Fatty acids, tall-oil, compds. with N-[3-(dimethylamino)propyl]tall-oil amides

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Feb to May 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

2018

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, tall-oil, compds. with N-[3-(dimethylamino)propyl]tall-oil amides

EC Number:

295-714-4

EC Name:

Fatty acids, tall-oil, compds. with N-[3-(dimethylamino)propyl]tall-oil amides

Cas Number:

92128-22-8

Molecular formula:

Not applicable (UVCB substance)

IUPAC Name:

Reaction products of tall-oil fatty acids and (3-aminopropyl)dimethylamine

Test material form:

liquid

Test animals

Species:

rat

Strain:

Wistar

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: 6-8 weeks
- Animal weights at study initiation: males: 177-223 g; females: 138-166 g
- Housing: in groups of up to 5 animals
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 11 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-21
- Humidity (%): 47-50
- Air changes (per hr): >= 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on oral exposure:

administration volume: 5 mL/kg bw (lowered to 2 mL/kg bw from Day 20 onwards)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

All concentrations and homogeneity analyzed in the formulations of Groups 2-4 were in agreement with target concentrations (i.e., mean accuracies between 90% and 110%), except for the formulation of Group 4 prepared for use in Week 1. Since the mean accuracy for the formulation of Group 4 prepared for use in Week 1 was slightly below the target concentration (i.e., 89% of target), the results were accepted.

In the Group 1 formulations, no test item was detected.

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

Duration of treatment / exposure:

at least 90 days

Frequency of treatment:

once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Group 1

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Remarks:

Group 2

Doses / concentrations 3

Dose / conc.:

200 mg/kg bw/day (actual dose received)

Remarks:

Group 3

Doses / concentrations 4

Dose / conc.:

800 mg/kg bw/day (actual dose received)

Remarks:

Group 4
from Day 18, this dose level was decreased to 400 mg/kg bw/day for the males

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

DOSE SELECTION RATIONALE:
The dose levels were selected based on the results of a 28-day repeated dose toxicity study with oral exposure to the test item in rats (Eiben, 2010), and in an attempt to produce graded responses to the test item. In the 28-day oral toxicity study only slight to moderate liver findings in males (reduced lipid content) and females (cytoplasmic changes of hepatocytes) at 800 mg/kg bodyweight (bw) were observed. The derived NOAEL was 200 mg/kg bw for both sexes. The high-dose level should produce some toxic effects, but not excessive lethality that would prevent meaningful evaluation. The mid-dose level is expected to produce minimal to moderate toxic effects. The low-dose level should produce no observable indications of toxicity. Thus, the doses of 0, 50, 200 and 800 mg/kg bw were chosen for the subchronic rat study.

Positive control:

none

Examinations

Observations and examinations performed and frequency:

MORTALITY / MORIBUNDITY CHECKS:
Throughout the study, animals were observed for general health/mortality and moribundity twice daily. Animals were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings. Animals showing pain, distress or discomfort which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ 2000/7). The circumstances of any death were recorded in detail.

CLINICAL OBSERVATIONS:
- Detailed Clinical Observations: The animals were removed from the cage, and a detailed clinical observation was performed weekly, beginning on Day 1, and on the day of necropsy. An unscheduled, additional Detailed Clinical Observation was performed for all animals on Day 3.
- Cage Side Clinical Observations: Cage side observations were performed once daily, beginning on Day 1 and lasting throughout the dosing period. These observations were performed 0 to 1 hours after dosing. Animals were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings. The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. In the data tables, the scored grades were reported, as well as the percentage of animals affected in summary tables.
- Arena Observations: Clinical observations were conducted in a standard arena beginning before the first administration of the test item and then once weekly throughout treatment period. These observations were conducted after dosing.

BODY WEIGHTS:
Animals were weighed individually weekly, starting on Day 1 (at predose). A fasted weight was recorded on the day of necropsy. In order to monitor the health status, the following animals were weighed additionally: all animals on Day 11, all Group 4 animals on Day 12, animal No. 43 (Control Group) on Day 12, animal No. 21 (Group 3) on Days 12 and 82, animal No. 24 (Group 3) on Day 12, animal No. 64 (Group 3) on Day 59, animal Nos. 67 and 69 (Group 3) on Day 12, animal No. 32 (Group 4) on Days 45 and 89, animal No. 33 (Group 4) on Day 34, animal No. 35 (Group 4) on Day 45.

FOOD CONSUMPTION:
Food consumption was quantitatively measured starting on Day 1 and continuing weekly throughout the dosing period. For Group 4 animals, an extra food consumption was measured on Day 17 to make sure food was available ad libitum.

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

OPHTHALMIC EXAMINATIONS:
The eyes were examined using an ophthalmoscope after application of a mydriatic agent (Tropicol 5 mg/ml solution, THEA Pharma, Wetteren, Belgium) during pretreatment in all animals, and in Week 13 in all Group 1, 3 and 4 (males only) animals.

FUNCTIONAL TESTS:
Functional tests were performed on the first 5 animals per sex per group during Week 12-13. These tests were performed after dosing and after completion of clinical observations. The following tests were performed (abbreviations mentioned in the respective tables are indicated between brackets):
- Hearing ability (HEARING) (Score 0 = normal/present, score 1 = abnormal/absent).
- Pupillary reflex (PUPIL L/R) (Score 0 = normal/present, score 1 = abnormal/absent).
- Static righting reflex (STATIC R) (Score 0 = normal/present, score 1 = abnormal/absent).
- Fore- and hind-limb grip strength, recorded as the mean of three measurements per animal (Series M4-10, Mark-10 Corporation, J.J. Bos, Gouda, The Netherlands).
- Locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system, Kinder Scientific LLC, Poway, USA). Total movements and ambulations were reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including
ambulations but also smaller or finer movements like grooming, weaving or movements of the head.

ESTROUS STAGE DETERMINATION:
On the day of necropsy a vaginal lavage was performed to determine the stage of the estrus for all females, except for the animals that were euthanized for humane reasons or died spontaneously. Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage. These data were only used for evaluation of extreme thyroid hormone effects in females and are not included in the results section.

HEMATOLOGY:
Blood was collected between 7.00 and 10.30 a.m. from the retro-orbital sinus under anesthesia using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) of fasted animals.
The following hematological parameters were determined on all animals per group near termination:
White blood cells (WBC), neutrophils, lymphocytes, monocytes, eosinophils, basophils, red blood cells, reticulocytes, red blood cell distributation width (RDW), mean corpuscular hemoglobin MCH), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular volume (MCV), hemoglobin, hematocrit, platelets, prothrombin time (PT), activated partial thromboplastin time (APTT).

CLINICAL CHEMISTRY:
The following parameters were determined on all animals per group near termination:
Alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT), alkaline phosphatase (ALP), total protein, albumin, total bilirubin, total cholesterol, HDL cholesterol, LDL cholesterol, creatinine, urea, glucose, potassium, sodium, chloride, calcium, inorganic phosphate, triiodothyronine (T3), thyroxine (T4), thyroid stimulating hormone (TSH).

Sacrifice and pathology:

NECROPSY:
- Scheduled Deaths: All animals were subjected to a complete necropsy examination, which included evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and
external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues.
- Unscheduled Deaths: A necropsy was conducted for animals that died on study, and specified tissues were saved. If necessary for humane reasons, study animals were euthanized as per Test Facility SOPs. These animals underwent necropsy, and specified tissues were retained. On Day 16, all Group 4 females were euthanized for ethical reasons. At necropsy, animals were subjected to a full post mortem examination and specified tissues were saved, but not weighed.

ORGAN WEIGHTS:
The following organs were weighed at necropsy for all scheduled euthanasia animals:
Brain, cervix, heart, liver, spleen, kidneys, thymus, adrenal glands, prostate, seminal vesicles, thyroid, epididymides, testes, ovaries and uterus.
Organ weights were not recorded for animals found dead or euthanized in poor condition or in extremis. Paired organs were weighed together. In the event of gross abnormalities, in addition to the combined weight, the weight of the aberrant organ was taken and recorded in the raw data. Organ weight as a percentage of body weight (using the terminal body weight) was calculated

HISTOPATHOLOGY:
The following organs/tissues were collected and fixed in 10 % neutral buffered formalin:
Animal identification, adrenals, aorta, femur, bone marrow, sternum, brain, cervix, epididymis, esophagus, eye, clitoral gland, harderian gland, lacrimal gland, mammary gland, parathyroid gland, pituitary gland, preputial gland, prostate, salivary gland, seminal vesicle gland, thyroid gland, gross lesions/masses, gut-associated lymphoid tissue, heart, kidney, large intestine (cecum, colon, rectum), larynx, liver, lung, nasopharynx, mandibular lymph nodes, mesenteric lymph nodes, skeletal muscle, optic nerve, sciatic nerve, ovary, pancreas, skin, small intestine (duodenum, ileum, jejunum), spinal cord, spleen, stomach, testis, thymus, tongue, trachea, urinary bladder, uterus, vagina.
Tissues identified above (except animal identification, nasopharynx, femur, bone marrow, clitoral gland, lacrimal gland, preputial gland and skeletal muscle) were embedded in paraffin (Klinipath, Duiven, The Netherlands), sectioned, mounted on glass slides, and stained with hematoxylin and eosin (Klinipath, Duiven, The Netherlands).
Histopathologic examination was performed on organs and tissues from all Group 1 and 4 animals, all Group 3 females, as well as the lung and mesenteric lymph node of Group 2 and 3 males, and thymus, ovaries, uterus, cervix and vagina from all Group 2 females, and all organs with macroscopic findings from all rats. The nasopharynx was examined from all found dead and preterminally euthanized Group 3 and 4 animals.

Other examinations:

none

Statistics:

STATISTICS FOR DATA COLLECTED IN PROVANTIS
- Parametric/Non-Parametric Levene’s test was used to assess the homogeneity of group variances. The groups were compared using an overall one-way ANOVA F-test if Levene’s test was not significant or the Kruskal-Wallis test if it was significant. If the overall F-test or Kruskal-Wallis test was found to be significant, then pairwise comparisons were conducted using Dunnett’s or Dunn’s test, respectively.
- ANCOVA: The data corresponding to a response variable of interest and to a related covariate were submitted to an analysis of covariance (ANCOVA), including only groups with at least three non-missing paired values and if found to be significant, then pairwise comparisons were conducted using Dunnett’s test.
- Incidence: A Fisher’s exact test was used to conduct pairwise group comparisons of interest.

STATISTICS FOR DATA COLLECTED / PROCESSED IN TOXDATA
- Parametric: Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test). For the motor activity data set (at least 3 groups) parametric (ANOVA) tests on group means were applied with Bonferroni correction for multiple testing. Mixed modelling techniques, comparing six different covariance structures, were used in order to select the best fitting statistical model.
- Non-Parametric: Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test).
- Incidence: An overall Fisher’s exact test was used to compare all groups. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test was significant.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Hunched posture and erected fur were observed in males and females at 50, 200 and 800/400 mg/kg/day between Days 3 and 92, and the incidence increased dose dependently.

In some female control animals, hunched posture, erected fur, abnormal respiratory rate and/or labored breathing were observed between Day 2 and 7. These signs resolved after Day 7 in all control animals.

Salivation seen after dosing among all animals was not considered toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response related to taste of the test item and/or vehicle rather than a sign of systemic toxicity.

Breathing with abnormal sounds and decreased activity observed among all animals were not considered toxicologically relevant, taking into account that it was observed at the same severity and incidence amongst all groups. This sign was considered to be a physiological response related to the formulation rather than a sign of systemic toxicity.

No findings were noted during the arena observations in this study.

Any other clinical signs noted 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 and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.

Mortality:

mortality observed, treatment-related

Description (incidence):

There were 21 preterminal deaths during the course of the study. Twelve occurred at 800 mg/kg/day, three at 800/400 mg/kg/day, four at 200 mg/kg/day and two at 50 mg/kg/day. For most preterminal animals, the combination of clinical observations (labored breathing or abnormal breathing sounds and/or distended abdomen), macroscopic findings (distention of stomach/intestines with gas), and histologic findings in the caudal nasal cavity/nasopharynx (occasionally lung and trachea) were consistent with gastro-esophageal reflux and aspiration as the cause of morbidity/mortality (Damsch S et al. (2011), Toxicol Pathol 39: 337-347 + 348-360).

At 800 mg/kg/day, two males and four females were send to necropsy between Days 7 and 15. In these animals, labored breathing, breathing with abnormal sounds, irregular or decreased respiratory rate, hunched posture, erected fur, thin appearance, decreased activity, abdominal distension and/or yellow staining around the anus were observed. Lower body weight gain and food consumption were also observed. Histologic findings of the nasopharynx included accumulation of material in the lumen (acellular, eosinophilic material, variable admixed with inflammatory cells), exudates with neutrophils/pyknotic nuclear debris, remodelling and fusion of the nasal turbinates, chronic or acute inflammation, and/or ulceration. For these preterminal animals, the combination of clinical observations (laboured breathing or abnormal breathing sounds and/or distended abdomen), macroscopic findings (distention of stomach/intestines with gas), and histologic findings in the caudal nasal cavity/nasopharynx (occasionally lung and trachea) were consistent with accidental aspiration of dose formulation, as a result of gastro-esophageal reflux after gavage dosing, and this was considered the cause of morbidity/mortality. Because of the high number of preterminal females at 800 mg/kg/day and the severity of the clinical signs observed in the majority of the remaining females, all females at 800 mg/kg/day were send to necropsy on Day 16. Three of these females, send to necropsy on Day 16, did not show any clinical signs, macroscopic findings, or microscopic findings.

Dose levels of Group 4 males were decreased from 800 to 400 mg/kg/day starting on Day 18, after a dosing holiday from Days 15 until 17. Dose volumes were lowered to 2 mL/kg from Day 20 onwards for all groups to try to prevent reflux reactions. After dose level was decreased to 400 mg/kg/day, two additional males were prematurely sacrificed on Days 34and 89 (Animal Nos. 33, 32, respectively) and one found dead on Day 89 (Animal Nos.36). In these animals labored breathing, abnormal breathing sounds, hunched posture, erected fur, thin appearance, decreased activity and/or abdominal distension were observed. Histologic findings of these animals included accumulation of material in the lumen (acellular, eosinophilic material, variable admixed with inflammatory cells), exudates with neutrophils/pyknotic nuclear debris, remodelling and fusion of the nasal turbinates, chronic or acute inflammation, and/or ulceration consistent with gastro-esophageal reflux and aspiration as the cause of morbidity/mortality.

At 200 mg/kg/day, two males and two females were preterminally sacrificed on Days 12, 22, 59 and 82 (Animal Nos. 69, 24, 64 and 21, respectively). In the preterminal animals, increased respiratory rate, labored breathing, breathing with abnormal sounds, hunched posture, irregular or decreased respiratory rate, decreased activity, erected fur, abdominal distension or yellow fur around the anus and/or thin appearance were observed on the day of sacrifice. Histologic findings of the nasopharynx of these animals included accumulation of material in the lumen (acellular, eosinophilic material, variable admixed with inflammatory cells), exudates with neutrophils/pyknotic nuclear debris, remodelling and fusion of the nasal turbinates, chronic or acute inflammation, and/or ulceration consistent with gastro-esophageal reflux and aspiration as the cause of morbidity/mortality.

At 50 mg/kg/day, two females were send to necropsy on Days 9 and 22 (Animal Nos. 59 and 54, respectively). Animal No. 54 showed slight body weight loss (-1%) before sacrifice on Day 22. In this animal, pleural and epicardial inflammation with plant material (correlated to adhesion of lungs the right lung lobes) and gelatinous white fluid in the thoracic cavity was consistent with gavage trauma as the cause of death. Animal No. 59 showed the combination of clinical observations (labored breathing or abnormal breathing sounds and/or distended abdomen), macroscopic findings (distention of stomach/intestines with gas), and histologic findings in the caudal nasal cavity/nasopharynx consistent with gastro-esophageal reflux and aspiration as the cause of morbidity.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weights and body weight gains of treated males at 50 mg/kg/day remained in the same range as controls over the study period.

A test item-related lower body weight was observed at 200 and 800/400 mg/kg/day, in males (with a maximum difference of -6.1 and -15.6%, respectively, of controls) and females (with a maximum difference of -5.4 and -13.7 %, respectively, of controls) from Day 8 onwards. Body weight gain was lower compared to controls in females at 50 mg/kg/day, and in males and females at 200 mg/kg/day and 800/400 mg/kg/day on several intervals.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption of treated animals at 50 mg/kg/day remained in the same range as controls over the study period.

At 200 and 800/400 mg/kg/day, a lower food consumption compared to control animals was observed in both males (with a maximum difference of -14.7% and -22.5% of controls, receptively) and females (with a maximum difference of -16.1% and -21.5% of controls, receptively).

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no relevant ophthalmology findings at pretreatment and in week 13.

The nature and incidence of ophthalmology findings noted during the Pretreatment Period and in Week 13 were similar among the groups, and occurred within the range considered normal for rats of this age and strain. These findings were therefore considered to be unrelated to treatment with the test item.

Haematological findings:

no effects observed

Description (incidence and severity):

No toxicologically relevant changes were noted in hematological parameters.

While few changes were statistically significant, the alterations in hematological parameters were unrelated to administration of the test item due to the minimal magnitude of the change, variation in direction of change and/or absence of a dose response.

No toxicologically relevant changes were noted in coagulation parameters.

While few changes were statistically significant, the alterations in coagulation parameters were unrelated to administration of the test item due to variation in direction of change and/or absence of a dose response.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant changes were noted in clinical chemistry parameters of the males at 50, 200 and 800/400 mg/kg/day and females at 50 mg/kg/day.

HDL and total cholesterol was lower in females (76% and 72% of controls) at 200 mg/kg/day, but was considered not toxicology relevant at this incidence and severity.

Any other statistically significant changes in clinical biochemistry parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.

No test item-related changes in thyroid hormones were noted in males at 50, 200 and 800/400 mg/kg/day and in females at 50 mg/kg/day.

Serum levels of thyroid stimulating hormone (TSH) were decreased in females (40% of controls) at 200 mg/kg/day, but occurred within the range considered normal for rats of this age and strain. These findings were therefore considered to be unrelated to treatment with the test item.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength was similar between control and high dose animals.

Motor activity (total movements and ambulations) was lower in the females at 200 mg/kg/day compared to control. In all other groups motor activity was comparable with controls. Furthermore, all groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no test item-related alterations in organ weights in terminally euthanized animals (males up to 800/400 mg/kg/day and females up to 200 mg/kg/day).

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item-related gross observations in terminally euthanized animals (males up to 800/400 mg/kg/day and females up to 200 mg/kg/day).

All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings after treatment with Fatty acids, tall-oil, compds. with N-[3-(dimethylamino)propyl]tall-oil amides were noted in the mesenteric lymph node of males (at 200 and 800/400 mg/kg/day), and in the thymus of females (at 200 mg/kg/day) and are summarized in Text Table 14.

In males, the incidence and severity of macrophage aggregates in the mesenteric lymph nodes at 800/400 mg/kg/day and 200 my/kg/day were slightly higher than the control males. In females, the incidence of this findings was comparable between the controls and the highest surviving dose group (200 mg/kg/day). This finding was not accompanied by any degenerative changes and was interpreted as non-adverse.

In females, there was a low incidence of minimal decreased lymphoid cellularity in the thymus (2/8) at 200 mg/kg/day, and a low incidence of minimal lymphoid single cell necrosis at 50 and 200 mg/kg/day (2/10 and 3/8, respectively). These minimal histologic changes, without significant thymus weight differences were interpreted as non-adverse.

Microscopic findings in the lungs were of low incidence and without a dose relationship. These included accumulation of pale basophilic material (variably mixed with macrophages) in the lumen of the bronchioles. Associated lung fields were sometimes collapsed/underinflated (recorded as atelectasis). While the identity of the material is uncertain, it may be the test item which has been aspirated.

There were no other test item-related histologic changes. Remaining histologic changes were considered to be incidental findings or within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

400 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: the combination of mortality, clinical signs, reduced food consumption, decreased body weights, macroscopic and histopathological findings at 800 mg/kg bw/day

Remarks on result:

other: the high dose level of males was decreased from 800 to 400 mg/kg bw/day starting on Day 18 due to severe effects consistent with accidental aspiration of dose formulation, as a result of gastro-esophageal reflux after gavage dosing

2

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: the combination of mortality, clinical signs, reduced food consumption, decreased body weights, macroscopic and histopathological findings at 800 mg/kg bw/day

Remarks on result:

other: all females of high dose group 800 mg/kg bw/day were preterminally necropsied on Day 16 due to severe effects consistent with accidental aspiration of dose formulation, as a result of gastro-esophageal reflux after gavage dosing

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

For further information on the results presented please see " Appendix 2 - Figures and Summary Tables" attached as background material in section "Overall remarks, attachments".

Applicant's summary and conclusion

Executive summary:

The test substance was administered in arachis oil orally by gavage to 10 male and 10 female Wistar rats per dose group in daily doses of 0, 50, 200 and 800 mg/kg bw according to OECD TG 408. Animals of the dose groups 0, 50 and 200 mg/kg bw were treated for at least 90 days. Based on mortality and clinical signs at 800 mg/kg bw, all females of this dose group were necropsied on Day 16 and the high dose level for males was decreased from 800 to 400 mg/kg bw starting at Day 18 after a dosing holiday between Days 15 and 17.

There were 21 preterminal deaths during the course of the study. Fifteen occurred at 800/400 mg/kg/day, four at 200 mg/kg/day and two at 50 mg/kg/day. At 800 mg/kg/day, two males and four females were send to necropsy between Days 7 and 15. In these animals, labored breathing, breathing with abnormal sounds, irregular or decreased respiratory rate, hunched posture, erected fur, thin appearance, decreased activity, abdominal distension and/or yellow staining around the anus were observed. Lower body weight gain and food consumption were also observed. Histologic findings of the nasopharynx included accumulation of material in the lumen (acellular, eosinophilic material, variable admixed with inflammatory cells), exudates with neutrophils/pyknotic nuclear debris, remodelling and fusion of the nasal turbinates, chronic or acute inflammation, and/or ulceration. For these preterminal animals, the combination of clinical observations (laboured breathing or abnormal breathing sounds and/or distended abdomen), macroscopic findings (distention of stomach/intestines with gas), and histologic findings in the caudal nasal cavity/nasopharynx (occasionally lung and trachea) were consistent with accidental aspiration of dose formulation, as a result of gastro-esophageal reflux after gavage dosing, and this was considered the cause of morbidity/mortality. Because of the high number of preterminal females at 800 mg/kg/day and the severity of the clinical signs observed in the majority of the remaining females, all females at 800 mg/kg/day were send to necropsy on Day 16. Three of these females, send to necropsy on Day 16, did not show any clinical signs, macroscopic findings, or microscopic findings. Hereafter, based on the early deaths, it was decided for the males to discontinue dosing with 800 mg/kg/day and continue with 400 mg/kg/day on Day 18, after a dosing holiday from Day 15 until Day 17.  Dose volumes were lowered to 2 ml/kg from Day 20 onwards for all groups to prevent reflux reactions.

 

After the dose level of Group 4 males was decreased to 400 mg/kg/day, three additional males were send to necropsy or found dead between Days 34 and 89. In these animals labored breathing, abnormal breathing sounds, hunched posture, erected fur, thin appearance, decreased activity and/or abdominal distension were observed. The combination of these clinical observations with macroscopic findings (distention of stomach/intestines with gas), and histologic findings in the caudal nasal cavity/nasopharynx (occasionally lung and trachea) were consistent with gastro-esophageal reflux and aspiration.

 

In the surviving males at 800/400 mg/kg/day, test item-related hunched posture, labored breathing, thin appearance, decreased activity and/or erected fur were observed between Days 3 and 92. These signs were not considered adverse because they were not supported by adverse morphologic changes. A test item-related decreased body weight gain was observed in males from Day 8 onwards, which correlated with decreased food consumption. The effects on body weight and food consumption were not considered to be detrimental to organ system function or vitality of the animals and were therefore considered non-adverse. A non-adverse test item-related increase in macrophage aggregates in the mesenteric lymph nodes were observed in males.

At 200 mg/kg/day, preterminal deaths due to reflux occurred in two males and two females with comparable findings as mentioned above. In the surviving animals, hunched posture and erected fur were observed between Days 3 and 92. A test item-related lower body weight compared to controls was observed in males and females from Day 11 onwards. Body weight gain was slightly lower compared to controls. These effects on body weight correlated with lower food consumption compared to control animals in males and females but these changes were considered to be non-adverse at the amplitude observed. Non-adverse test item-related microscopic findings were present in the mesenteric lymph nodes (increased macrophage aggregates) of males, and thymus (minimal decreased lymphoid cellularity and minimal lymphoid single cell necrosis) of a few females.

At 50 mg/kg/day, one female was preterminally sacrificed with findings indicating reflux as the cause of death. In one other preterminally sacrificed female, pleural and epicardial inflammation with plant material (correlated to adhesion of lungs of the right lung lobes) and gelatinous white fluid in the thoracic cavity was consistent with gavage trauma as the cause of death. In the surviving animals, hunched posture and erected fur were observed between Days 3 and 92. Body weight gain was slightly lower compared to control in females starting on Day 22 and considered non-adverse.

No treatment-related changes were noted in any of the remaining parameters investigated in this study (i.e. ophthalmoscopy, functional observations, hematology, coagulation, thyroid hormones, estrous stage determination, macroscopic examination and organ weights).

In conclusion, test item-related morbidity/mortality, secondary to gastro-esophageal reflux, were present at treatment with 50 mg/kg/day (female only), 200 mg/kg/day, and 800/400 mg/kg/day Fatty acids, tall-oil, compds. with N-[3-(dimethylamino)propyl]tall-oil amides. This is considered to be a species and route specific issue (i.e., rat, oral gavage) which is not likely to occur by other routes or in other animal species. Because of the high number of preterminal females at 800 mg/kg/day and the severity of the clinical signs observed in the majority of the remaining females, all females at 800 mg/kg/day were send to necropsy preterminally. In animals that survived to the end of the study, non-adverse test item-related changes were noted in the mesenteric lymph nodes of males at 200 and 800/400 mg/kg/day (increased macrophage aggregates, up to mild) and the thymus of females at 50 and 200 mg/kg/day (minimal decreased lymphoid cellularity or single cell necrosis). Based on these results, the no-observed-adverse-effect level (NOAEL) was considered to be 400 mg/kg/day for males and 200 mg/kg/day for females.