Fatty acids, C18 unsat, reaction products with diethylenetriamine

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

• NOAEL (reproductive toxicity) = at least 100 mg/kg bw/day - OECD 422 - NOTOX 491556 (2010)


• NOAEL (parental toxicity) = 30 mg/kg bw/day - OECD 422 - NOTOX 491556 (2010)


• Study Ongoing, Reporting expected Q4 2022: Oral OECD 443 (Cohorts 1A 1B + 3), Charles River Laboratories 20257161 (2021)

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 September 2009 - 15 October 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study has been performed according to OECD 422 guidelines and GLP principles.

Reason / purpose for cross-reference:

reference to same study

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

Species:

rat

Strain:

other: Crl:WI(Han).

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: Approximately 12 weeks.
At start treatment, animals were approximately 12 weeks old instead of approximately 10 weeks. A slight deviation in age does not affect the study integrity. Mating started shortly after the animals had attained full sexual maturity according to the OECD 422 guideline.
- Fasting period before study: no
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon cages (MIV type, height 18 cm). This also accounts for the Recovery males for the complete treatment period.
Mating: Females were caged together with Main males on a one-to-one-basis in Macrolon cages (MIII type, height 18 cm).
Post-mating: Main males were housed in their home cage (Macrolon cages, MIV type, height 18 cm) with a maximum of 5 animals/sex/cage. Females were individually housed in Macrolon cages (MIII type, height 18 cm).
Lactation: Pups were kept with the dam until termination in Macrolon cages (MIII type, height 18 cm).
General: Sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) was supplied. Certificates of analysis were examined and then retained in the NOTOX archives. During activity monitoring, animals were housed individually in Macrolon cages (MIII type; height 15 cm) with sterilised sawdust as bedding material. No cage-enrichment was provided during overnight activity monitoring.

- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: At least 5 days prior to start of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.7 – 22.0°C
- Humidity (%): 28 - 79%
Temporary deviations from the minimum level of relative humidity occurred in the animal room. Laboratory historical data do not indicate an effect of the deviations.
- Air changes (per hr): approximately 15 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours artificial light and 12 hours darkness per day. Temporary fluctuations from the light/dark cycle (with a maximum of 1 hour and 6 minutes) occurred due to performance of functional observations in the room.

On Day 2 or 4 of lactation, the maximum allowed deviation from the dark period of 1 hour was exceeded with a maximum of approximately 6 minutes for the 5 selected females with live pups per group. These temporary deviations from the light/dark cycle were considered not to have affected the study outcome.

IN-LIFE DATES: From: 01 September 2009 to 15 October 2009

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenised to a visually acceptable level. Adjustment was made for density of the test substance and specific gravity of the vehicle (1.036) .

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at NOTOX.
- Concentration in vehicle: 2, 6 and 20 mg/mL

- Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: A maximum of 13 instead of 14 days was allowed for mating. All females had mated within this period
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum.
- After successful mating each pregnant female was caged individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted on a single occasion during treatment phase (02 October 2009) according to a validated method (NOTOX project 491557). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature was also determined (highest and lowest concentration).

The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration for solutions. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.

RESULTS:
The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%).
The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%). Formulations at the entire range were stable when stored at room temperature for at least 6 hours.

Duration of treatment / exposure:

Males were exposed for 28 days, i.e. 2 weeks prior to mating, during mating, and up to termination or up to the day prior to start of the recovery period for Recovery males. Females were exposed for at least 42 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation.

Frequency of treatment:

Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.

Details on study schedule:

- Age at mating of the mated animals in the study: 14 weeks

Remarks:

Doses / Concentrations:
0, 10, 30 and 100 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

10 and an extra 5 males for Group 1 and 4. The study included a recovery phase for males only. These animals were not mated and, consequently, were not used for the assessment of reproduction/developmental toxicity.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were selected based on the results of the dose range finding/MTD study (NOTOX Project 491555). See endpoint study record 7.5.1: Repeated dose toxicity: oral. notox 491555.
- Rationale for animal assignment (if not random): 5 animals/sex/group were randomly selected at allocation for functional observations, clinical pathology, macroscopic examination (full list), organ weights (full list) and histopathology.
Males: the first 5 males per group for main and recovery
Females: the first 5 females (with live offspring only)

Positive control:

no

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily (early morning/late afternoon)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from start of treatment onwards, between approximately 1 and 2 hours after dosing and once daily during the recovery period, detailed clinical observations were made in all animals. Once prior to start of treatment and at weekly intervals this was also performed outside the home cage in a standard arena. Arena observations were not performed when the animals were mating, or housed individually.

On Day 27 of the repro period, clinical signs of females were inadvertently recorded under the online data collection project number for males (NOTOX project 491961). For males a second observation was conducted on this day, together with clinical observations of the females. Females: clinical observations were conducted; no clinical signs were noted for the females. Males: additional information.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.

FOOD CONSUMPTION: Yes
Weekly, for males and females. Food consumption was not recorded during the mating period, except for Recovery males. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.

FOOD EFFICIENCY: Yes
(Average food consumption (per animal per day)/average body weight per cage) X 1000

WATER CONSUMPTION: No
Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination, between 7.00 and 10.30 a.m.
- Anaesthetic used for blood collection: Yes, iso-flurane
- Animals fasted: Yes, but water was available
- How many animals: 5 animals/sex/group (females with live offspring only).
- Parameters examined were: White blood cells, Differential leucocyte count (neutrophils, lymphocytes, monocytes,eosinophils, basophils), Red blood cells, Reticulocytes, Red blood cell distribution width, Haemoglobin, Haematocrit, Mean corpuscular volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, Platelets, Prothrombin time, Activated Partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination, between 7.00 and 10.30 a.m.
- Anaesthetic used for blood collection: Yes, iso-flurane
- Animals fasted: Yes, but water was available
- How many animals: 5 animals/sex/group (females with live offspring only).
- Parameters examined were: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Total Protein, Albumin, Total Bilirubin, Urea, Creatinine, Glucose, Cholesterol, Sodium, Potassium, Chloride, Calcium, Inorganic Phosphate, Bile acids.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: The selected males were tested during Week 4 of treatment and the selected females were tested during lactation (all before blood sampling).
- Dose groups that were examined: all
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex, grip strength and motor activity.

OTHER:
General reproduction data:
Male number paired with, mating date, confirmation of pregnancy, and delivery day was recorded. Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of these females was examined to detect signs of abortion or premature birth. Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.

Oestrous cyclicity (parental animals):

not determined

Sperm parameters (parental animals):

testis weight, epididymis weight.
For 5 males of the control and high dose group, slides of the testes were prepared to examine staging of spermatogenesis.

Litter observations:

PARAMETERS EXAMINED
Each litter was examined to determine the following, if practically possible:

Mortality / Viability:
The numbers of live and dead pups at the First Litter Check (= check at Day 1 of lactation) and daily thereafter were determined. If possible, defects or cause of death were evaluated.

Clinical signs:
At least once daily, detailed clinical observations were made in all animals.

Body weights:
Live pups were weighed on Days 1 and 4 of lactation.

Sex:
Sex was determined for all pups on Days 1 and 4 of lactation (by assessment of the ano-genital distance).

GROSS EXAMINATION OF DEAD PUPS: Yes
All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated.

Postmortem examinations (parental animals):

SACRIFICE
All animals were fasted overnight (with a maximum of approximately 21 hours) prior to planned necropsy, but water was provided. Animals surviving to scheduled necropsy were anaesthetised using iso-flurane (Abbott Laboratories Ltd., Hoofddorp, The Netherlands) and subsequently exsanguinated.

Necropsy was conducted on the following days:
- Females which delivered: Lactation Day 5.
- Females which failed to deliver: Post-coitum Day 26-27 (females with evidence of mating)
- Males (Main): Following completion of the mating period (a minimum of 28 days of dose administration).
- Males (Recovery): After a recovery phase of 14 days.

Several animals were necropsied later than after a maximum of 20 hours fasting, i.e. with a maximum of 1 hour and 59 minutes. The fasting period was only slightly longer and was considered not to have adversely affected the clinical laboratory, macroscopic or microscopic findings.

GROSS NECROPSY
All parental animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded. The number of former implantation sites and corpora lutea were recorded.

Samples of the following tissues and organs were collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands):

Selected 5 animals/sex/group and all Recovery males: Identification marks: not processed, Ovaries. Adrenal glands, Pancreas, Aorta, Peyer's patches (jejunum, ileum) if detectable, Brain (cerebellum, mid-brain, cortex), Pituitary gland, Caecum, Preputial gland, Cervix, Prostate gland, Clitoral gland, Rectum, Colon, (Salivary glands - mandibular, sublingual), Duodenum, Sciatic nerve, Epididymides*, Seminal vesicles including coagulating gland, Eyes with optic nerve (if detectable) and Harderian gland*, Skeletal muscle, (Skin), (Female mammary gland area), Spinal cord (cervical, midthoracic, lumbar), Femur including joint, Spleen, Heart, Sternum with bone marrow, Ileum, Stomach, Jejunum, Testes*, Kidneys, Thymus, (Lacrimal gland, exorbital), Thyroid including parathyroid (if detectable), (Larynx), (Tongue), Liver, Trachea, Lung, infused with formalin, Urinary bladder, Lymph nodes (mandibular, mesenteric), Uterus, (Nasopharynx), Vagina, Oesophagus, All gross lesions.

All remaining animals and females which failed to deliver$:

Identification marks: not processed, Prostate gland, Cervix, Seminal vesicles including coagulating glands, Clitoral gland, Testes*, Epididymides*, Uterus, Ovaries, Vagina, Preputial gland, All gross lesions.

* Fixed in modified Davidson's solution (prepared at NOTOX using Formaldehyde 37-40%, Ethanol, Acetic acid (glacial)(all Merck, Darmstadt, Germany) and Milli-Ro water (Millipore Corporation, Bedford, USA)) and transferred to formalin after fixation for at least 24 hours.
$ In case no macroscopically visible implantation sites were present, nongravid uteri were stained using the Salewski technique (Salewski, 1964) in order to detect any former implantation sites (Salewski staining prepared at NOTOX using Ammoniumsulfide-solution 20% (Merck, Darmstadt, Germany) and Milli-Ro water (Millipore Corporation, Bedford, USA)).

Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.

ORGAN WEIGHTS: Yes
The following organ weights and terminal body weight were recorded from the following animals on the scheduled day of necropsy:

Selected 5 animals/sex/group and all Recovery males: Adrenal glands, Spleen, Brain, Testes, Epididymides, Thymus, Heart, Uterus (including cervix),
Kidneys, Prostate*, Liver, Seminal vesicles including coagulating glands*, Ovaries, Thyroid including parathyroid*.
* weighed when fixed for at least 24 hours.

All remaining males: Epididymides, Testes.

HISTOTECHNOLOGY:
All organ and tissue samples, as defined under Histopathology (following), were processed, embedded and cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin (Klinipath, Duiven, The Netherlands).

Of the selected 5 males/group of the control and high dose Main group additional slides of the testes were prepared to examine staging of spermatogenesis. The testes were processed, sectioned at 3-4 micrometers, and stained with PAS/haematoxylin (Klinipath, Duiven, The Netherlands).

HISTOPATHOLOGY: Yes
The following slides were examined by a pathologist:
-The preserved organs and tissues of the selected 5 Main animals/sex of Groups 1 and 4.
-Mesenteric lymph nodes and ileum of selected Group 2 and 3 males and females and Recovery group 1 and 4 males, liver of selected Group 2 and 3 males and Recovery group 1 and 4 males and thymus of selected Group 2 and 3 females (based on (potentially) treatment-related histopathological changes in Group 4).
-The additional slides of the testes of the selected 5 Main males of Groups 1 and 4 to examine staging of spermatogenesis.
-The reproductive organs (cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles, testis, uterus, and vagina) of all Main animals that failed to conceive:
Group 1: One male and one female
Group 2: One male and one female
-All gross lesions of all animals (all dose groups).

Inadvertently, a few tissues were not available for histopathology. Reasons included that these tissues were not discernable at necropsy or trimming, or were erroneously not collected at necropsy. Tissues were listed in raw data and pathology report. Sufficient data was available for evaluation.

All abnormalities were described and included in the report. An attempt was made to correlate gross observations with microscopic findings.

Postmortem examinations (offspring):

SACRIFICE
Pups were killed by decapitation on Day 5 of lactation.

GROSS NECROPSY
All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk.

HISTOPATHOLOGY / ORGAN WEIGTHS
no

Statistics:

The following statistical methods were used to analyse the data:
-If the variables could be assumed to follow a normal distribution, the Dunnett-test (Dunnett, 1955) (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
-The Steel-test (Miller, 1981) (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
-The Fisher Exact-test (Fisher, 1950) was applied to frequency data.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.

Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations might have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Reproductive indices:

For each group, the following calculations were performed:

Percentage mated: Number of females mated/Number of females paired x 100

Fertility index: Number of pregnant females/Number of females paired x 100

Conception index: Number of pregnant females/Number of females mated x 100

Gestation index: Number of females bearing live pups/Number of pregnant females x 100

Duration of gestation: Number of days between confirmation of mating and the beginning of parturition.

Offspring viability indices:

Percentage live males at First Litter Check:
Number of live male pups at First Litter Check/Number of live pups at First Litter Check x 100

Percentage live females at First Litter Check:
Number of live female pups at First Litter Check/Number of live pups at First Litter Check x 100

Percentage of postnatal loss Days 0-4 of lactation:
Number of dead pups on Day 4 of lactation/Number of live pups at First Litter Check x 100

Viability index (%):
Number of live pups on Day 4 of lactation/Number of pups born alive x 100

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No mortality occurred during the study period.

There were no clinical signs of toxicity noted during the observation period.

One male and two females at 100 mg/kg/day showed lethargy, flat posture and/or piloerection on one or several days during treatment. Given the low incidence of these findings among these animals and absence of these findings among other animals of the same dose group, this was considered no be of no toxicological relevance.

Other incidental findings included salivation, rales, alopecia, a wound and scabs. These were considered to be within the normal range of biological variation for rats of this age and strain, and considered signs of no toxicological relevance.

No clinical signs were noted among control males and males at 10 and 30 mg/kg/day.

BODY WEIGHT AND WEIGHT GAIN (PARENTAL ANIMALS)
No toxicologically significant changes in body weight (gain) occurred.

Males at 100 mg/kg/day showed a slight, but statistically significant, lower mean body weight and body weight gain during the treatment period, which recovered to control levels during the recovery phase. Some of these males showed (notable) weight loss during the premating period, which (largely) recovered during the mating/repro period. Overall, these changes were slight and reversible in nature. The statistically significant lower body weight gain on Day 4 of Lactation occurred in the absence of a dose- and time related trend, and was very slight in nature. Hence, these variations in body weight (gain) were not considered to be of toxicological relevance.

Body weight and body weight gain of males at 10 and 30 mg/kg/day was similar to control levels throughout the observation period.

FOOD CONSUMPTION (PARENTAL ANIMALS)
No toxicologically significant changes in food consumption before or after allowance for body weight occurred.

Food consumption before or after allowance for body weight of males at 100 mg/kg/day appeared slightly lower than controls during the premating and mating/repro phase. However during the recovery phase food intake levels were similar to controls. Therefore, these changes were not considered to be of toxicological relevance.

Males at 10 and 30 mg/kg/day, and females at 10, 30 and 100 mg/kg/day showed normal food consumption levels, before or after allowance for body weight. The statistically significant lower food consumption before or after allowance for body weight of females at 100 mg/kg/day over Days 4-7 of the post coitum phase was of a very slight nature and within the range considered normal for rats of this age and strain. Food intake of these females remained similar to control levels during the remainder of the post coitum and lactation phase.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No toxicologically significant effects on reproductive parameters, fertility index and conception rate were noted.

Precoital time and number of corpora lutea and implantation sites were unaffected by treatment.

ORGAN WEIGHTS
Males at 100 mg/kg/day showed a statistically significant lower prostate and seminal vesicle weight, and lower prostate to body weight ratio at the end of the treatment period. These changes were absent at the end of the recovery period for males.

Organ weights and organ to body weight ratios of males at 10 and 30 mg/kg/day and of females at 10, 30 and 100 mg/kg/day were similar to those of control animals.

GROSS PATHOLOGY
Necropsy did not reveal any toxicologically relevant alterations.

Incidental findings included yellowish-soft nodules on the epididymides, a hard nodule on the liver, reduced size of the preputial glands, prostate or thymus, red discolouration of the mesenteric lymph nodes, diaphragmatic hernia of the left median lobe of the liver, red foci on the lungs or kidneys, a cyst on the kidneys, watery-clear cysts on the ovaries, a red-brown focus on the clitoral glands, yellowish discolouration of the papillary process of the liver, a black focus on the thymus and alopecia. The incidence of these findings was within the background range of findings that are encountered among rats of this age and strain, and did not show a dose-related incidence trend. These necropsy findings were therefore considered to be of no toxicological significance.

No macroscopic abnormalities were observed among males at 30 mg/kg/day.

HISTOPATHOLOGY:
The following microscopic findings were considered to be related to treatment:
- Ileum: foamy macrophage foci in 5/5 males (minimal to slight) and in 5/5 females (minimal to slight) at 100 mg/kg/day.
- Mesenteric lymph node: slightly increased incidence and degree of macrophage foci in males and females at 100 mg/kg/day.
- Thymus: increased incidence of lymphoid atrophy in females at 100 mg/kg/day.
At the end of the 14-day recovery period for males, foamy macrophage foci in the ileum had completely resolved, whilst macrophage foci in the mesenteric lymph node persisted at slightly increased incidence and severity, with the mean severity grade being higher than encountered at the end of treatment.

All other microscopic findings recorded were considered to be within the normal range of background pathology encountered in Wistar rats of this age and strain.

No abnormalities were seen in the reproductive organs of non-fertile animals which could account for infertility.

The assessment of the integrity of the spermatogenetic cycle did not provide any evidence of impaired spermatogenesis.

REPRODUCTIVE DATA
No toxicologically significant effects on reproductive parameters, fertility index and conception rate were noted.

Precoital time and number of corpora lutea and implantation sites were unaffected by treatment.

No deficiencies in maternal care were observed. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No signs of difficult or prolonged parturition were noted among the pregnant females.

No toxicologically significant effects on number of females with live pups, gestation index, duration of gestation were noted.

Dose descriptor:

NOAEL

Effect level:

>= 30 - < 100 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on the increased incidence/severity of macrophage foci in the mesenteric lymph node at both the end of treatment and recovery period in males.

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

DEVELOPMENTAL DATA
No toxicologically significant effects on early postnatal pup development (body weight, clinical signs, viability index and external macroscopy) were noted.

The incidence of dead/missing pups among the dose groups was within the range considered normal for this type of study. Incidental macroscopic findings among these pups included absence of milk in the stomach and autolysis. No relationship with treatment was established for these deaths and they were considered to be of no toxicological significance.

Incidental clinical symptoms and macroscopic findings among surviving pups consisted of a small size and absence of milk in the stomach. No relationship with treatment was established for these observations and they were considered to be within the normal biological variation for rats of this age and strain.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 100 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No reproductive/developmental toxicity was observed at any dose level.

Reproductive effects observed:

not specified

Conclusions:

No reproductive/developmental toxicity was observed at any dose level. A reproductive/developmental NOAEL of 100 mg/kg/day was derived.

Executive summary:

Tall oil diethylenetriamine imidazoline was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 10, 30 and 100 mg/kg/day. The males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 28 days). There was a 2 week recovery period for 5 animals of Group 1 and 4. The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation.

 

Formulation analysis showed that the formulations were prepared accurately and homogeneously and were stable for at least 6 hours at room temperature.

 

Parental results:

 

The changes in clinical biochemistry parameters in animals at 100 mg/kg/day at the end of the treatment period were generally slight in nature (i.e. mostly within the normal range) and were fully reversible after a 14-day recovery period in males. Moreover, no morphological changes were observed that would support these changes which included higher alanine and aspartate aminotransferase activity and creatinine level in males, and slightly lower total protein and urea level males and females respectively. Therefore, these changes were not considered to be of toxicological relevance.

 

The lower prostate and seminal vesicle weight, and lower prostate to body weight ratio at the end of the treatment period in males at 100 mg/kg/day were absent at the end of the recovery period in males, and were not supported by any histopathological lesions or reproductive toxicity. No toxicological relevance was ascribed to these findings.

 

Histopathology revealed foamy macrophage foci in the ileum of all selected males and females at 100 mg/kg/day, a slightly increased incidence and degree of macrophage foci in the mesenteric lymph node of both sexes at 100 mg/kg/day, and increased incidence of lymphoid atrophy in the thymus of females at 100 mg/kg/day. At the end of the 14-day recovery period for males, foamy macrophage foci in the ileum had completely resolved, whilst macrophage foci in the mesenteric lymph node persisted at higher severity than observed at the end of treatment. Given the persistence of this finding it was considered to be of an adverse nature.

 

No toxicologically significant changes were noted in any of the remaining parental parameters investigated in this study (i.e. clinical appearance, functional observations, body weight, food consumption, haematology and macroscopic examination).

 

Reproductive/Developmental results:

 

No reproductive/developmental toxicity was observed at any dose level.

 

Based on the increased incidence/severity of macrophage foci in the mesenteric lymph node at both the end of treatment and recovery period in males, a parental No Observed Adverse Effect Level (NOAEL) of 30 mg/kg/day was derived.

 

A reproductive/developmental NOAEL of 100 mg/kg/day was derived.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The endpoint is currently concluded based on a single study assigned a Klimisch rating of 1: reliable without restriction. This has been undertaken according to GLP and an accepted OECD TG for this endpoint (TG422). A GLP OECD 443 (EOGRTS, Cohorts 1A 1B and 3) is ongoing following ECHA Decision on Compliance Check CCH-D-2114488748-25-01/F. Reporting is expected Q4 2022.
Study results are consistent results within the whole group of Amidoamine/imidazolines (AAI), indicating a no concerns for reproduction toxicity.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Summary of studies

 

OECD 422 (NOTOX, 491556, 2010)

Fatty acids, C18 unsaturated, reaction products with diethylenetriamine (AAI-DETA, the test item) was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 10, 30 and 100 mg/kg/day. The males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 28 days). There was a 2-week recovery period for 5 animals of Group 1 and 4. The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation.

 

Parental results:

Lower prostate and seminal vesicle weights, and lower prostate to body weight ratio reported at the end of the treatment period in males at 100 mg/kg bw/day were absent at the end of the recovery period and were not supported by any histopathological lesions or reproductive toxicity. No toxicological relevance was ascribed to these findings.

 

Observed changes in clinical biochemistry parameters (higher alanine and aspartate aminotransferase activity and creatinine level in males, and lower total protein and urea level in males and females respectively) in animals at 100 mg/kg/day at the end of the treatment period were slight in and were fully reversible after a 14-day recovery period in males. Moreover, no morphological changes were observed that would support these changes.

 

Histopathology revealed an increased incidence and severity of foamy macrophage foci in the mesenteric lymph node at the end of treatment in all selected males and females at 100 mg/kg bw/day, persisting in males after the after the recovery period. This was considered to be adverse. An increased incidence of lymphoid atrophy was also observed in the thymus of females at 100 mg/kg bw/day.

 

No toxicologically significant changes were noted in any of the remaining parental parameters investigated i.e. clinical appearance, functional observations, body weight, food consumption, haematology and macroscopic examination.

 

A parental NOAEL of 30 mg/kg bw/day was concluded, based on the increased incidence and severity of foamy macrophage foci in the mesenteric lymph node at the end of treatment (persistent in males after the recovery period).

 

Reproductive/Developmental results:

No reproductive/developmental toxicity was observed up to the highest dose tested (100 mg/kg bw/day).

A reproductive/developmental NOAEL of 100 mg/kg bw/day was derived. 

 

OECD 443 - EOGRTS Cohorts 1A 1B + 3 (Charles River Laboratories, 20257161)

Following an ECHA decision (15 November 2019), an Extended One Generation Reproductive toxicity study (Annex X, Section 8.7.3.; test method: OECD TG 443) in rats, oral route with the registered substance was commenced. The study design includes: Ten weeks premating exposure duration for the parental (P0) generation; Dose level setting with the aim to induce systemic toxicity at the highest dose level; Cohort 1A (Reproductive toxicity); Cohort 1B (Reproductive toxicity) without extension to mate the Cohort 1B animals to produce the F2 generation); and Cohort 3 (Developmental immunotoxicity). The study is ongoing at time of submission, and will be reported upon finalisation of reporting (expected: Q1 2023).

 

Other relevant studies in the AAI category

Similar OECD 422 studies have been performed on other substances from the AAI category, specifically on: i) Fatty acids, C18 unsaturated, reaction products with tetraethylenepentamine (AAI-TEPA) and ii) Fatty acids, C18 unsaturated, reaction products with pentaethylenehexamine (AAI-PEHA). No indication of concern for reproductive or developmental toxicity was observed in these studies. For AAI-TEPA, the NOAEL derived was >100 mg/kg bw/day; for AAI-PEHA, the NOAEL derived was ≥ 300 mg/kg bw/day. 

 

Data from repeated dose toxicity studies from the category of AAI substances, including 90-day studies in rats and dogs on a similar substance, suggest low toxicity and have shown no adverse effects on reproductive organs.  A 90-day repeated dose study on AAI-DETA showed no indications of possible reproductive toxicity up to the highest dose of 100 mg/kg bw/day (NOAEL ~ 730 mg/kg-bw/day). No treatment related changes in estrous cycle length were noted across the dose groups during the period in which estrous cycle length was determined (Day 72 up to and including Day 92), and histopathological examination of the male and female reproductive organs (including spermatogenesis staging) did not show treatment-related lesions.

Justification for selection of Effect on fertility via inhalation route:
Likelihood of exposures via inhalation is low considering the high boiling point (> 300 °C) and very low vapour pressure (0.00017 mPa at 25°C). The potential for inhalation is not significant to justify a study. Furthermore, as the substance is classified as corrosive, such testing should normally not be conducted.

Justification for selection of Effect on fertility via dermal route:
All substances from the category of Amidoamine/imidazolines (AAI) are corrosive to the skin and are not expected to easily pass the skin. The skin is therefore not a preferred route when studying reproductive toxicity.

Effects on developmental toxicity

Description of key information

• NOAEL (developmental) = at least 100 mg/kg/day | NOAEL (parenteral) = 30 mg/kg bw/day - OECD 422, rat, Oral - NOTOX 491556 (2010)


• NOAEL (developmental) = at least 150 mg/kg/day | NOAEL (maternal) = at least 50 mg/kg/day - OECD 414 - Rat, Oral - WIL Research, 503870 (2014)


• NOAEL (developmental) = at least 50 mg/kg/day | NOAEL (maternal) = 15 mg/kg/day - OECD 414, rabbit, oral - Charles River Laboratories 20257165 (2021)* 

*Conclusion based on data from 12 litters, due to maternal systemic toxicity leading to mortalities. See discussion for further information.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 Jan 2021 - 18 March 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted in accordance with international guidelines and in accordance with GLP. All guideline validity criteria were met. Any deviations are detailed (table 01). Following consideration, none were considered to affect the integrity of the study or evaluation of results.

Reason / purpose for cross-reference:

reference to same study

Remarks:

Reference to Dose Range Finding study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

1998

Deviations:

yes

Remarks:

The deviations neither affected the overall interpretation of study findings nor compromised the integrity of the study. Detailed within 'Any other information on materials and methods incl. tables'.

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

2008

Deviations:

yes

Remarks:

The deviations neither affected the overall interpretation of study findings nor compromised the integrity of the study. Detailed within 'Any other information on materials and methods incl. tables'.

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

2018

Deviations:

yes

Remarks:

The deviations neither affected the overall interpretation of study findings nor compromised the integrity of the study. Detailed within 'Any other information on materials and methods incl. tables'.

Principles of method if other than guideline:

Guideline study conducted to GLP.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature protected from light container flushed with nitrogen
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Stability for at least 24 hours at room temperature under normal laboratory light conditions, for at least 8 days in the refrigerator and for at least 3 weeks in the freezer, is confirmed over the concentration range 1 to 200 mg/mL (solutions), Test Facility Study No. 20257162.
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis: test item handling using amber glassware or wrap containers in aluminum-foil

TREATMENT OF TEST MATERIAL PRIOR TO TESTING: None

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (Chatillon sur Chalaronne, France)
- Age at study initiation: 18-20 weeks old
- Weight at study initiation: 2873 – 4158 g females
- Fasting period before study: None
- Housing: labelled cages with perforated floors (Ebeco, Germany, dimensions 67 x 62 x 55 cm)
- Diet (e.g. ad libitum): Restricted access to pelleted diet for rabbits (KLIBA NAFAG Rabbit Diet 3409 maintenance and breeding, from Kliba NAFAG Granovit AG, Kaiseraugst, Switzerland). From post-coitum Days 14-17 onwards (for the different subgroups, respectively), celery was daily provided to all animals in order to stimulate food consumption.
- Water (e.g. ad libitum): Municipal tap water ad libitum
- Acclimation period: 5 Days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-19 ˚C
- Humidity (%): 49-52 %
- Air changes (per hr):
- Photoperiod (12 hrs dark / 12 hrs light):

IN-LIFE DATES: From: 01 Mar 2021 To: 26 Mar 2021

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Remarks:

Merck, Darmstadt, Germany | Batch: Progly12 and Progly14 | specific gravity: 1.036

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item and vehicle were administered to the appropriate animals by once daily oral gavage 7 days a week from Day 7 to Day 28 post-coitum, inclusive. The dose volume for each animal were based on the most recent body weight measurement. The doses were given using a plastic catheter attached to a plastic disposable syringe.
The dosing formulations were stirred continuously during dose administration.
Dose pot identification via Provantis were used as additional check to verify the dosing procedure according to Standard Operating Procedures.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Propylene glycol (Merck, Darmstadt, Germany). Test item selection confirmed and justified during tolerability and dose-range finding studies.
- Concentration in vehicle: see experimental design (table 3)
- Amount of vehicle (if gavage): dose volume for each animal based on the most recent body weight measurement. See experimental design (table 3)
- Lot/batch no. (if required): Progly12 and Progly14
- Purity: N/A

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The objective of the analytical study was to determine the accuracy of preparation and homogeneity of the test item in formulations. Accuracy and homogeneity were determined for formulations prepared for use during treatment in Week 1.
The chemical analyses were conducted as specified below. Samples and remaining formulation were protected from light using amber glassware.
Duplicate samples (approximately 500 mg), which were taken from the formulations using a pipette, were accurately weighed into volumetric flasks of 10 mL (Group 1 and Group 2),
20 mL (Group 3) or 50 mL (Group 4). For determination of accuracy, samples were taken at middle position (50% height) or at top, middle and bottom position (90%, 50% and 10% height). The samples taken at 90%, 50% and 10% height were also used for the determination of the homogeneity of the formulations.
The volumetric flasks were filled up to the mark with methanol. The solutions of Groups 2-4 were further diluted with methanol by a factor of 10000 (Group 2 and Group 3) or 15000 (Group 4) to obtain concentrations within the calibration range.
Accuracy: No test item was detected in the Group 1 formulation.
The concentrations analyzed in the formulations of Group 2, Group 3 and Group 4 were after correction for the mean recovery of the corresponding QC samples, in agreement with target concentrations (i.e. mean accuracies between 90% and 110%).
Homogeneity: The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

Details on mating procedure:

- Impregnation procedure: purchased timed pregnant

Duration of treatment / exposure:

The test item and vehicle were administered to the appropriate animals by once daily oral gavage 7 days a week from Day 7 to Day 28 post-coitum, inclusive.

Frequency of treatment:

Daily gavage

Duration of test:

24. Feb - 26 Mar 2021 (29 days per animal, staggered arrival 24-26 Feb)

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

5 mg/kg bw/day (nominal)

Dose / conc.:

15 mg/kg bw/day (nominal)

Dose / conc.:

50 mg/kg bw/day (nominal)

No. of animals per sex per dose:

22

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on the results of the dose range finder, and in an attempt to produce graded responses to the test item.
- Rationale for animal assignment (if not random): N/A
- Fasting period before blood sampling for (rat) dam thyroid hormones: N/A
- Time of day for (rat) dam blood sampling: N/A
- Other: N/A

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Mortality/Moribundity Checks At least twice daily throughout the study.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily, starting on Day 7 post-coitum up to the day prior to necropsy. During the dosing period, this observation will be performed postdose.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed on Days 7, 9, 12, 15, 18, 21, 24, 27 and 29 post-coitum. In addition, data on body weight Day 0 post-coitum (i.e. the day of mating) were provided by the Supplier (non-GLP) and included in the report.

FOOD CONSUMPTION: YEs
- Food consumption of animals were measured daily from Day 3 post-coitum onwards.


WATER CONSUMPTION: Yes
- Regular basis throughout the study. Water consumption was monitored by visual inspection of the water bottles.


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 29 post-coitum
- Organs examined:

OTHER: N/A

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: The number and distribution of live and dead fetuses.

Blood sampling:

N/A

Fetal examinations:

- External examinations: Yes: [ all per litter ]
- Soft tissue examinations: Yes: [ all per litter ]
- Skeletal examinations: Yes: [ half per litter ]
- Head examinations: Yes: [ half per litter ]
- Anogenital distance of all live rodent pups: N/A

Statistics:

STATISTICAL ANALYSIS : All statistical analyses were performed within the respective study phase, unless otherwise noted. Numerical data collected on scheduled occasions were summarized and statistically analyzed as indicated below according to sex and occasion. See additional information on materials and methods.

Indices:

Litter Variables
Sex Ratio (% males): (No. male fetuses x 100) / Total no. of fetuses |
Pre-Implantation Loss (%): ((No. of corpora lutea – no. of implants) x 100) / No. of corpora lutea |
PostImplantation Loss (%): ((No. of implants – no. of live fetuses) x 100) / No. of implants |
Litter % of Fetuses with Abnormalities: (No. of fetuses in litter with a given finding x 100) / No. of fetuses in litter examined

Historical control data:

Yes, HCD held by laboratory.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Note: Individual findings of females that died preterm are given under Mortality. Decreased feces production was observed for most females in all groups, including control. This finding was considered to be unrelated to treatment with the test item, as it occurred in both treated and control animals at comparable incidence.
Erected fur was incidentally observed in one or two females surviving until scheduled necropsy in the control group and at 5, 15 and 50 mg/kg/day, respectively.
Other clinical observations recorded (i.e. scabs, salivation, loss of fur, swollen or discolored forepaw, hunched posture) occurred within the range of background findings to be expected for rabbits of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered to be unrelated to treatment with the test item.

Mortality:

mortality observed, treatment-related

Description (incidence):

A total of 13 females did not survive until scheduled necropsy: two females at 5 mg/kg/day, two at 15 mg/kg/day and nine at 50 mg/kg/day.
Of these premature deaths, five at 50 mg/kg/day (Female Nos. 68, 70, 71, 73 and 82) and one at 15 mg/kg/day (Female No. 49) were considered to be related to treatment with the test item. All females showed strongly reduced or no food intake, along with body weight loss (5-10%) and reduced feces output. Other clinical signs included erected fur, pallor, thin appearance, abnormal feces (pale, mucoid, small in size) and/or anorexia. See table 04. For both females at 5 mg/kg/day (Nos. 37 and 40) and all other females at 50 mg/kg/day (Nos. 67, 74, 79 and 81), the cause of death was not clear, but was likely to be secondary to other influences such as the gavage procedure rather than to the test item. For all of these females, labored breathing (with abnormal sounds), salivation, erected fur and/or pallor were noted (shortly) after dosing. For some of these females, red fluid was noted on the dosing tube (data taken from study daybook). No other (relevant) signs or changes in food intake and body weight were noted for these animals. Macroscopic findings included an enlarged gallbladder, small and large intestines and/or colon distended with gas, thoracic cavity filled with dark, brown watery, gritty fluid or frothy, white content in the trachea, multifocal dark, red foci or pale discoloration in the lungs, pale heart, irregular surface, gelatinous content, mucoid white content, a multifocal crater and/or black multifocal foci in the stomach (fundus wall), accessory lobe (liver), cysts in the oviduct, ectopic splenic tissue, and/or pale discoloration of the kidney).
Finally, Female No. 56 at 15 mg/kg/day was sacrificed in extremis on post-coitum Day 12 which was considered unrelated to treatment with the test item, as findings/observations commenced prior to start of treatment. As such it was concluded that the animal was not able to acclimatize to the test facility within the standard five days. Food intake of this animal was absent from Day 3 post-coitum, resulting in a body weight loss of 13% on Day 12 (compared to Day 0). In-life, reduced feces production, erected fur and a swollen forepaw were noted for this female. At necropsy, dark, black multifocal foci in the stomach (fundus) and ectopic splenic tissue were present. See Table 04.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Note: Individual findings of females that died preterm are given in Section 'Mortality'. At 50 mg/kg/day, slight mean body weight loss (-1% and up to 10% for individual animals) was observed between post-coitum Days 7-9 followed by a reduced body weight gain between post-coitum Days 9-24 (not statistically significant), when compared to the concurrent control group. This was mainly attributed to Female Nos. 68, 70, 71 and 73, that were sacrificed in this period (see Mortality & table 04). Normal body weight gain was noted over post-coitum Days 24-29. This resulted in a reduced absolute mean body weight between post- coitum Days 18-24 (up to 6-7% lower than control). At the end of treatment (on post-coitum Day 29), mean body weight was 5% lower than control (not statistically significant).
At 15 mg/kg/day, a slight reduced body weight gain was noted between post-coitum Days 7-9 (no statistical significance achieved), which recovered to control levels between post-coitum Days 9-29. Therefore, this was considered to be of no toxicological significance.
No changes in body weight and body weight gain were noted at 5 mg/kg/day.
Mean body weight loss (corrected for the weight of gravid uterus), was observed in all groups including controls, without a dose-related trend and was therefore considered to be unaffected by treatment with the test item. See Table 05, and Figure 1 of attached results.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Note: Individual findings of females that died preterm are given in Section 'Mortality'.
Mean food consumption was decreased at 50 mg/kg/day (up to 30% lower, when compared to the concurrent control group) from post-coitum Days 7 onwards (statistical significance achieved between post-coitum Days 7-12 only), which recovered to similar levels over post-coitum Days 18-29 when compared to control.
At 15 mg/kg/day, slightly lower mean food consumption was observed at the start of treatment between post-coitum Days 7-9 (11% of control; no statistical significance achieved) with complete recovery thereafter. Therefore, this was considered of no toxicological relevance.
Note: From post-coitum Days 14, 15 16 or 17 onwards, daily celery was offered to all females in addition to the standard food to stimulate appetite. For some females, recovery of food consumption was noted. See Table 06, and Figure 2 of attached results.

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Note: Individual findings of females that died preterm are given in Section Mortality. Macroscopic observations at scheduled necropsy did not reveal any alterations that were considered to have arisen as a result of treatment with the test item.
A crater in the stomach (fundus) was noted in 2/22, 5/22, 2/22 and 3/22 females of the control, 5, 15 and 50 mg/kg/day groups, respectively.
Other findings noted among control and treated animals are occasionally seen among rabbits used in these types of study and in the absence of correlated microscopic findings they were considered changes of no toxicological significance.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

At microscopic examination mild erosions in the stomach (fundus) were recorded for a single female of the control, 5 and 15 mg/kg/day groups, respectively. This was regarded the microscopic correlate to the craters in the fundus recorded at necropsy. One additional erosion was found in the cardia of the stomach of one female at 15 mg/kg/day.
No microscopic correlates could be found for the recorded craters in the fundus of the remaining animals.
The remainder of the recorded microscopic findings in the stomach were within the range of background pathology encountered in female rabbits subjected to a Prenatal Developmental Toxicity Study. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

The lower mean number of implantation sites at 5, 15 and 50 mg/kg/day was mainly attributed to relatively high values in the control group when compared to historical control data. As values remained well within the range of historical control data, these changes were considered to be unrelated to treatment with the test item.
Mean pre-implantation loss was higher at 50 mg/kg/day when compared with mean concurrent control values (15.82 vs. 5.47% in the control group). As treatment was initiated after the implantation period was completed, this was considered to be unrelated to treatment with the test item.
The number of corpora lutea, post-implantation loss and early and late resorptions were comparable between all groups and within the range of normal biological variation. See table 07 - 08.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

The number of corpora lutea, post-implantation loss and early and late resorptions were comparable between all groups and within the range of normal biological variation.

Early or late resorptions:

no effects observed

Description (incidence and severity):

The number of corpora lutea, post-implantation loss and early and late resorptions were comparable between all groups and within the range of normal biological variation.

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Details on maternal toxic effects:

All females, including those that were sacrificed or died preterm were found to be pregnant, except for Female No. 16 (control group), Nos. 29, 36 and 42 (5 mg/kg/day), Nos. 47, 51 and 66 (15 mg/kg/day) and Nos. 78 and 81 (50 mg/kg/day). Excluding of females that were sacrificed/died prior to scheduled necropsy and the non-pregnant females resulted in a total of 21, 17, 17 and 12 pregnant females with viable litters for evaluation in the control, 5, 15 and 50 mg/kg/day groups, respectively.
Note: Although a minimum of 16 litters is required for evaluation of developmental data according to the guidelines, the 12 litters available at 50 mg/kg/day were included for evaluation in this study. As such, results at 50 mg/kg/day should be interpreted with caution.
See table 07 - 09.

Key result

Dose descriptor:

NOAEL

Effect level:

15 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

mortality

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

There was no test item-related effect on fetal body weights (both sexes) noted following treatment up to 50 mg/kg/day. Fetal weight was comparable between the control and test groups and within the range of biological variation. See table 09.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

See table 08.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The male:female ratio was unaffected by treatment up to 50 mg/kg/day. The apparent lower number of male fetuses at 5 mg/kg/day was considered to be unrelated to treatment with the test item as this finding was not dose-related. See table 08.

Changes in litter size and weights:

effects observed, non-treatment-related

Description (incidence and severity):

Mean litter sizes were 10.7, 9.2, 9.2 and 8.6 live fetuses/litter for the control, 5, 15 and
50 mg/kg/day groups, respectively. The lower mean litter size at 5, 15 and 50 mg/kg/day was
mainly attributed to relatively high values in the control group when compared to historical control data . As values remained well within the range of historical control data and no clear dose-related response was noted, these changes were considered unrelated to treatment with the test item. See table 08 - 09.

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

External malformations were observed in two fetuses. One fetus at 50 mg/kg/day was noted with a small, open eye and absent pinna and one fetus at 15 mg/kg/day was found with a hyperflexed hindlimb. Due to the lack of a dose-related response and single observations in different dose groups, the recorded external malformations were considered chance findings and not related to treatment with the test item.
No external variations were observed in any groups.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Skeletal malformations were noted in the skull, ribs and vertebra. The most frequently observed skeletal malformations were supernumerary lumbar vertebra in 4 (4) and 2 (2) fetuses (litters) of the control and 5 mg/kg/day groups, respectively. Due to the occurrence of these findings in the control group, they were considered incidental and not related to treatment with the test item.
All other skeletal malformations and variations occurred in the absence of a dose-related trend and/or were also found in the control group and considered not related to treatment with the test item.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Visceral malformations were observed in 1 (1), 7 (4), 1 (1) and 3 (2) fetuses (litters) of the control, 5, 15 and 50 mg/kg/day groups, respectively. The malformations principally concerned the heart and major vessels, but also the kidney and brain.
Of the visceral malformations observed, retroesophageal subclavian arteries were seen across different groups. As this finding was noted also in the control group and no dose-related response was observed, this was considered to be unrelated to treatment with the test item. Additionally, four fetuses in one litter (Litter No. 25) at 5 mg/kg/day were observed with tetralogy of fallot and two fetuses in one litter (Litter No. 76) at 50 mg/kg/day were observed with malpositioned kidneys. As each malformation was identified within one litter only and no dose-related response was observed, these findings were considered a familiar cause and not related to treatment with the test item.
The remaining visceral malformations affecting the heart and major vessels were noted in one single fetus at 5 mg/kg/day (Litter No. 39, Fetus R9). Therefore, these findings were considered to be chance findings.
Visceral variations occurred across a variety of structures, the low incidences and group distribution of these findings did not indicate any test item-related effects.

Details on embryotoxic / teratogenic effects:

Summaries of Fetal Abnormalities by Finding and by Classification are included in attachment.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: based on data from 12 litters (due to systemic toxicity leading to maternal mortalities).

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Table 04: Test Item-Related Premature Decedents

Group	Female No.	Day of No. Sacrifice	Findings
Group 3: 15 mg/kg/day	49	Sacrificed in extremis on Day 19 PC	-  Extremely low FC for 9consecutive days -  BW loss of 6% compared to BW Day 9 PC -  Decreased feces output from Day 10 PC onwards, pale feces output on Day 17-19 PC, erected fur on Day 18 and 19 and anorexia on Day 18 (recorded by the veterinarian). -  Dark, red discoloration of the stomach (fundus) at necropsy -  Gravid with 12 live embryos
Group 4: 50 mg/kg/day	68	Sacrificed in extremis on Day 24 PC	-  Extremely low/absent FC for 8 consecutive days -  BW loss of 5% compared to BW Day 7 PC -  Decreased feces output from Day 9 PC onwards, absent feces output on Days 23 and 24, pale feces on Days 22-24 and erected fur on Days 20-24 PC -  No macroscopic findings at necropsy -  Gravid with 6 live fetuses and 5 late resorptions examined
	70	Sacrificed in extremis on Day 20 PC	-  Extremely low/absent FC for 7 consecutive days -  BW loss of 7%compared to BW Day 7 PC -  Decreased feces output from Day 13 PC onwards, pale feces on Days 19 and 20 PC and erected fur on Day 20 PC -  Enlarged gallbladder and ectopic splenic tissue at necropsy -  Gravid with 12 live embryos
	71	Sacrificed in extremis on Day 20 PC	- Extremely low/absent FC for 7 consecutive days - BW loss of 10% compared to BW Day 7 PC •                -  Decreased feces output from Day 9 PC onwards, mucoid feces and reduced in size on Day 20 PC, erected fur from Day 15 PC onwards and thin and pallor on Days 19 and 20 PC •                -  Enlarged gallbladder and a watery content in the cecum and colon at necropsy •                -  Gravid with 11 live embryos
	73	Sacrificed in extremis on Day 24 PC	- Extremely low/absent FC for 8 consecutive days - BW loss of 6%comparedtoBWDay7PC - Decreased feces output on Day 11 PC and from Day 14 PC onwards, absent feces on Days 21-24, erected fur and thin appearance on Day 24 PC - A prominent lobular architecture of the liver and mucoid white content in the wall of the stomach at necropsy - Gravid with 9 live fetuses
	82	Sacrificed in extremis on Day 20 PC	- Extremely low/absent FC for 8 consecutive days - BWlossof5%comparedtoBWDay7PC - Decreased feces output from Day 10 PC onwards, pale feces reduced in size on Day 20 PC and erected fur on Days 18-20 PC - No macroscopic findings at necropsy - Gravid with 8 live embryos and 1 early resorption

PC = post-coitum, FC = food consumption, BW = body weight.

 

Table 05: Summary of Body Weight Gains (g): Gestation

Bodyweight Gain (Interval)		Day(s) Relative to Mating (Litter: A)
Sex: Female		7 → 9 [G]	9 → 12 [G1]	12 → 15 [G]	15 → 18 [G1]	18 → 21 [G]	21 → 24 [G]	24 → 27 [G]	27 → 29	7 → 29
0 mg/kg/day Group 1	Mean SD N	39.0 46.2 21	32.0 34.1 21	48.6 49.7 21	42.2 83.2 21	49.3 39.0 21	62.6 41.3 21	35.7 61.7 21	31.4 41.1 21	340.9 162.9 21
5 mg/kg/day Group 1	Mean SD N	29.1 43.4 19	71.1 64.6 19	93.9 65.7 18	28.2 46.7 18	39.1 45.5 18	48.1 53.5 17	61.9 53.3 17	49.4 49.1 17	415.7 198.5 17
15 mg/kg/day Group 1	Mean SD N	3.7 74.5 19	45.5 56.7 19	73.5 62.9 18	8.4 58.2 18	48.1 54.4 17	55.4 50.5 17	17.0 62.3 17	57.6 37.8 17	346.8 172.5 17
50 mg/kg/day Group 1	Mean SD N	-23.1** 63.9 20	21.0 90.6 20	38.9 90.5 20	14.9 92.2 18	28.6 78.3 15	46.9 57.9 15	60.2 65.8 13	49.1 54.5 12	326.6 193.7 12

[G] - Anova & Dunnett: ** = p ≤ 0.01

[G1] - Kruskal-Wallis & Dunn

 

Table 06: Summary of Food Consumption: Gestation

Food Mean Daily Consumption (g/animal/day)		Day(s) Relative to Mating (Litter: A)
Sex: Female		7 → 9 [G]	9 → 12 [G1]	12 → 15 [G]	15 → 18 [G1]	18 → 21 [G]	21 → 24 [G]	24 → 27 [G]	27 → 29	7 → 29
0 mg/kg/day Group 1	Mean SD N	133.14 26.09 21	126.90 21.44 21	80.24 43.04 21	96.10 39.71 21	115.63 28.42 21	100.59 24.12 21	86.30 27.27 21	89.07 36.02 21	104.18 20.40 21
5 mg/kg/day Group 1	Mean SD N % Diff	138.03 18.90 19 3.67	132.18 22.47 19 4.15	101.33 43.63 19 26.29	121.07 28.66 18 25.99	133.15 20.75 18 15.15	108.08 28.48 17 -2.27	93.41 27.86 17 8.24	92.00 29.44 17 3.29	114.41 20.25 17 9.84
15 mg/kg/day Group 1	Mean SD N % Diff	188.13 40.38 19 -11.27	113.46 44.89 19 -10.60	99.65 45.03 18 24.19	97.70 53.39 18 1.67	116.82 31.79 17 1.03	114.39 26.03 17 3.44	90.57 34.24 17 4.94	94.68 28.10 17 6.29	109.30 26.00 17 4.93
50 mg/kg/day Group 1	Mean SD N % Diff	100.78** 25.07 20 -30.09	88.72** 34.12 20 -30.09	66.02 34.86 20 -17.72	69.59 46.08 18 -27.58	107.51 50.61 15 -7.03	106.33 45.09 15 -3.85	95.03 27.23 13 10.11	89.17 36.10 12 0.11	99.62 17.03 12 -4.37

[G] - Anova & Dunnett: ** = p ≤ 0.01

[G1] - Kruskal-Wallis & Dunn

 

Table 07: Summary of Maternal Performance and Mortality

		0 mg/kg/day Group 1	5 mg/kg/day Group 1	15 mg/kg/day Group 1	50 mg/kg/day Group 1
Group Size - Females		22	22	22	22
Number of Females Pregnant [f]	N+ve %	21 95.5	19 86.4	19 86.4	20 90.9
Female with Live Fetuses [f]	N+ve %	21 100.0	18 94.7	19 100.0	19 95.0
Total Resorptions [f]	N+ve %	0 0.0	0 0.0	0 0.0	0 0.0
Female with all Nonviable [f]	N+ve %	0 0.0	1 5.3	0 0.0	1 5.0
Terminal Euthanasia [f]	N+ve %	22 100.0	20 90.9	20 90.9	13** 59.1**
Unscheduled Death/Euthanasia [f]	N+ve %	0 0.0	2 9.1	2 9.1	9** 40.9**
Found Dead [f]	N+ve %	0 0.0	1 4.5	0 0.0	1 4.5
Unscheduled Euthanasia [f]	N+ve %	0 0.0	1 4.5	2 9.1	8** 36.4**
Delivered [f]	N+ve %	0 0.0	0 0.0	0 0.0	0 0.0

[f] - Fisher's Exact: ** = p ≤ 0.01

 

Table 08: Summary of Ovarian and Uterine Examinations and Litter Observations

Days relative to mating		0 mg/kg/day Group 1	5 mg/kg/day Group 2	15 mg/kg/day Group 3	50 mg/kg/day Group 4
Female with live fetuses	N +ve %	21 100	17 100.0	17 100.0	12 100.0
Number of corpora lutea (k)	Mean SD N % diff	12.0 1.9 21 -	11.2 2.7 17 -6.0	11.1 2.0 17 -7.5	11.6 3.3 12 -3.1
Number of Implantations (k)	Mean SD N % diff	11.2 1.6 21 -	10.1 2.4 17 -10.5	9.9 2.4 17 -12.1	9.4 2.6 12 -16.2
Pre-implantation loss (%) (k)	Mean SD N % diff	5.47 7.63 21 -	9.29 14.07 17 70.04	10.74 11.69 17 96.52	15.82 23.75 12 189.41
Total number of fetuses (k)	Mean SD N % diff	10.7 1.5 21 -	9.2 2.4 17 -13.4	9.2 2.0 17 -13.4	8.7 3.0 12 -18.8
Number of libe fetuses	Mean SD N % diff	10.7 1.5 21 -	9.2 2.4 17 -13.4	9.2 2.0 17 -13;4	8.6 2.9 12 -19.5
Number of dead fetuses (k)	Mean SD N % diff	0.0 0.0 21 -	0.0 0.0 17 -	0.0 0.0 17 -	0.1 0.3 12 -
Number of early resorptions (k)	Mean SD N % diff	0.3 0.7 21 -	0.5 0.8 17 58.8	0.5 0.9 17 41.2	0.6 1.2 12 75.0
Number of late resorptions (k)	Mean SD N % diff	0.2 0.4 21 -	0.3 0.8 17 23.5	0.2 0.7 17 -25.9	0.2 0.4 12 -30.0
Total Number of Resorptions [k]	Mean SD N % diff	0.6 0.7 21 -	0.8 1.2 17 44.1	0.6 1.1 17 13.2	0.8 1.5 12 31.3
Post-implantation Loss (%) [k]	Mean SD N % diff	4.92 5.92 21 -	8.12 12.70 17 64.86	5.70 9.60 17 15.81	9.61 14.43 12 95.12
Number of Live Male Fetuses [k]	Mean SD N % diff	5.6 1.4 21 -	3.9* 2.0 17 -29.9	4.9 2.2 17 -12.1	4.3 1.9 12 -24.4
Number of Live Female Fetuses [k]	Mean SD N % diff	5.0 1.9 21 -	5.3 2.8 17 5.9	4.3 1.4 17 -14.1	4.3 1.7 12 -13.3
Live Male Fetus/Litter (%) [k]	Mean SD N % diff	53.35 14.30 21 -	44.74 23.81 17 -16.15	51.95 17.25 17 -2.63	47.82 12.53 12 -10.36
Live Female Fetuses/Litter (%) [k]	Mean SD N % diff	46.21 14.76 21 -	55.26 23.81 17 19.58	48.05 17.26 17 3.97	51.34 13.01 12 11.10
Mean Fetal Weight males (g) [G]	Mean SD N % diff	38.37 3.61 21 -	39.40 6.70 17 2.67	39.74 5.16 17 3.57	41.17 5.37 12 7.29

[k] - Kruskal-Wallis & Dunn: * = p ≤ 0.05

[G] - Anova & Dunnett

 

Table 09: Summary of Ovarian and Uterine Examinations and Litter Observations

Sex: Female Day(s) Relative to Mating (Litter: A)		0 mg/kg/day Group 1	5 mg/kg/day Group 2	15 mg/kg/day Group 3	50 mg/kg/day Group 4
Mean Fetal Weight females (g) [G]         Mean Fetal Weight all (g) [G]	Mean SD N %Diff   Mean SD N %Diff	37.36 4.18 21 - 3   37.89 3.76 21 -	38.42 3.91 16 2.82   39.34 4.99 17 3.81	40.00 5.02 17 7.06   39.91 4.73 17 5.33	39.46 4.39 12 5.61   40.23 4.60 12 6.16

Conclusions:

Based on the results of this prenatal developmental toxicity study in time-mated female New Zealand White rabbits, the maternal and developmental No Observed Adverse Effect Levels (NOAELs) for Fatty acids, C18 (unsaturated), reaction products with diethylenetriamine were established:
Maternal NOAEL: 15 mg/kg/day (based on test item-related mortality, due to reduced food intake and lower body weight gain at
50 mg/kg/day). | Developmental NOAEL: At least 50 mg/kg/day (based on data from 12 litters).

Executive summary:

Objective

The objectives of this study were to determine the potential of Fatty acids, C18 (unsaturated), reaction products with diethylenetriamine (the test item) to induce developmental toxicity after maternal exposure during the critical period of organogenesis and to characterize maternal toxicity at the exposure levels tested when given orally by gavage to time-mated female New Zealand White rabbits from Day 7 to 28 post- coitum, inclusive. In addition, the No Observed Adverse Effect Levels (NOAELs) for maternal toxicity and developmental toxicity were evaluated.

 

Study Design

The dose levels in this study were selected to be 0, 5, 15, 50 mg/kg/day, based on the results of the Dose Range Finder (Test Facility Reference No. 20257164). The study design is outlined within table 04.

Chemical analyses of formulations were conducted once during the study to assess accuracy and homogeneity.

The following parameters and end points were evaluated in this study for the F0-generation: mortality/moribundity, clinical signs, body weights, food consumption, macroscopic examination, uterine contents, corpora lutea, implantation sites and pre- and post-implantation loss.

In addition, the following parameters were determined for the F1-generation: the number of live and dead fetuses, fetal body weights, sex ratio, external, visceral and skeletal malformations and developmental variations.

Formulation analyses confirmed that formulations of test item in Propylene glycol were prepared accurately and homogenously.

 

Maternal Toxicity

Treatment with the test item resulted in a significant level of maternal toxicity at 50 mg/kg/day.

At this high dose level, five females were euthanized in extremis between post-coitum Days 17 and 24. These females showed severely reduced or no food consumption for at least 7 consecutive days and a significant body weight loss (5-10% compared to body weight at start of treatment) during the treatment period. Clinical signs of toxicity noted among these females included erected fur, thin appearance, pallor, and/or pale or mucoid feces, feces reduced in size and amount.

At 15 mg/kg/day, one female was sacrificed in extremis on Day 19, based on similar findings as observed for the animals at 50 mg/kg/day (reduced or absent food consumption, body weight loss and reduced, pale feces production, erected fur and anorexia). Due to this single occurrence at 15 mg/kg/day, which incidentally also occurs in controls, this sacrifice was not considered to be toxicologically relevant.

In addition, two females at 5 mg/kg/day, one female at 15 mg/kg/day and three females at 50 mg/kg/day died or were sacrificed in extremis between post-coitum Days 13 and 23, due to severe breathing issues and quick deterioration of condition. For some of these animals, red fluid was noted on the dosing tube and at necropsy macroscopic observations related to the dosing issues were observed for most of the females. Therefore, the cause of death for these females was considered to be gavage procedure-related, rather than test item-related.

Finally, the preterm sacrifice of one female at 15 mg/kg/day was considered unrelated to treatment with the test item, as the findings that led to early sacrifice were already present prior to start of treatment (reduced feces production, erected fur and a swollen forepaw).

Overall, a lower mean food consumption (up to 30%) was noted in females at 50 mg/kg/day between post-coitum Days 7-18 (statistically significantly over Days 7-12 post-coitum), which appeared to recover to control levels over post-coitum Days 18-29. As a result, body weight loss (up to 10% for individual animals) was noted for most females treated at 50 mg/kg/day between post-coitum Days 7-9 followed by a reduced body weight gain between post-coitum Days 9-24 in individual females. As a result, five of these females were euthanized in extremis between post-coitum Days 17-24. In pregnant females surviving until scheduled necropsy, normal body weight gain was noted from post-coitum Day 9 (n=8) or 12 (n=4) onwards. Despite the apparent recovery, mean body weight was 5% lower than control on post-coitum Day 29. Given the magnitude of the observed effects on body weight gain and food consumption, resulting in sacrifice of five females at 50 mg/kg/day, these effects were considered adverse.

Findings for the stomach included macroscopic craters in the fundus and were noted in 2/22, 5/22, 2/22 and 3/22 females of the control, 5, 15 and 50 mg/kg/day groups, respectively. At microscopic examination of the control group and the 5 and 15 mg/kg/day treated groups, focal mild erosions in the fundus were found in a single animal, this was regarded to be the microscopic correlate for the craters. In most rabbits there was no microscopic correlate. These erosions were not seen in the rabbits at 50 mg/kg/day and not regarded a test item-effect. As such, they are suggested to be mainly related to the gavage procedure (tip of gavage touching fundus on insertion).

After exclusion of the females sacrificed/died prior to scheduled necropsy (No. 40 at 5 mg/kg/day, Nos. 49 and 56 at 15 mg/kg/day and Nos. 67, 68, 70, 71, 73, 74, 79, 81 and 82 at 50 mg/kg/day) and the non-pregnant females (No. 16 of control, Nos. 29, 36 and 42 at 5 mg/kg/day, Nos. 47, 51 and 66 at 15 mg/kg/day and No. 78 at 50 mg/kg/day), a total of 21, 17, 17 and 12 pregnant females were available with viable litters for developmental evaluation in the control, 5, 15 and 50 mg/kg/day groups, respectively.

The number of corpora lutea, implantation sites, viable or dead fetuses, early or late resorptions, and pre- and post-implantation loss were considered not to be affected by treatment with the test item up to 50 mg/kg/day.

 

Developmental Toxicity

Note: The total number of litters with viable fetuses available for evaluation was 21, 17, 17 and 12 in the control, 5, 15 and 50 mg/kg/day groups, respectively. Although a minimum of 16 litters is required for evaluation of developmental data according to the guidelines, for completeness of information the 12 litters available at 50 mg/kg/day were included for evaluation in this study. As such, the results at 50 mg/kg/day should be interpreted with caution.

No test item-related changes were noted up to 50 mg/kg/day in any of the developmental parameters investigated in this study (i.e. litter size, sex ratio, fetal body weights, external, visceral and skeletal malformations and developmental variations).

In conclusion, based on the results of this prenatal developmental toxicity study in time-mated female New Zealand White rabbits, the maternal and developmental No Observed Adverse Effect Levels (NOAELs) for the test item were established:

Maternal NOAEL: 15 mg/kg/day (based on test item-related mortality, due to reduced food intake and lower body weight gain at

50 mg/kg/day).
Developmental NOAEL: At least 50 mg/kg/day (based on data from 12 litters).

 

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 November - 19 December 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Wistar (Han)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Sex: females, nulliparous, nonpregnant and untreated fenimals were used at initiation of the study. (Stock male rats Crl:WI(Han) (outbred, SPF-Quality) were used for mating with the females. After mating these males were placed back in their stock and might be used in further studies.)
- Age at study initiation: Approximately 11 weeks.
- Weight at study initiation: Body weight variation was within +/- 20% of the sex mean (214 grams).
- Fasting period before study: no
- Housing:
Acclimatization: Animals were housed in groups of 5 animals/cage in Macrolon cages.
Mating: Females were caged together with stock males on a one-to-one-basis or two-to-one-basis in Macrolon cages.
Post-coitum: Females were individually housed in Macrolon cages.
General: Sterilised sawdust as bedding material and paper as cage enrichment/nesting material were supplied.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days

Environmental controls for the animal room were set to maintain 18 to 24°C (range of actual daily mean: 19.6-20.2°C), a relative humidity of 40 to 70% (range of actual daily mean: 48-50%), approximately 15 room air changes/hour, and a 12-hour light/12-hour dark cycle. Any variations to these conditions were recorded in the raw data and were considered not to have had any effect on the outcome of the study.

IN-LIFE DATES: From: 18 November - 19 December 2013

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for the specific gravity of the vehicle (1.036) and test substance. No correction was made for the purity/composition of the test substance.
- Storage conditions of formulations: At ambient temperature.

VEHICLE
- Justification for use and choice of vehicle: Based on Project 491556 (combined 28-day repro screening study with same batch of test substance).

DOSE VOLUME:
5 ml/kg body weight. Actual dose volumes were calculated according to the latest body weight

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 8 days in the refrigerator under nitrogen was also determined (highest and lowest concentration). Additional stability measurements over 6 hours were performed on concentrations of 2 and 30 mg/kg. The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration for solutions. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.

Details on mating procedure:

- M/F ratio per cage: 1/1.
- Age at start of mating of the animals in the study: Approximately 12 weeks
- Length of cohabitation: until sufficient mated females had been obtained for each dose group.
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage and/or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated.

Duration of treatment / exposure:

Duration of treatment: From Days 6 to 19 post-coitum, inclusive.

Frequency of treatment:

Once daily for 7 d/w.

Duration of test:

Duration of test: From Day 0 to 20 post-coitum, inclusive.

No. of animals per sex per dose:

22

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale: Dose levels were based on a MTD study (NOTOX Project 491555) and a combined 28-day/repro screening study (NOTOX Project 491556). In the MTD study, 3 rats/sex/group were exposed for 10 days at 50 and 250 mg/kg. At 250 mg/kg, toxicity consisted of clinical signs, decreased body weights and food consumption, and decreased weights of the thymus and spleen for one female. In the combined 28-day/repro screening study, 10 rats/sex/group (plus 5 recovery males in the control and high dose group) were exposed at 10, 30 and 100 mg/kg. Histopathology revealed foamy macrophage foci in the ileum of all selected males and females at 100 mg/kg/day, a slightly increased incidence and degree of macrophage foci in the mesenteric lymph node of both sexes at 100 mg/kg/day, and increased incidence of lymphoid atrophy in the thymus of females at 100 mg/kg/day. At the end of the 14-day recovery period for males, foamy macrophage foci in the ileum had completely resolved, whilst macrophage foci in the mesenteric lymph node persisted at higher severity than observed at the end of treatment. Given the persistence of this finding it was considered to be of an adverse nature.

Maternal examinations:

CAGE SIDE OBSERVATIONS
- Time schedule: At least twice daily.

DETAILED CLINICAL OBSERVATIONS
- Time schedule: At least once daily from Day 0 post-coitum onwards up to the day prior to necropsy. The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity.

BODY WEIGHT
- Time schedule for examinations: Days 0, 3, 6, 9, 12, 15, 17 and 20 post-coitum.

FOOD CONSUMPTION
- Time schedule for examinations: Days 0-3, 3-6, 6-9, 9-12, 12-15, 15-17 and 17-20 post-coitum.

WATER CONSUMPTION
No. Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

GENERAL REPRODUCTION DATA
- Mating date and confirmation of pregnancy were recorded.
- Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of these females was examined to detect signs of abortion or premature birth.

POST-MORTEM EXAMINATIONS
- Sacrifice on Day 20 post-coitumusing an oxygen/carbon dioxide procedure and subsequently subjected to a to an external, thoracic and abdominal examination.

Ovaries and uterine content:

Each ovary and uterine horn of animals surviving to planned necropsy were dissected and examined
as quickly as possible to determine:
- The number of corpora lutea.
- The weight of the (gravid) uterus.
- The number and distribution of live and dead fetuses.
- The number and distribution of embryo-fetal deaths.
- The weight of each fetus.
- The sex of each fetus from the ano-genital distance (during necropsy) and also from gonadal inspections (during further fetal examination).
- Externally visible macroscopic fetal abnormalities.

Fetal examinations:

External, visceral and skeletal fetal findings were recorded as developmental variations or malformations.
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: Groups 1 and 4
- Head examinations: Yes: half per litter

Statistics:

The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Mann Whitney test was used to compare mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and postimplantation loss, and sex distribution.
- Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test (Ref. 10) to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test was used to compare the compound-treated groups to the control group.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and post-implantation loss.

Indices:

For each litter the following calculations were performed:
Pre-implantation loss (%) = (number of corpora lutea - number of implantation sites) / number of corpora lutea x 100
Post-implantation loss (%) = (number of implantation sites - number of live fetuses) / number of implantation sites x 100
The fetal developmental findings were summarized by: 1) presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the group; and 2) considering the litter as the basic unit for comparison, calculating the number of affected fetuses as a mean litter proportion on a total group basis, where:
Viable fetuses affected / litter (%) = number of viable fetuses affected/litter / number of viable fetuses/litter x 100

Details on maternal toxic effects:

Details on maternal toxic effects:
MORTALITY:
No mortality occurred during the study period.

CLINICAL SIGNS:
At 150 mg/kg, one female (no. 85) showed hunched posture, piloerection and reduced faeces production during the last two days of treatment. This animal also showed affected body weight, food consumption and macroscopic findings, and therefore these clinical signs were regarded treatment related. At 150 mg/kg, eight additional animals also showed clinical signs (rales, piloerection and/or pale faeces). However, as these signs were only noted for 1-3 days, these signs were not considered toxicologically relevant. Incidental findings that were noted for the other groups consisted of black or red discolouration of the tail, rales, alopecia, and pale faeces. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological relevance.

BODY WEIGHTS:
Reduced body weights and body weight gain from Day 12 post-coitum onwards, and lower body weight gain corrected for gravid uterus weight were noted for approximately half of the animals at 150 mg/kg. Two animals (nos. 85 and 86) were most severely affected; female no. 86 gained only 2% and female no. 85 lost 18% by the end of the treatment period. At 15 and 50 mg/kg, body weights and body weight gain (also body weight gain corrected for uterine weight) remained in the same range as controls over the treatment period.

FOOD CONSUMPTION:
At 150 mg/kg, food consumption was statistically significantly reduced during the complete treatment period (Days 6-20 post-coitum). Food consumption of female nos. 85 and 86 was severely reduced from Day 12 post-coitum onwards. Food consumption before or after allowance for body weight was similar between controls and animals treated at 15 and 50 mg/kg.

MACROSCOPIC EXAMINATION:
At 150 mg/kg, two females (nos. 85 and 86) had the gastro-intestinal tract distended with gas and small thymus and spleen. In addition, one of these also showed irregular surface of the stomach and was emaciated. The incidence of other incidental findings among control and treated animals was within the background range of findings that are encountered among rats of this age and strain, and did not show a dose-related incidence trend. These necropsy findings were therefore not considered to be toxicologically relevant, and included several reddish foci on the thymus, uterus containing fluid, alopecia, and pelvic dilation of the kidneys.

MATERNAL PREGNANCY DATA:
There were 22, 21, 21, and 18 pregnant females in the control, 15, 50 and 150 mg/kg groups, respectively, with 22, 21, 21, and 18 litters available for evaluation. No significant differences were observed between control and treated groups regarding the number of corpora lutea, implantation sites, viable or dead fetuses, early or late resorptions, or pre- and post-implantation loss.

Key result

Dose descriptor:

NOAEL

Effect level:

> 50 - < 150 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
FETAL FINDINGS
LITTER SIZE
No treatment related effect on litter size was noted up to 150 mg/kg. The mean number of viable fetuses per litter was 11.3, 11.5, 11.9 and 11.3 in the control, 15, 50 and 150 mg/kg groups, respectively.

SEX RATIO
There were no treatment-related effects on the sex ratio of the fetuses.

FETAL BODY WEIGHT
No toxicologically relevant change was noted for fetal body weights up to 150 mg/kg. One litter at 150 mg/kg showed a very low mean fetal body weight (i.e. 2.0 gram). This was considered secondary to the health status of this dam. Body weights of fetuses (sexes combined) were 3.5, 3.4, 3.5 and 3.3 grams for the control, 15, 50 and 150 mg/kg groups, respectively.

FETAL MORPHOLOGICAL EXAMINATIONS
EXTERNAL MALFORMATIONS AND VARIATIONS
The numbers of fetuses (litters) available for external, visceral and skeletal morphological evaluation were 248(22), 241(21), 249(21) and 204(18) in Groups 1, 2, 3, and 4, respectively. Soft tissue cephalic examination was done for approximately half of the fetuses of all groups and skeletal examinations were done for all the fetuses of Groups 1 and 4. Malformations were observed in 6(5), 0(0)*, 4(3)* and 5(4) fetuses (litters) in these same respective dose groups.
* Note: For Group 2 and 3 treated at 15 and 50 mg/kg, these numbers of malformations were based on external, visceral and soft tissue cephalic examination; no skeletal examination had been performed for these groups.

EXTERNAL MALFORMATIONS AND VARIATIONS
There were no treatment related effects on fetal external morphology up to 150 mg/kg. Two external malformations were noted in this study. One fetus of the control group had a cleft palate (over the entire length) and one fetus at 50 mg/kg had exencephaly (absence of part of the cranial vault, with the brain protruding outside the skull). One external variation (i.e. subcutaneous edema in the neck) was noted for one fetus at 150 mg/kg. At this single occurrence in the control, mid and high dose group, these findings were not considered to be treatment related.

VISCERAL MALFORMATIONS AND VARIATIONS
There were no treatment related effects on fetal visceral morphology up to 150 mg/kg. Visceral malformations were observed in four fetuses (three litters). Internal hydrocephaly was noted for one fetus of the control group and one fetus at 50 mg/kg. At 50 mg/kg, one fetus had a small eye and one fetus showed persistent truncus arteriosus and absent eyes. At this low incidence and without a dose response relationship, these malformations were not considered treatment related. In addition, all findings (except persistent truncus arteriosus) were noted before in historical control fetuses. As persistent truncus arteriousus was noted for only one fetus at the mid dose group, it was considered a chance finding and not related to treatment. Visceral variations noted for fetuses in the control and/or treated group(s) were small supernumerary liver lobe, dilated ureter, liver appendix, partially undescended thymus horns, and convoluted ureter. These variations were not considered to be treatment related, because they occurred infrequently, in the absence of a dose related trend and/or at frequencies within the historical control range.


SKELETAL MALFORMATIONS AND VARIATIONS
There were no treatment related effects on fetal skeletal morphology up to 150 mg/kg. Skeletal malformations were observed in four fetuses (three litters) of the control group and five fetuses (four litters) at 150 mg/kg. One control fetus showed fusion of the sternebrae (nos. 4 and 5 with thread-like ossification bridge) with severely malaligned sternebrae (no. 4; slight no. 3). As this concerned a control fetus, it was not treatment related. Bent limb bones were noted for three control fetuses (two litters) and five high dose fetuses (four litters). One of these high dose fetuses also showed sternoschisis (nos. 1-6). Bent limb bones have been noted in 17 historical control fetuses (15 litters) with a maximum incidence of 2.0%. The litter incidence of the concurrent control group is 1.2% and 3.6% at 150 mg/kg. Even though the incidence at 150 mg/kg is outside the historical control range, these findings were not considered treatment related as the absolute number of affected fetuses at 150 mg/kg was only slightly above the concurrent control group (5 fetuses at 150 mg/kg versus 3 control fetuses). Moreover, bent limb bones are considered reversible (Ref. 12) and are therefore not adverse. In addition, as sternoschisis was noted for a single fetus, and as it was seen previously in the historical control data (2 fetuses), it was considered a chance finding and not toxicologically relevant. Skeletal variations noted for fetuses in the control and/or high dose group were unossified or reduced ossification of several bones (skull, entire sternum, vertebral centra, vertebral arches, pubis, ischium, ribs, sternebrae nos. 1, 2, 3, 4 5 and/or 6, vertebral centra, hyoid, metacarpals and/or metatarsals), 14th full or rudimentary ribs, bent ribs, 7th cervical full or rudimentary ribs, cervical centrum no. 1 ossified, slight or moderate malaligned sternebrae, caudal shift of pelvic girdle, and branched sternebrae. These variations were not considered to be treatment related, because they occurred at similar or higher frequencies in the control group, occurred infrequently and/or occurred at frequencies within the historical control range.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 150 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

FORMULATION ANALYSIS:

Accuracy of preparation: The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). A small response at the retention time of the test substance was observed in the chromatograms of the Group 1 formulation. It was not considered to derive from the formulation since a similar response was obtained in the analytical blanks.

Homogeneity: The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

Stability: Formulations at the entire range were stable when stored at room temperature under normal laboratory light conditions for at least 6 hours.

Conclusions:

The prenatal developmental toxicity study (OECD 414) on AAI-DETA resulted to a maternal NOAEL of 50 mg/kg. The developmental NOAEL was at least 150 mg/kg.

Executive summary:

Fatty acids, C18 unsat, reaction products with diethylenetriamine (AAI-DETA)was evaluated for developmental toxicity/teratogenicity in a study performed according to theOECD 414 guideline and in compliance to GLP. Eighty-eight mated female Wistar Han rats were assigned to four dose groups. The test item was administered once daily by oral gavage from Days 6 to 19 post-coitum at doses of 15, 50 and 150 mg/kg.

Females were checked daily for the presence of clinical signs. Food consumption and body weight were determined at periodic intervals. Formulations prepared on one day during treatment were analyzed for accuracy, homogeneity and stability. On an additional occasion, extra stability measurements were performed.

All animals surviving to Day 20 post-coitum were subjected to an examination post-mortem and external, thoracic and abdominal macroscopic findings were recorded. A laparo-hysterectomy was performed on each surviving female of the groups. The uteri, placentae and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations and corpora lutea were recorded. Gravid uterine weights were recorded, and corrected body weights (changes) were calculated. The fetuses were weighed, sexed and examined for external, visceral and skeletal malformations and developmental variations. All live fetuses were euthanized. One half of the fetuses were decapitated and the heads were fixed in Bouin’s fixative, all fetuses were dissected and examined for visceral anomalies and subsequently fixed in 96% aqueous ethanol and stained with Alizarin Red S. Skeletal examinations were performed for all fetuses of de control and high dose group..

 

Accuracy, homogeneity and stability of formulations were demonstrated by analyses.

Maternal findings:

Maternal toxicity was noted at 150 mg/kg, and consisted of reduced body weight gain and food consumption. The two dams that were most affected, also showed corroborative macroscopic abnormalities (gastro-intestinal tract distended with gas, small thymus and spleen, irregular surface of the stomach and/or emaciation), and one of these showed clinical signs (hunched posture, piloerection and reduced faeces) during the last two days of treatment. No maternal toxicity was observed in the 15 and 50 mg/kg groups.

Developmental findings:

No developmental toxicity was observed up to the highest dose levels tested (150 mg/kg).

Based on the results in this prenatal developmental toxicity study the maternal No Observed Adverse Effect Level (NOAEL) for Fatty acids, C18 unsat, reaction products with diethylenetriamine was established as being 50 mg/kg. The developmental NOAEL was at least 150 mg/kg.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

150 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The endpoint is concluded based on a weight of evidence appraisal. All studies are assigned a Klimisch rating of 1: reliable without restriction. Aside from range finding studies, all have been undertaken according to GLP. All studies have been designed based on an accepted OECD TG for this endpoint (TG414). The database for this endpoint met all relevant data requirements under REACH for the respective tonnage band. Consistent results from all studies within the whole group of Amidoamine/imidazolines (AAI), indicating a no concerns for developmental toxicity. The lower NOAEL derived from the rabbit OECD 414 is a consequence of systemic maternal toxicity, which led to lower dose setting in comparison to the rodent study. The NOAEL from the rat OECD 414 (at least 150 mg/kg bw/day) is considered to be the most sensitive descriptor for this endpoint.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Summary of studies

In considering effects on development, studies in the rat and rabbit performed with Fatty acids, C18 unsaturated, reaction products with diethylenetriamine (AAI-DETA) using oral (gavage) dosing were evaluated. These are outlined in brief below:

• OECD 422 (NOTOX, 491556, 2010)


• OECD 414 in first species - rat (WIL, 503870, 2014)


• OECD 414 in second species – rabbit – Tolerability and Range Finder (Charles River Laboratories, 20257163 & 20257164, 2021)


• OECD 414 in second species - rabbit (Charles River Laboratories, 20257165, 2021)

Other relevant studies in the AAI category are also considered in to place AAI-DETA in context of the other members of the group. Each study is outlined in brief below, followed by discussion of salient findings, and a conclusion on selection of most sensitive descriptor (NOAEL) for this endpoint.

 

OECD 422 (NOTOX, 491556, 2010)

AAI-DETA was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 10, 30 and 100 mg/kg/day. The males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 28 days). There was a 2-week recovery period for 5 animals of Group 1 and 4. The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation. 

 

Reproductive/Developmental results:

No reproductive/developmental toxicity was observed up to the highest dose tested (100 mg/kg bw/day).

A reproductive/developmental NOAEL of 100 mg/kg/day was derived. 

 

OECD 414 in first species - rat (WIL, 503870, 2014)

The test item was evaluated for developmental toxicity/teratogenicity in a study performed according to the OECD 414 guideline and in compliance to GLP. Eighty-eight mated female Wistar Han rats were assigned to four dose groups. The test item was administered once daily by oral gavage from Days 6 to 19 post-coitum at doses of 15, 50 and 150 mg/kg.

 

Maternal findings:

Maternal toxicity was noted at 150 mg/kg, and consisted of reduced body weight gain and food consumption. At 25 and 50 mg/kg/day, mean food consumption was reduced (up to 39 and 51% lower than control, respectively) from post-coitum Days 9-12 onwards until Day 29 post-coitum. Body weight gain was reduced from Day 12 post-coitum onwards, and lower body weight gain corrected for gravid uterus weight was present in approximately half of the animals at 150 mg/kg bw/day. The reduced body weight gain resulted in a statistically significantly decreased body weight at 50 mg/kg/day from post-coitum Day 12 onwards (up to 14% at the end of treatment). The two dams that were most affected also showed corroborative macroscopic abnormalities (gastro-intestinal tract distended with gas, small thymus and spleen, irregular surface of the stomach and/or emaciation), and one of these showed clinical signs (hunched posture, piloerection and reduced faeces) during the last two days of treatment. No maternal toxicity was observed in the 15 and 50 mg/kg groups.

A maternal NOAEL of 50 mg/kg bw/day was derived.

 

Developmental findings:

No developmental toxicity was observed up to the highest dose levels tested (150 mg/kg).

A developmental NOAEL of 150 mg/kg bw/day was derived.

 

OECD 414 in second species – rabbit – Tolerability and Range Finder (Charles River Laboratories, 20257163 & 20257164, 2021) 

A non-GLP tolerability study (Test Facility Study No. 20257163) in non-pregnant rabbits, and a dose range finding study in pregnant rabbits were undertaken to inform selection of dose levels for the OECD 414 study.  In the tolerability study, two groups of 3 females were dosed via oral gavage at 50 mg/kg/day (starting dose level) and subsequently at 100 and 75 mg/kg/day. Dosing duration was once daily for 7 days. A wash-out period of at least 7 days was maintained for each group between Periods 1 and 2. Based on evaluation of key parameters (mortality, clinical observations, body weight, food consumption and macroscopic examination), dose levels selected for the dose-range finding study (DRF) in pregnant rabbits were 0, 12.5, 25 and 50 mg/kg/day.

 

The DRF in pregnant animals (Test Facility Study No. 20257164) followed closely the design for the OECD 414 study. Four groups of 6 females were exposed to 0, 12.5, 25 and 50 mg/kg/day for Days 7 to 28 post-coitum inclusive by oral gavage.

 

Maternal findings:

In total, two females were sacrificed in extremis prior to scheduled necropsy (one animal in the 12.5 mg/kg bw/day group, and one animal in the 25 mg/kg bw/day group); neither were considered to be test item-related. At 25 and 50 mg/kg/day, mean food consumption was reduced (up to 39 and 51% lower than control, respectively) from post-coitum Days 9-12 and until Day 29 post-coitum. Consequently, a reduced body weight gain was observed for the 25 and 50 mg/kg/day-treated females between post-coitum Days 7-29. This resulted in a statistically significantly decreased body weight at 25 and 50 mg/kg/day from post-coitum Day 12 onwards (up to 14% at the end of treatment at 50 mg/kg bw/day; not significant at 25 mg/kg/day). Adjusted body weight gain was dose dependently lower than control but did not reach statistical significance (-23.7 gram in control vs -86.4, -157.7 and -291.1 gram, respectively). Despite incidental observations, no clinical or macroscopic observations of toxicological relevance were identified.

The numbers of pregnant females, corpora lutea and implantation sites, and pre- and post- implantation loss in the control and test item groups were similar and in the range of normal biological variation.

 

Developmental findings:

Litter sizes were within normal limits for all groups. Although not statistically significant, mean fetal body weights at 50 mg/kg/day were lower compared to control values (mean of 35.03 g vs 38.48 g control mean). This observation was consistent with that of reduced body weight gain / body weight in dams. External examination of the foetuses did not show any test item-related abnormalities.

 

OECD 414 in second species - rabbit (Charles River Laboratories, 20257165, 2021)

This study evaluated the potential for the test item to induce developmental toxicity after maternal exposure during the critical period of organogenesis and to characterize maternal toxicity at the exposure levels tested when given orally by gavage to time-mated female New Zealand White rabbits from Day 7 to 28 post- coitum, inclusive. The test item was administered once daily via oral gavage to groups of 22 females at doses of 0, 5, 15, 50 mg/kg/day (based on the results of Tolerability and Dose Range Finding studies, and in accordance with ECHA’s advice on dose selection.

 

Maternal findings:

Treatment with the test item resulted in a significant level of maternal toxicity at 50 mg/kg/day. At this dose level, five females were euthanized in extremis between post-coitum Days 17 and 24. These females showed severely reduced or no food consumption for at least 7 consecutive days and a significant body weight loss (5-10% compared to body weight at start of treatment) during the treatment period. Clinical signs of toxicity noted among these females included erected fur, thin appearance, pallor, and/or pale or mucoid faeces, faeces reduced in size and amount. Three further females at 50 mg/kg/day died or were sacrificed in extremis between post-coitum Days 13 and 23, due to severe breathing issues and quick deterioration of condition.

Two further females at 15 mg/kg bw/day were sacrificed in extremis; neither was considered to be toxicologically relevant. A further 6 animals died or were sacrificed in extremis due to gavage procedure-related issues (two females at 5 mg/kg/day, one female at 15 mg/kg/day and three females at 50 mg/kg/day).

Overall, a lower mean food consumption (up to 30%) was noted in females at 50 mg/kg/day between post-coitum Days 7-18, which appeared to recover to control levels over post-coitum Days 18-29. As a result, body weight loss (up to 10% for individual animals) was noted for most females treated at 50 mg/kg/day between post-coitum Days 7-9, followed by a reduced body weight gain between post-coitum Days 9-24 in individual females. This resulted in five animals being sacrificed in extremis between post-coitum Days 17-24. In pregnant females surviving until scheduled necropsy, normal body weight gain was noted from post-coitum Day 9-12 onwards. Despite the apparent recovery, mean body weight was 5% lower than control on post-coitum Day 29. Given the magnitude of the observed effects on body weight gain and food consumption, and associated mortalities, these effects were considered adverse.

After exclusion of the females sacrificed/died prior to scheduled necropsy a total of 21, 17, 17 and 12 pregnant females were available with viable litters for developmental evaluation in the control, 5, 15 and 50 mg/kg/day groups, respectively. Although a minimum of 16 litters is required to ensure sufficient statistical power in evaluation of developmental data according to the guidelines, for completeness of information the 12 litters available at 50 mg/kg/day were included for evaluation in this study. The results at 50 mg/kg/day were interpreted with caution. Taking this into account, it was clear that the number of corpora lutea, implantation sites, viable or dead foetuses, early or late resorptions, and pre- and post-implantation loss were considered not to be affected by treatment with the test item up to 50 mg/kg/day.

Based on the general toxicity leading to reduced food consumption and body weight loss / reduced body weight gain, a maternal NOAEL of 15 mg/kg bw/day was derived.

 

Developmental findings:

No test item-related changes were noted up to 50 mg/kg/day in any of the developmental parameters investigated in this study (i.e. litter size, sex ratio, fetal body weights, external, visceral and skeletal malformations and developmental variations).

 

A developmental NOAEL of at least 50 mg/kg bw/day was derived*.

*Based on data from 12 litters.

 

Other relevant studies in the AAI category

Reproductive and Developmental screening studies (OECD 422) have been performed on other substances from the AAI category, specifically on: i) Fatty acids, C18 unsaturated, reaction products with tetraethylenepentamine (AAI-TEPA) and ii) Fatty acids, C18 unsaturated, reaction products with pentaethylenehexamine (AAI-PEHA). No indication of concern for reproductive or developmental toxicity was observed in these studies. For AAI-TEPA, the NOAEL derived was >100 mg/kg bw/day; for AAI-PEHA, the NOAEL derived was ≥ 300 mg/kg bw/day. 

 

Discussion

The database meets all relevant data requirements under REACH for the respective tonnage band. All studies on AAI-DETA have been assigned a Klimisch rating of 1: reliable without restriction. Aside from tolerability/range finding study in rabbits, all have been undertaken according to GLP. All studies were designed based on an accepted OECD TG for this endpoint.

 

Due to general maternal toxicity within the OECD 414 rabbit study (which led to significant mortalities in the 50 mg/kg/day, high dose group), the total number of litters with viable foetuses available for evaluation was 21, 17, 17 and 12 in the control, 5, 15 and 50 mg/kg/day groups, respectively. Of the 10 animals lost from the 50 m/kg/day group, only 5 were considered to be directly related to the test item. Of the others, 1 survived but was found to be non-pregnant, whilst 4 were sacrificed due to effects likely to be secondary to other influences such as the gavage procedure rather than to the test item.

 

It should be noted that dose setting for the OECD 414 in rabbits was extremely difficult, due to a number of factors:

1. the test item's inherent corrosivity;


2. requirement for the use of Propylene Glycol as a vehicle (which poses certain challenges for dose volume);


3. the fact that rabbits are known for increased susceptibility to gastrointestinal disturbances (compared to rodents); and


4. the steep dose-response toxicity observed in tolerability and DRF studies (with non-pregnant and pregnant animals, respectively) with AAI-DETA.

In design and conduct of the study, full consideration was afforded to management of vehicle and dose volume, the tolerability and range finding studies were carefully designed and executed (from which indication of ‘some developmental and/or maternal toxicity i.e. clinical signs of a decrease in body weight’ were observed in the high dose, in accordance with ECHA’s advice on dose setting for OECD 414), and on-study interventions such as ensuring sufficient dietary fibre intake (via provision of fresh vegetables in the diet), this combination of factors led to significant mortalities in the final OECD 414 study by comparison to the DRF phase. As there was no difference between study protocols (aside from intervention to add fresh vegetables when severe depressions in food consumption became apparent), this outcome could be a result of the increased number of animals per group (22 v. 6), or to other external factors, such as variability in sensitivity (e.g. to G.I. disturbance) between different batches of animals from the supplier.

 

It is acknowledged that the number of litters ultimately available for evaluation within the 50 mg/kg/day group was lower than the 16 recommended according to the test guideline to ensure statistical power in evaluation. However, for completeness of information the 12 litters available at 50 mg/kg/day were included for evaluation, to maximise learning from those animals sacrificed to science during the study. Interpretation of outcomes was undertaken with appropriate caution.

 

In all studies considered, no test-item related findings were reported following hysterectomy examination of maternal animals (examination of uteri, placentae and ovaries, and the numbers of foetuses, early and late resorptions, total implantations and corpora lutea).

 

Within all studies, litter sizes were within normal limits for all groups, and external examination of the foetuses did not show any test item-related abnormalities. Furthermore, visceral and skeletal examinations

 

Comparison of developmental parameters between studies reveals one inconsistency: a reported reduction in mean fetal body weights at 50 mg/kg/day compared to control values within the in the rabbit OECD 414 DRF, which was not replicated in the main rabbit OECD 414 study. Further appraisal reveals that this observation did not achieve statistical significance. As the finding was not repeated within the larger main OECD 414 study, it can be considered not to be a finding of relevance.

 

Conclusion

In conclusion, results from all studies on AAI-DETA and within the wider group of Amidoamine/imidazolines (AAI) consistently indicate no effects in developmental parameters, and as such no concern for specific developmental toxicity. Although the significant maternal toxicity within the rabbit OECD 414 study resulted in lower than desired numbers of litters with viable foetuses for examination, the thoroughly conducted developmental examination gave absolutely no indication of treatment-related effects. As such, the NOAEL derived from the rabbit study is only lower than those in the rodent studies as systemic toxicity meant it was not possible to test at higher dose levels. 

 

Effect on developmental toxicity: via oral route:
Results from all guideline studies on AAI-DETA and within the wider group of Amidoamine/imidazolines (AAI) consistently indicate no concerns for developmental toxicity. The most relevant descriptor for this endpoint and route of exposure is derived from the rodent OECD 414 (GLP, Guideline, Klimisch 1, 2010) as at least 150 mg/kg/day.

Effect on developmental toxicity: via inhalation route:
Likelihood of exposures via inhalation is low considering the high boiling point (> 300 °C) and very low vapour pressure (0.00017 mPa at 25°C). The potential for inhalation is not significant to justify this study. Furthermore, as the substance is classified as corrosive, such testing is not normally conducted.

Effect on developmental toxicity: via dermal route:
All substances from the group of Amidoamine/imidazolines (AAI) are corrosive to the skin and are not expected to easily pass the skin. The skin is therefore not a preferred route when studying developmental toxicity. Use is limited to industrial and professional users where because of its corrosive and sensitising properties sufficient measures will be taken to prevent dermal exposures.

Mode of Action Analysis / Human Relevance Framework

Not relevant. No adverse effects were observed for all measurable parameters on developmental and reproductive toxicity at the maximum dose tested. As such, it can be concluded the test item does not cause reproductive or developmental toxicity.

Justification for classification or non-classification

The database of relevant studies available for Fatty acids, C18 unsaturated, reaction products with diethylenetriamine (AAI-DETA) includes OECD 414 studies in both first and second species (rat, rabbit), and an OECD 422 repeated dose toxicity with reproductive and developmental toxicity screening study. An OECD 443 study (EOGRTS, Cohorts 1A 1B + 3) is ongoing, and will be reported on completion. None of the studies completed to date report evidence for specific reproduction or developmental toxicity. Furthermore, all available data from the group of AAI substances, including a 90-day study in dogs on a similar substance, indicate low toxicity and no adverse effects on reproductive organs.

 

NOAEL (reproduction) = at least 100 mg/kg bw/day (OECD 422, NOTOX, 491556, 2010)

NOAEL(developmental) = at least 150mg/kg bw/day (WIL Research, 503870, 2014)  

 

As no reproductive or developmental toxicity were observed under the conditions of the studies, the substance does not meet the criteria for classification in accordance with Regulation (EC) No 1272/2008 (CLP) on Classification, Labelling and Packaging of Substances and Mixtures.