Fatty acids, C16-18, reaction products with diethylenetriamine

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

NOAEL > 1000 mg/kg bw/day

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

other: read across from analogue substance

Adequacy of study:

key study

Study period:

2017

Reliability:

2 (reliable with restrictions)

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.

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

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

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.

Route of administration:

oral: gavage

Vehicle:

other: 1% methylcellulose in 0.1% Tween 80

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.

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.

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.

Positive control:

no positive control included

Parental animals: Observations and examinations:

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.

Oestrous cyclicity (parental animals):

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.

Sperm parameters (parental animals):

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.

Litter observations:

No signs were recorded that were considered to be related to parental treatment with Amidoamine (UVCB). 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).
There was no effect of parental treatment upon offspring group mean body weight gain.
The ano-genital distances of offspring were unaffected by parental treatment.

Postmortem examinations (parental animals):

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

Postmortem examinations (offspring):

The macroscopic examination of offspring revealed no test item related lesions.

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.

Reproductive indices:

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.

Offspring viability indices:

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.

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):

no effects observed

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:

no effects observed

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:

no effects observed

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:

no effects observed

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:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

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.

Reproductive function: oestrous cycle:

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.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

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.

Reproductive performance:

no effects observed

Description (incidence and severity):

Estrous cyclicity, pre-coital interval, gestation length, mating performance and fertility were unaffected by treatment.
Each uterine horn: number of implantation sites was counted and confirmed if none were visible at visual inspection.

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.

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:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

haematology

clinical biochemistry

urinalysis

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

histopathology: neoplastic

reproductive function (oestrous cycle)

reproductive function (sperm measures)

reproductive performance

Key result

Critical effects observed:

no

Immunological findings:

not examined

Other effects:

no effects observed

Clinical signs:

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.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality 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).

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no effect of parental treatment upon offspring group mean body weight gain.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

effects observed, non-treatment-related

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.

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

The macroscopic examination of offspring revealed no test item related lesions.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

The ano-genital distances of offspring were unaffected by parental treatment.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

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.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat. (total fraction)

Sex:

male/female

Basis for effect level:

viability

clinical signs

mortality

body weight and weight gain

clinical biochemistry

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

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.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

NOAEL > 1000 mg/kg bw/day

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

other: read across from analogue substance

Adequacy of study:

key study

Study period:

2017

Reliability:

2 (reliable with restrictions)

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.

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 422

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

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

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.

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.

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.

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.

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.

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" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): 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.

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

Key result

Abnormalities:

no effects observed

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)
FetalPupBodyWeightChanges: no effects observed
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.

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

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

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.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

Reproductive toxicity includes adverse effects on sexual function and fertility in sexually adult males and females animals, as well as developmental toxicity in the offspring. However, developmental toxicity essentially means all the adverse effects induced during pregnancy that can be manifested at any point of the life span of the animal, which might in turn bring to structural abnormality, altered growth and/or organs development, functional deficiency, even death.

Table 3.7.1(a) of Annex I of EC Regulation 1272/2008 states that to classify compounds "for category 2 suspected human reproductive toxicant, reproductive effects shall have been observed in the absence of other toxic effects, or if occurring together with other toxic effects the adverse effect on reproduction is considered not to be a secondary non-specific consequence of the other toxic effects". To this extent the screening study performed on analogue substance 1 does not provide any indication of direct adverse effect on reproduction. In fact up to the dose of 1000 mg/kg bw/day no effects were observed in both genders on reproduction, nor parental toxicity was detected. Moreover, no developmental toxic effects in the offspring were observed at all doses.

In conclusion, since no adverse effects on reproduction were observed, classification for reproductive/developmental toxicity is not warranted under Regulation 1272/2008.

Based on the read across considerations same non classification apply to Stearamide DETA.