Fatty acids, C18-unsatd., reaction products with ammonia-ethanolamine reaction by-products

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

No data available.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

01 September 2009 - 15 October 2009

Reliability:

2 (reliable with restrictions)

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:

The available data available within the group of Amidoamines/imidazolines (AAI) substances indicate that for AAI substances based on shorter polyethyleneamines (EA), higher toxicity is observed compared to AAI based on longer EA. The forming of imidazoline itself does not seem to play a significant role. For cross-reading purposes between substances of AAI, Fatty acid reaction product with diethylene-triamine (AAI-DETA) therefore represents the worst case. In series of 28-day and combined repeated dose/reproduction screening toxicity studies (OECD 422) AAI-DETA has shown the highest level of toxicity. (See also document in support of category justification)

Use of the results obtained from studies with AAI-DETA also represent a conservative approach in the evaluation of Fatty acids, C18 unsaturated, reaction product with ammonia-ethanolamine reaction by-products (AAI-AEA). AAI-AEA differs from other substances in the AAI group that are based on fatty acids with polyamines, in that it contains a high amount of hydroxylated polyethylene amines, causing it to display more neutral OH groups, rather than basic primary amine groups. As toxicity is much related to the presence of free primary amine groups, it can be expected that in specifically in aspects ofacute toxicity related to direct chemical interactions, this substance shows less effects compared to other AAI substances, and specifically compared to AAI-DETA which represents the worst case in the AAI category

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 lower total protein and urea level in 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:

Consistent results from all studies within the whole group of Amidoamine/imidazolines (AAI), indicating no concerns for reproduction toxicity. (See also document in support of category justification).

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

The available data available within the group of Amidoamines/imidazolines (AAI) substances indicate that for AAI substances based on shorter polyethyleneamines (EA), higher toxicity is observed compared to AAI based on longer EA. The forming of imidazoline itself does not seem to play a significant role. For cross-reading purposes between substances of AAI, Fatty acid reaction product with diethylene-triamine (AAI-DETA) therefore represents the worst case. In series of 28-day and combined repeated dose/reproduction screening toxicity studies (OECD 422) AAI-DETA has shown the highest level of toxicity. (See also document in support of category justification)

Use of the results obtained from studies with AAI-DETA also represent a conservative approach in the evaluation of Fatty acids, C18 unsaturated, reaction product with ammonia-ethanolamine reaction by-products (AAI-AEA). AAI-AEA differs from other substances in the AAI group that are based on fatty acids with polyamines, in that it contains a high amount of hydroxylated polyethylene amines, causing it to display more neutral OH groups, rather than basic primary amine groups. As toxicity is much related to the presence of free primary amine groups, it can be expected that in specifically in aspects ofacute toxicity related to direct chemical interactions, this substance shows less effects compared to other AAI substances, and specifically compared to AAI-DETA which represents the worst case in the AAI category.

 

No reproductive/developmental toxicity was observed at any of the dose levels in an OECD 422 study with Fatty acid reaction product with diethylene-triamine (AAI-DETA), and thus a reproduction/developmental NOAEL of 100 mg/kg/day was determined. Similar OECD 422 studies have been performed on other substances from the group of AAI: on Fatty acid reaction product with tetraethylene-pentamine (AAI-TEPA) and on Fatty acid reaction product with pentaethylene-hexamine (AAI-PEHA). No indication of concern for reproductive or developmental toxicity was observed in these studies. Also OECD 414 developmental toxicity studies in rat on AAI-DETA and a similar substance showed no concern for developmental effects.

 

Data from repeated dose toxicity studies from the group 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. Specifically a 90-day repeated dose study on AAI-EDTAshowed no indications of possible reproductive toxicity up to the highest dose of 100 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.

 

Also the low likelihood of exposure can be considered as its use is limited to industrial and professional users where following its irritating properties to the skin and corrosive effects on the eyes will provide for sufficient protection measures to prevent exposure. The likelihood of exposures via inhalation is low considering the high boiling point (> 300 °C) and very low vapour pressure (8.4x10^-8Pa at 25°C) and use applications that do not involve the forming of aerosols, particles or droplets of an inhalable size.

In view of low potential of exposures in combination with an overall low level of toxicity, and a total lack of effects observed in reproductive parameters from developmental toxicity and reproduction screening studies within the group of AAI, and no effects on reproductive organs observed in available repeated dose studies, a 2-generation study is not considered necessary.

Short description of key information:
NOAEL for reproduction/developmental of 100 mg/kg/day was established from a combined repeated dose/reproduction screening toxicity study with AAI-DETA.
Repeated dose studies, including a 90-day study on AAI-DETA, do not indicate any concerns for reproductive tissues (estrous cycle length and histopathological examination of the male and female reproductive organs, including spermatogenesis staging). In combination with low exposures no further reproduction studies are considered necessary.

Justification for selection of Effect on fertility via oral route:
Most relevant study available.

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 (8.4x10-8 Pa at 25°C) and use applications that do not involve the forming of aerosols, particles or droplets of an inhalable size. The potential for inhalation is not significant to justify this study

Justification for selection of Effect on fertility via dermal route:
The substance is irritating to the skin and is not expected to easily pass the skin. The skin is therefore not a preferred route when studying repeated dose systemic toxicity.

Effects on developmental toxicity

Description of key information

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. Also no developmental toxicity was observed in a OECD 414 study on a similar substance.
No developmental toxicity was observed in an OECD 422 screening study with AAI-DETA or other substances from the AAI category.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

18 November - 19 December 2013

Reliability:

2 (reliable with restrictions)

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.

Remarks:

Doses / Concentrations:
0, 15, 50, 150 mg/kg bw/day
Basis:
actual ingested

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.

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

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: developmental 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.

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:

The available data available within the group of Amidoamines/imidazolines (AAI) substances indicate that for AAI substances based on shorter polyethyleneamines (EA), higher toxicity is observed compared to AAI based on longer EA. The forming of imidazoline itself does not seem to play a significant role. For cross-reading purposes between substances of AAI, Fatty acid reaction product with diethylene-triamine (AAI-DETA) therefore represents the worst case. In series of 28-day and combined repeated dose/reproduction screening toxicity studies (OECD 422) AAI-DETA has shown the highest level of toxicity. (See also document in support of category justification)

Use of the results obtained from studies with AAI-DETA also represent a conservative approach in the evaluation of Fatty acids, C18 unsaturated, reaction product with ammonia-ethanolamine reaction by-products (AAI-AEA). AAI-AEA differs from other substances in the AAI group that are based on fatty acids with polyamines, in that it contains a high amount of hydroxylated polyethylene amines, causing it to display more neutral OH groups, rather than basic primary amine groups. As toxicity is much related to the presence of free primary amine groups, it can be expected that in specifically in aspects of acute toxicity related to direct chemical interactions, this substance shows less effects compared to other AAI substances, and specifically compared to AAI-DETA which represents the worst case in the AAI category

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:

Recent OECD 414 guideline study of high validity. Consistent results from all studies within the whole group of Amidoamine/imidazolines (AAI), indicating a no concerns for developmental toxicity. (See also document in support of category justification).

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

The available data available within the group of Amidoamines/imidazolines (AAI) substances indicate that for AAI substances based on shorter polyethyleneamines (EA), higher toxicity is observed compared to AAI based on longer EA. The forming of imidazoline itself does not seem to play a significant role. For cross-reading purposes between substances of AAI, Fatty acid reaction product with diethylene-triamine (AAI-DETA) therefore represents the worst case. In series of 28-day and combined repeated dose/reproduction screening toxicity studies (OECD 422) AAI-DETA has shown the highest level of toxicity. (See also document in support of category justification)

Use of the results obtained from studies with AAI-DETA also represent a conservative approach in the evaluation of Fatty acids, C18 unsaturated, reaction product with ammonia-ethanolamine reaction by-products (AAI-AEA). AAI-AEA differs from other substances in the AAI group that are based on fatty acids with polyamines, in that it contains a high amount of hydroxylated polyethylene amines, causing it to display more neutral OH groups, rather than basic primary amine groups. As toxicity is much related to the presence of free primary amine groups, it can be expected that in specifically in aspects ofacute toxicity related to direct chemical interactions, this substance shows less effects compared to other AAI substances, and specifically compared to AAI-DETA which represents the worst case in the AAI category.

 

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. All animals surviving to Day 20 post-coitum were subjected to an examination post-mortem and external, thoracic and abdominal macroscopic findings were recorded. 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.

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).

 

No developmental toxicity was observed in an OECD 422 screening study with AAI-DETA. Similar OECD 422 studies have been performed on other substances from the group of AAI: on Fatty acid reaction product with tetraethylene-pentamine (AAI-TEPA) and on Fatty acid reaction product with pentaethylene-hexamine (AAI-PEHA). No indication of concern for reproductive or developmental toxicity was observed in these studies up to the highest dose tested. Also an OECD 414 developmental toxicity in rat on a similar substance showed no concern for developmental effects.

 

Also the low likelihood of exposure can be considered as its use is limited to industrial and professional users where following its irritating properties to the skin and corrosive effects on the eyes will provide for sufficient protection measures to prevent exposure. The likelihood of exposures via inhalation is low considering the high boiling point (> 300 °C) and very low vapour pressure (8.4x10^-8Pa at 25°C) and use applications that do not involve the forming of aerosols, particles or droplets of an inhalable size.

In view of low potential of exposures in combination with an overall low level of toxicity, and a total lack of effects observed in reproductive parameters from developmental toxicity and reproduction screening studies within the group of AAI, and no effects on reproductive organs observed in available repeated dose studies, further developmental toxicity studies in a second species is not indicated.

Justification for selection of Effect on developmental toxicity: via oral route:
Most valid and relevant study available.

Justification for selection of 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 (8.4x10-8 Pa at 25°C). The potential for inhalation is not significant to justify this study.

Justification for selection of Effect on developmental toxicity: via dermal route:
The substance is irritating to the skin and is not expected to easily pass the skin. The skin is therefore not a preferred route when studying repeated dose systemic toxicity.

Justification for classification or non-classification

The database of relevant studies available for the group of Amidoamine/imidazolines (AAI) include various OECD 422 studies and an OECD 414 study, that all show no concerns regarding reproduction or developmental toxicity. Also all already 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.

 

Lactation: no adequate information.