans penis, levator Ani plus Bulbo Cavernosus (LABC) muscle complex, skin with mammary gland, ovaries (2), prostate, seminal vesicles with coagulating glands, testes (2), thyroids, uterus (incl. cervix and oviducts), vagina.

For the assessment of the testes, a detailed qualitative examination was made, 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, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells in the lumen. Any cell- or stage-specificity of testicular findings was noted.

-In addition, the following organs or parts of organs of the selected 20 adult males and 20 adult females (see section above) were fixed in 10% formalin:
Abnormalities
Adrenal gland (2)
Bone marrow (os femoris)
Brain (cerebrum, cerebellum, pons)
Eyes
Heart (including auricular and ventricular regions)
Intestine, small (duodenum, jejunum, ileum, incl. Peyer's patches)
Intestine, large (colon, rectum)
Kidney and ureter (2)
Liver (section from 2 lobes)
Lungs (section from two major lobes incl. bronchi)
Lymph node (1, left acillary), Lymph node (1, mesenteric)
Nerve (sciatic)
Peyer’s Patch
Pituitary
Skeletal muscle
Sklin with mammary gland
Spinal cord (transverse and longitudinal sections at the cervical level)
Spleen
Sternum (with marrow)
Stomach
Trachea
Thyroid (incl. parathyroids)
Thymus
Urinary bladder

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

There we no signs seen at the routine examination that were considered to be related to treatment with Amidoamine and no premature deaths occurred during the study.

Other effects:

no effects observed

Description (incidence and severity):

There we no signs seen at the routine examination that were considered to be related to treatment with Amidoamine and no premature deaths occurred during the study.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat. (total fraction)

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: neoplastic

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

urinalysis

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

It was concluded that the oral administration of Amidoamine (UVCB) to parental Han Wistar rats at dose levels of 100, 330 or 1000 mg/kg/day for two weeks before pairing, during pairing and then up to termination of the males after five weeks of treatment and females on Day 14 of lactation was well tolerated, with no adverse effect of treatment identified. Reproductive performance, fertility, litter size and offspring survival and growth were unaffected by parental treatment and, in the context of this study, Amidoamine (UVCB) showed no evidence of being an endocrine disruptor. The no-observed-adverse-effect level (NOAEL) of Amidoamine (UVCB) for systemic toxicity and also for reproductive/developmental toxicity was considered to be 1000 mg/kg/day.

Executive summary:

The aim of the study was to obtain information on possible effects of the test item on general toxicity, reproduction and/or development according to OECD guideline 422.

The oral administration of Amidoamine (UVCB) to parental Han Wistar rats at dose levels of 100, 330 or 1000 mg/kg/day for two weeks before pairing, during pairing and then up to termination of the males after five weeks of treatment and females on Day 14 of lactation was well tolerated, with no premature deaths. The clinical condition of the animals, their behavior in the arena, sensory reactivity, grip strength, motor activity, body weight gain and food intake were all unaffected by treatment and no treatment-related abnormalities were identified histopathologically.

In this study there were some inter-group differences from controls that attained statistical significance, particularly at the hematological investigation, where there were some minor differences from controls in respect of the cellular components of the peripheral blood. There were minor increases of neutrophil, lymphocyte and monocyte counts in females receiving 100, 330 or 1000 mg/kg/day with a similar effect seen upon the neutrophil and monocyte counts of males at these dose levels. In view of the minimal extent of the differences from controls, the absence of any histopathological finding that would account for the trends (such as inflammatory change in any tissue) and the absence a clear dose-response, the variations of leucocyte numbers were considered to represent normal variation and, consequently, were of no biological significance and unrelated to treatment.

There was no histopathological correlate for the slightly low heart weights in males given 330 or 1000 mg/kg/day or the low liver weights in males given 1000 mg/kg/day, and no similar changes were observed in the females, therefore these changes were considered to be fortuitous and of no consequence.

This study included a screen for reproductive/developmental effects and the results obtained were unremarkable. Estrous cycles, pre-coital interval, mating performance, fertility, litter size and gestation length were unaffected by treatment. The clinical condition of the offspring, their survival, growth, sex ratio, ano-genital distance on Day 1 of age and male nipple counts on Day 13 of age showed no adverse effects of parental treatment. There were also no signs in the decedent offspring, or offspring at termination on Day 13 of age that were considered to be related to parental treatment.

The study design also included an assessment of endocrine disruptor relevant endpoints. This objective was met by including the measurement of the hormone thyroxine (T4) in adult males and in offspring at Day 13 of age, by evaluating changes in adult organ weight and gross organ pathology of endocrine-sensitive organs and, because some developmental stages (e.g. gestational and neo-natal) are particularly sensitive to endocrine effects, an external examination of all offspring, measurement of the ano-genital distance of offspring on Day 1 of age and nipple counts for male offspring on Day 13 of age. No adverse effect of treatment was evident on the circulating levels of thyroxine. No significant changes were identified at the microscopic examination of thyroid, adrenal and pituitary glands and the reproductive organs. All offspring were macroscopically normal; in particular no effects were seen on the external genitalia. Ano-genital distances and male nipple counts were not adversely affected by treatment. It was therefore concluded that, in the context of this study, Amidoamine (UVCB) showed no evidence of being an endocrine disruptor.