Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

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.
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
Details on test material:
- Name of test material (as cited in study report): Tall oil reaction products with tetraethylene-pentamine
(Amidoamine/Imidazoline)
- Molecular weight (if other than submission substance): 461
- Substance type: Clear slightly viscous amber liquid (determined at NOTOX)
- Physical state: liquid
- Analytical purity: see Certificate of Analysis
- Impurities (identity and concentrations): Free fatty acid = 1.6 % , Tetraethylene pentamine = 1.3 %
- Composition of test material: see Certificate of Analysis.
- Purity test date: 11 May 2009
- Lot/batch No.: S000925
- Expiration date of the lot/batch: 07 March 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: 950 kg/m3 at 20°C
pH (1% in water, indicative range): 11.3 – 11.2 (determined at NOTOX)
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: 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.

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: 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.
Doses / concentrations
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

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 (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

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:
no effects observed
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

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.

Effect levels (P0)

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.

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:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Details on results (F1)

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.

Effect levels (F1)

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.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

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.