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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
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:
other: OECD Guideline 422
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Triglycerides, C16-18 (even numbered), reaction products with diethylenetriamine
EC Number:
810-543-2
Cas Number:
1618093-67-6
Molecular formula:
not applicable
IUPAC Name:
Triglycerides, C16-18 (even numbered), reaction products with diethylenetriamine
Test material form:
solid: flakes
Details on test material:
Amidoamine 2 (UVCB based on hydrogenated tallow not hydrogenated palm oil):
- Name of test material (as cited in study report): FCM (DETA)M C16-18 TLW hydrogenated (Amidoamine 2 (UVCB based on hydrogenated tallow not hydrogenated palm oil))
- Substance type: Amidoamine
- Chemical name (old): Amides, from diethylenetriamine and hydrogenated tallow
- Chemical name (new): Triglycerides, C16-18 (even-numbered), reaction products with diethylenetriamine
- CAS 68920-82-1
- Physical state: pale yellowish solid at 20 °C
- Batch No.: PU22340011
- Expiry date of batch: 21 August 2014
- 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.

Source substance:

Amidoamine (UVCB)
Pulcra ID: DE07_2014_012_BEL66 (amidoamine without emulsifier)
Former chemical name: Amides, from diethylenetriamine and hydrogenated palm oil
Former CAS No.: 1618093-67-6
New chemical name: Triglycerides, C16-18 (even-numbered), reaction products with diethylenetriamine
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

Target substance:

Amidoamine 2 (UVCB, low nitrogen):
Former chemical name: Amides, from diethylenetriamine and hydrogenated palm oil
CAS No.: 1618093-67-6
New chemical name: Glycerides, C16-18 (even numbered) and their amidation products with diethylenetriamine
Physical state: pale yellowish solid at 20 °C
Batch No.: PU50070067
Purity: 100 % (UVCB)
Storage condition of test material: Room temperature, protected from light
Stability: stable under test conditions

Test animals

Species:
rat
Strain:
Wistar
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.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 1% methylcellulose in 0.1% Tween 80
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.
Details on mating procedure:
Pairing commenced: After a minimum of two weeks of treatment.
Male/female ratio: 1:1 from within the same treatment groups.
Duration of pairing: Up to two weeks.
Daily checks for evidence of mating: Ejected copulation plugs in cage tray and sperm in the vaginal smear.
Day 0 of gestation: When positive evidence of mating was detected.
Male/female separation: Day when mating evidence was detected.
Pre-coital interval: Calculated for each female as the time between first pairing and evidence of mating.
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 / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
330 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
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.

Examinations

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: maternal animals

General toxicity (maternal animals)

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.
Dermal irritation (if dermal study):
not examined
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.
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 were 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):
There were 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
Neuropathological findings:
no effects observed
Description (incidence and severity):
There were 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 were 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 were 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.

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
not examined
Total litter losses by resorption:
not examined
Early or late resorptions:
not examined
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
All females allocated to study showed normal 4/5 day estrous cycles during the acclimatization period. Estrous cyclicity, pre-coital interval, gestation length and index, mating performance and fertility were considered unaffected by treatment with Amidoamine (UVCB). All females showed diestrus at termination.

Migrated Data from removed field(s)
Field "Effects on pregnancy duration : no effects observed
Field "Description (incidence and severity): All females allocated to study showed normal 4/5 day estrous cycles during the acclimatization period.

Estrous cyclicity, pre-coital interval, gestation length and index, mating performance and fertility were considered unaffected by treatment with Amidoamine (UVCB). All females showed diestrus at termination.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
All females allocated to study showed normal 4/5 day estrous cycles during the acclimatization period.
Estrous cyclicity, pre-coital interval, gestation length and index, mating performance and fertility were considered unaffected by treatment with Amidoamine (UVCB).
All females showed diestrus at termination
Other effects:
no effects observed
Details on maternal toxic effects:
All females allocated to study showed normal 4/5 day estrous cycles during the acclimatization period.
Estrous cyclicity, pre-coital interval, gestation length and index, mating performance and fertility were considered unaffected by treatment with Amidoamine (UVCB). All females showed diestrus at termination.



Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat. (total fraction)
Basis for effect level:
behaviour (functional findings)
body weight and weight gain
clinical biochemistry
clinical signs
dead fetuses
effects on pregnancy duration
gross pathology
haematology
histopathology: neoplastic
histopathology: non-neoplastic
maternal abnormalities
mortality
ophthalmological examination
organ weights and organ / body weight ratios
urinalysis

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
not examined
Description (incidence and severity):
There was no effect of parental treatment upon offspring group mean body weight gain.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes": no effects observed
Field "Description (incidence and severity)": There was no effect of parental treatment upon offspring group mean body weight gain.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
All females were pregnant and had a live litter on Day 13 of lactation. Litter size, offspring survival to Day 13 of age and sex ratio were unaffected by parental treatment with Amidoamine (UVCB).
Changes in sex ratio:
no effects observed
Description (incidence and severity):
All females were pregnant and had a live litter on Day 13 of lactation. Litter size, offspring survival to Day 13 of age and sex ratio were unaffected by parental treatment with Amidoamine (UVCB).
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
The clinical condition, litter size, sex ratio, survival indices and body weight gain of offspring were unaffected by parental treatment.
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
The clinical condition, litter size, sex ratio, survival indices and body weight gain of offspring were unaffected by parental treatment.
External malformations:
no effects observed
Description (incidence and severity):
The macroscopic examination of offspring revealed no test item related lesions.
The ano-genital distances of offspring were unaffected by parental treatment.
Skeletal malformations:
no effects observed
Description (incidence and severity):
The macroscopic examination of offspring revealed no test item related lesions.
Visceral malformations:
no effects observed
Description (incidence and severity):
The macroscopic examination of offspring revealed no test item related lesions.
Other effects:
no effects observed
Description (incidence and severity):
There was no effect of treatment on the circulating levels of thyroxine (T4) in adult males or in offspring on Day 13 of age.
The group mean T4 concentration for female offspring on Day 13 of age derived from F0 parents treated at 1000 mg/kg/day was marginally high, when compared with controls, but all individual values were below the highest control value and in both the control group and the high dose group, three individual offspring had T4 levels greater than 60000 pg/mL.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat. (total fraction)
Sex:
male/female
Basis for effect level:
changes in litter size and weights
changes in postnatal survival
external malformations
skeletal malformations
visceral malformations

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental 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.