Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01 September 2009 - 15 October 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study has been performed according to OECD 422 guidelines and GLP principles.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Tall oil diethylenetriamine imidazoline
- Molecular weight (if other than submission substance): 359
- Substance type: Clear slightly viscous amber liquid (determined at NOTOX)
- Physical state: liquid
- Analytical purity: see Certificate of Analysis
- Impurities (identity and concentrations): Free diethylenetriamine 3.6% and free fatty acid 2.0%.
- Composition of test material: see Certificate of Analysis.
- Purity test date: 11 May 2009
- Lot/batch No.: S000922
- Expiration date of the lot/batch: 02 July 2017
- Stability under test conditions: see Details on analytical verification of doses or concentrations.
- Storage condition of test material: At room temperature in the dark under nitrogen
- Other:
Specific Gravity / Density: 980 kg/m3 at 20°C
pH : 10-12 at concentration of 75%
Solubility in Propylene glycol: Unknown

Test animals

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

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

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

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

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenised to a visually acceptable level. Adjustment was made for density of the test substance and specific gravity of the vehicle (1.036) .

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

- Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: A maximum of 13 instead of 14 days was allowed for mating. All females had mated within this period
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum.
- After successful mating each pregnant female was caged individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on a single occasion during treatment phase (02 October 2009) according to a validated method (NOTOX project 491557). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature was also determined (highest and lowest concentration).

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

RESULTS:
The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%).
The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation = 10%). Formulations at the entire range were stable when stored at room temperature for at least 6 hours.
Duration of treatment / exposure:
Males were exposed for 28 days, i.e. 2 weeks prior to mating, during mating, and up to termination or up to the day prior to start of the recovery period for Recovery males. Females were exposed for at least 42 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation.
Frequency of treatment:
Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.

Details on study schedule:
- Age at mating of the mated animals in the study: 14 weeks
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 10, 30 and 100 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
10 and an extra 5 males for Group 1 and 4. The study included a recovery phase for males only. These animals were not mated and, consequently, were not used for the assessment of reproduction/developmental toxicity.

Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected based on the results of the dose range finding/MTD study (NOTOX Project 491555). See endpoint study record 7.5.1: Repeated dose toxicity: oral. notox 491555.
- Rationale for animal assignment (if not random): 5 animals/sex/group were randomly selected at allocation for functional observations, clinical pathology, macroscopic examination (full list), organ weights (full list) and histopathology.
Males: the first 5 males per group for main and recovery
Females: the first 5 females (with live offspring only)
Positive control:
no

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily (early morning/late afternoon)

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

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

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

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

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

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

OPHTHALMOSCOPIC EXAMINATION: No

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

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

URINALYSIS: No

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

OTHER:
General reproduction data:
Male number paired with, mating date, confirmation of pregnancy, and delivery day was recorded. Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of these females was examined to detect signs of abortion or premature birth. Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.
Oestrous cyclicity (parental animals):
not determined

Sperm parameters (parental animals):
testis weight, epididymis weight.
For 5 males of the control and high dose group, slides of the testes were prepared to examine staging of spermatogenesis.



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

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

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

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

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

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

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

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

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

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

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

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

All remaining animals and females which failed to deliver$:

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

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

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

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

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

All remaining males: Epididymides, Testes.

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

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

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

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

All abnormalities were described and included in the report. An attempt was made to correlate gross observations with microscopic findings.
Postmortem examinations (offspring):
SACRIFICE
Pups were killed by decapitation on Day 5 of lactation.

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

HISTOPATHOLOGY / ORGAN WEIGTHS
no
Statistics:
The following statistical methods were used to analyse the data:
-If the variables could be assumed to follow a normal distribution, the Dunnett-test (Dunnett, 1955) (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
-The Steel-test (Miller, 1981) (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
-The Fisher Exact-test (Fisher, 1950) was applied to frequency data.

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

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

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

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

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

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

Duration of gestation: Number of days between confirmation of mating and the beginning of parturition.
Offspring viability indices:
Percentage live males at First Litter Check:
Number of live male pups at First Litter Check/Number of live pups at First Litter Check x 100

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

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

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

GROSS PATHOLOGY
Necropsy did not reveal any toxicologically relevant alterations.

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

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

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

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

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

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

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

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

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

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

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
>= 30 - < 100 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on the increased incidence/severity of macrophage foci in the mesenteric lymph node at both the end of treatment and recovery period in males.

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

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

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

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

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 100 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No reproductive/developmental toxicity was observed at any dose level.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

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

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

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

Parental results:

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

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

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

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

Reproductive/Developmental results:

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

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

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

As explained in the category justification, For cross-reading in general use is made with data of same or lower EA-length where available, and that of Tall oil + DETA representing the worst case.This dossier is for the substance "Fatty acids C18 unsat, reaction products with triethylenetetramine" (or TO + TETA). As for the substance itself no toxicological information is available, cross-reading has been applied to TO + DETA.