Registration Dossier

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

NOAEL for reproduction/developmental of 300 mg/kg/day was established from a combined repeated dose/reproduction toxicity study with Fatty acid reaction product with tetraethylene-pentamine (FA+TEPA).

Furthermore, there is a 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. In combination with low exposures, possible risks are sufficiently controlled: very low vapour pressure of 0.00017 mPa at 25°C based on FA+DETA which is considered to have the highest vapour pressure within the AAI, use limited to industrial and professional users in applications that do not involve the forming of aerosols, particles or droplets of an inhalable size, and where following its corrosive and sensitising properties will provide for sufficient protection measures to prevent exposure.

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:
27 August 2009 - 14 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:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
other: Crl:WI(Han)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: At start treatment the animals were 12 weeks old instead of 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 accounted 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) were 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): 19.8 – 22.4°C
- Humidity (%): 30 - 81%
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) occurred due to performance of functional observations in the room.

On Day 4 of lactation, the maximum allowed deviation from the dark period of 1 hour was exceeded with a maximum of approximately 6 minutes. These temporary deviations from the light/dark cycle were considered not to have affected the study outcome.

IN-LIFE DATES: From: 27 August 2009 To: 14 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: 6, 20 and 60mg/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: Females were caged together with Main males on a one-to-one-basis in Macrolon cages (MIII type, height 18 cm).
- Length of cohabitation: A maximum of 13 days was allowed for mating. After 13 days of mating, females who had not shown evidence of mating were separated from their males.

A maximum of 13 instead of 14 days was allowed for mating. One additional day of mating would probably not have resulted in successful mating. In addition, sufficient litters were obtained in the dose group.

- 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. In order to confirm the initial result of vaginal smear evaluation, vaginal smears were re-evaluated for all females, including staging of the estrous cycle.
- Further matings after two unsuccessful attempts: no
- 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 (05 October 2009) according to a validated method (NOTOX project 491570). 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 the day prior to start of the recovery period for Recovery males. Females were exposed for at least 41 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: Approximately 14 weeks.
Remarks:
Doses / Concentrations:
0, 30, 100 and 300 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 based on results of a 9-day oral range finding study with Tall oil reaction products with tetraethylene-pentamine by daily gavage in the rat (NOTOX project 491569). See attachment in the result section of this file.
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 (between approximately 1 and 2 hours after dosing from Day 8 pre-mating onwards) 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 17 of treatment (i.e. Day 3 of the Repro period) and on Day 11 of the Recovery period, clinical signs of males were not recorded online. Sufficient clinical observations were conducted for adequate interpretation of the study data.

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)x1000.

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 test.

No functional observations were conducted for one female at 30 mg/kg/day. Sufficient functional observation test results were available for adequate interpretation of the study results.

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):
In order to confirm the initial result of vaginal smear evaluation, vaginal smears were re-evaluated for all females, including staging of the estrous cycle.
Sperm parameters (parental animals):
Parameters examined in all male 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:
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):
Animals were fasted overnight (with a maximum of approximately 23 hours) prior to planned necropsy, but water was provided. Eight Females that appeared visually non-pregnant (based on body weights and palpation) underwent scheduled necropsy without having been fasted. 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 5-7.
Females which failed to deliver :
- Females with evidence of mating: Post-coitum Day 25-26
- Females with no evidence of mating: 21 days after the last day of the mating period.
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.

Most females were necropsied later than after a maximum of 20 hours fasting, i.e. with a maximum of approximately 3 hours. The fasting period was only slightly longer and was considered not to have adversely affected the clinical laboratory, macroscopic or microscopic findings.

Eight Females that appeared visually non-pregnant (based on body weights and palpation) underwent scheduled necropsy without having been fasted. Since no blood was collected from these animals, it was considered that the non-fasting of these animals did not adversely affect data interpretation.

GROSS PATHOLOGY: Yes
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, (Larynx), Thyroid including parathyroid (if detectable), (Lacrimal gland, exorbital), (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:
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.

For three animals no terminal body weight was determined. Sufficient terminal body weight data were available for evaluation.

HISTOTECHNOLOGY: Yes
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.
- The additional slides of the testes of the selected 5 Main males of Groups 1 and 4 to examine staging of spermatogenesis.
- All gross lesions of all animals (all dose groups).
- The reproductive organs* of all animals that failed to mate, conceive, sire or deliver healthy pups:
Group 1: Two males and two females (failed to conceive)
Group 2: Five males and five females (failed to conceive)
Group 3: One male and one female (failed to conceive), one male and one female (failed to mate)
Group 4: One male and one female (failed to conceive), one male and one female (failed to mate)

* Reproductive organs included cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles, testis, uterus, and vagina.

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

The nodule on the epididymides of one animal (Group 1) was not available for histopathology (not found at trimming). Sufficient data was available for evaluation.
Postmortem examinations (offspring):
SACRIFICE
Pups were killed by decapitation on Day 5-7 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. If possible, defects or cause of death were evaluated.

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 mating = Number of females mated/Number of females paired x 100

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

Conception rate = 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 lactation = Number of dead pups on Day 4 lactation/Number of live pups at First Litter Check x 100

Viability index = Number of live pups on Day 4 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:
no effects observed
Other effects:
not examined
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No clinical signs of toxicity were noted during the observation period.

Incidental findings that were noted included alopecia, salivation, rales and scabs. 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 significance.

No clinical signs were noted among control males and females at 100 mg/kg/day.

No mortality occurred during the study period.

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

Slightly lower mean body weight and body weight gain was noted for males at 300 mg/kg/day throughout the study period (statistically significant on most occasions), but means remained within the range considered normal for rats of this age and strain. Body weight (gain) remained slightly lower during the recovery period but showed a trend towards normalizing to control levels. Therefore, these changes were considered not to be of toxicological relevance.

A minor statistically significant lower body weight gain was also recorded for males at 100 mg/kg/day at the end of the Repro (i.e. mating) period, but since this change was also slight (within normal ranges) and because body weight (gain) at 300 mg/kg/day essentially recovered to control levels, this was also considered not to be a toxicologically relevant change.

Mean body weight and body weight gain for both sexes at 30 mg/kg/day were considered to have been unaffected by treatment.

No toxicologically significant changes in food consumption before and after allowance for body weight occurred.

Slightly lower (statistically significant) food consumption before and/or after allowance for body weight was recorded for males and females at 300 mg/kg/day during the first week of the premating period. However, food intake was similar to control levels during the remainder of the observation period. The lower relative food consumption of males at 100 mg/kg/day over Days 8-15 of the premating period and (relative) food intake of females at 300 mg/kg/day (both statistically significant) during lactation occurred in the absence of a dose-related trend and was very slight in nature. Therefore, no toxicological relevance was ascribed to these changes.

REPRODUCTIVE DATA (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 considered to be unaffected by treatment.

The mean number of implantation sites at 300 mg/kg/day appeared slightly lower than controls without achieving a level of statistical significance. This apparent change occurred in the absence of a clear group response, and was due to a few lower values from individual females. Moreover, the mean number of live pups on Day 1 of lactation was within normal ranges, and there were no treatment-related histopathological correlates. Therefore, this finding was considered to be of no toxicological relevance.

A relatively high number of non-pregnant females was observed in the study. These females were initially confirmed as being pregnant based on presence of sperm cells in the vaginal smears. Based on body weight development/palpation during post-coitum several of these females were considered to be non-pregnant. As such, the selection of females for parameters such as clinical biochemistry and histopathology was adjusted to obtain 5 females with live pups per group. One other non-selected female was determined to be non-pregnant at a later stage. Re-evaluation of the vaginal smears of all females, including estrous cycle staging, confirmed that that these females were non-pregnant since these animals were not in the estrus phase and hence were unable to become pregnant. For two of the females, the presence of sperm cells was confirmed and no estrous stage could be determined based on the high number of sperm cells present on the smear.

The distribution of non-pregnant females did not show a dose-response relationship, nor were any supportive treatment-related histopathological lesions noted. Moreover, Groups 1, 3 and 4 contained 8 pregnant females that delivered live pups (i.e. the minimal number of pregnant females required per group as specified in the OECD 422 guideline), and at least 5 females with live pups could be selected per dose group for parameters including clinical biochemistry and histopathology. The lower number of pregnant females in Group 2 (5 out of 10) did not adversely affect the interpretation of the study results, taking into account the absence of any evidence of reproductive toxicity up to the highest dose. Overall, it was concluded that an adequate evaluation of and conclusion on the study results was possible.

DEVELOPMENTAL DATA
No toxicologically relevant effects on number of females with live pups, gestation index, duration of gestation and early postnatal pup development (body weight, clinical signs, viability index and external macroscopy) were noted.

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

ORGAN WEIGHTS (PARENTAL ANIMALS)
A statistically significant lower heart weight and heart to body weight ratio was measured for males and females at 300 mg/kg/day (not statistically significant for heart to body weight ratio of males and within range considered normal for rats of this age and strain). Seminal vesicle weight and seminal vesicle to body weight ratio was lower with statistical significance at 300 mg/kg/day, but also remained within range considered normal for rats of this age and strain. Prostate weight at 300 mg/kg/day also appeared lower when compared to controls, but this occurred without statistical significance. These changes had resolved at the end of the recovery phase.

The lower liver weight of males at 100 and 300 mg/kg/day (statistically significant at 300 mg/kg/day) was considered to be related to the slightly lower terminal body weights since liver to body weight ratios were similar to control levels.

GROSS PATHOLOGY (PARENTAL ANIMALS)
Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment.

Incidental findings included a yellowish focus or nodule on the epididymides, reduced size of the testes, epididymides, preputial glands and/or thymus, greenish nodules on the clitoral glands, red foci on the thymus or lungs, scab formation, alopecia, exophthalmus, pelvic dilation, a black-brown focus on the preputial glands, enlargement of the iliac lymph node or clitoral glands and fluid in the uterus. 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.

HISTOPATHOLOGY (PARENTAL ANIMALS)
No toxicologically significant histopathological abnormalities were noted.

No treatment-related histopathological findings were noted among the 22 animals which failed to mate or conceive, nor did the incidence of these cases show a dose-related trend. Except for one male at 300 mg/kg/day, which had a bilateral severe degree of seminiferous tubular atrophy in the testes with resultant very severe oligospermia in the epididymides, there were no microscopic findings in any of the other animals suspected of infertility which could explain their lack of reproductive performance.

The remaining recorded microscopic findings were within the range of background pathology encountered in Wistar rats of this age in this type of study and occurred at similar incidences and severity in both control and treated rats. The spermatogenic staging profiles were normal for all group 1 and group 4 males evaluated.
Dose descriptor:
NOAEL
Effect level:
>= 300 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No toxological signs up to 300 mg/kg bw/day.
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:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
DEVELOPMENTAL DATA:
No toxicologically relevant effects on early postnatal pup development (body weight, clinical signs, viability index and external macroscopy) were noted.

One pup of female no. 85 (300 mg/kg/day) was found dead on Day 1 of lactation. Necropsy showed absence of milk in the stomach. Incidental clinical symptoms or macroscopic findings of surviving pups consisted of small size, pale appearance and a missing tail. No relationship with treatment was established for these observations and they were considered to be of no toxicological significance.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 300 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No toxological signs up to 300 mg/kg bw/day.
Reproductive effects observed:
not specified
Conclusions:
Based on these results, a parental, reproductive and developmental No Observed Adverse Effect Level (NOAEL) of 300 mg/kg/day was determined.
Executive summary:

Tall oil reaction products with tetraethylene-pentamine (Amidoamine/Imidazoline) was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 30, 100 and 300 mg/kg/day. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for at least 28 days). The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 41-48 days).

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 at the end of treatment were generally slight in nature and had normalized at the end of the recovery period. These changes consisted of higher alanine and aspartate aminotransferase activity in both sexes at 300 mg/kg/day, higher aspartate aminotransferase activity in males at 100 mg/kg/day, higher inorganic phosphate level in males at 300 mg/kg/day, and higher chloride level in females at 300 mg/kg/day. No macroscopic or histopathological lesions were observed that would support these variations. Therefore, these variations in clinical biochemistry parameters were considered to be of no toxicological relevance.

The lower (relative) heart weight in both sexes at 300 mg/kg/day, and lower (relative) seminal vesicle and prostate weight at 300 mg/kg/day generally remained within the range considered normal for rats of this age and strain. Moreover, these changes had resolved at the end of the recovery phase and no histopathological correlates were found. Also, the lower seminal vesicle weight was not reflective of reproductive toxicity. Therefore, these organ weight changes were considered to be of no toxicological relevance.

No treatment-related changes were noted in any of the remaining parental parameters investigated in this study (i.e. clinical appearance, functional observations, haematology, macroscopic and microscopic examination).

Reproductive/Developmental results:

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

Based on these results, a parental, reproductive and developmental No Observed Adverse Effect Level (NOAEL) of 300 mg/kg/day was determined.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
300 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 a 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 for the group of Amidoamines/imidazolines (AAI) substances indicate that cross-reading between substances in this group is justified. The data shows that for AAI substances based on shorter polyethyleneamines (EA), relatively higher toxicity is observed compared to AAI based on longer EA. The forming of imidazoline itself does not seem to play a significant role. Therefore, Fatty acid reaction product with diethylene-triamine (FA+DETA) represents worst case for the group of Amidoamines/imidazolines (AAI). (See also document in support of category justification).

 

No reproductive/developmental toxicity was observed at any of the dose levels in an OECD 422 study with Fatty acid reaction product with tetraethylene-pentamine (FA+TEPA), and thus a reproduction/developmental NOAEL of 300 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 diethylene-triamine (FA+DETA) and on Fatty acid reaction product with pentaethylene-hexamine (FA+PEHA). No indication of concern for reproductive or developmental toxicity was observed in these studies up to the highest dose tested. 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 FA+EDTA showed 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. (See also document in support of category justification).

In combination with low exposures, possible risks are sufficiently controlled: FA+TEPA has a very low vapour pressure of 0.00017 mPa at 25°C based on FA+DETA which is considered to have the highest vapour pressure within the AAI, use limited to industrial and professional users in applications that do not involve the forming of aerosols, particles or droplets of an inhalable size, and where following its corrosive and sensitising properties will provide for sufficient protection measures to prevent exposure.

Effects on developmental toxicity

Description of key information

No developmental toxicity was observed in an OECD 422 screening study with Fatty acid reaction product with tetraethylene-pentamine (AAI-TEPA); No developmental toxicity was observed in a developmental toxicity study (OECD 414) study on FA+DETA used for read-across.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
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.
Justification for type of information:
Also see category read across justification document attached to chapter 13
REPORTING FORMAT FOR THE ANALOGUE APPROACH
(Tall oil diethylenetriamine imidazoline, CAS no. 68442-97-7 has been registered as Fatty acids, C18 unsat, reaction products with diethylenetriamine, CAS no.: 1226892-43-8. Both CAS numbers thus describe the same substance.)
Read across is done from (source) (Tall oil reaction products with diethylenetriamine, CAS 1226892-43-8 (FA+DETA) to (target) Fatty acids C18 unsat, reaction products with tetraethylenepentamine, CAS 1226892-45-0 (FA+TEPA)

These amidoamine/imidazolines are made from fatty acid and polyethyleneamines. The manufacturing process is a one-step process with formation of amide and imidazoline structures. To promote imidazoline formation from the amide, the reaction mixture is heated to temperatures above 180ºC. The resulting product therefore is a mixture of the amide structure of the fatty acid and the polyethyleneamine and its imidazoline.
Both substances FA+DETA and FA+TEPA are manufactured using the same fatty acid source. The difference between the two is only related to the length of the polyethyleneamines: diethylene-triamine versus tetraethylene-pentamine.
As both have the similar chemical structure and contain the same functional groups (only with a difference in number of ethyleneamines), they are expected to show similar chemical reactivity and have very comparable physicochemical properties. Consequently, they share the same mechanism of action. This is applicable to all constituents of these UVCBs.

The available data show that substances of the AAI group are of low toxicity, and an increase in the duration from 28 days to 90-days does not results to increased toxicity. The only difference between FA+DETA and the other AAI with higher polyethyleneamine groups, is the occurrence of foamy macrophages in mesenteric lymph nodes which is only observed in studies with FA+DETA, but not with the higher polyethyleneamines. However, this is shown to have no impact on fetal development.
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
other: Wistar (Han)
Details on test animals 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:
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

No developmental toxicity was observed in an OECD 422 screening study with Fatty acid reaction product with tetraethylene-pentamine (FA+TEPA).

Similar OECD 422 studies have been performed on other substances from the group of AAI: on Fatty acid reaction product with diethylene-triamine (FA+DETA) and on Fatty acid reaction product with pentaethylene-hexamine (FA+PEHA). No indication of concern for reproductive or developmental toxicity was observed in these studies up to the highest dose tested.

 

The available data available for the group of Amidoamines/imidazolines (AAI) substances indicate that cross-reading between substances in this group is justified. (See document in support of category justification). The data shows that for AAI substances based on shorter polyethyleneamines (EA), relatively higher toxicity is observed compared to AAI based on longer EA. The forming of imidazoline itself does not seem to play a significant role.

To strengthen the data for the group of AAI, a full prenatal developmental toxicity study in rats according to OECD 414 is therefore performed on FA+DETA as in the group of AAI this substance is expected have the highest potential for toxicity.

 

Fatty acids, C18 unsat, reaction products with diethylenetriamine (FA+DETA) 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.

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 analysed 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).

 

 

The low likelihood of exposure follows from its use limited to industrial and professional users where following its corrosive and sensitising properties 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 (0.00017 mPa at 25°C, based on FA+DETA which is considered to have the highest vapour pressure within the AAI) 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 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.