Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
one-generation reproductive toxicity
Remarks:
based on generations indicated in Effect levels (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 August 2009 - 19 October 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study has been performed according to OECD 422 guidelines and GLP principles.
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
other: Crl:WI(Han).
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: 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): 19.6 – 22.0°C
- Humidity (%): 31 - 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) occurred due to performance of functional observations in the room.

On Day 3 or 4 of lactation, the maximum allowed deviation from the dark period of 1 hour was exceeded with a maximum of approximately 9 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 25 August 2009 to 19 October 2009
Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenised to a visually acceptable level. Adjustment was made for density of the test substance and specific gravity of 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: Following a minimum of 14 days of exposure for the Main males and Main females, one Main female was cohabitated with one Main male of the same treatment group, avoiding sibling mating (Charles River supplied non-litter mates).

A maximum of 13 instead of 14 days was allowed for mating. All females had mated before the end of the 13-day mating 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.
- 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 according to a validated method (NOTOX project 491638). 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. One female (Group 3) was not dosed during littering.
Frequency of treatment:
Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.
Details on study schedule:
- Age at mating of the mated animals in the study: Approximately 14 weeks.
Remarks:
Doses / Concentrations:
0, 30, 100 and 300 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
10 and an extra 5 males for Group 1 and 4. The study included a recovery phase for males only. These animals were not mated and, consequently, were not used for the assessment of reproduction/developmental toxicity.

Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected in consultation with the Sponsor based on the results of a 10-day pilot study (NOTOX Project 491888; see attached file) and taking into account the results of a MTD-study with Tall oil diethylenetriamine imidazoline (NOTOX Project 491555; see endpoint study record 7.5.1: repeated dose toxicity: oral.rat_NOTOX 491555 ) and a 10-day pilot study with Tall oil reaction products with tetraethylene-pentamine (NOTOX Project 491569; see endpoint study record 7.5.1: repeated dose toxicity: oral.NOTOX 491568).
Positive control:
no
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily (early morning/late afternoon).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from start of treatment onwards (immediately after dosing from Days 1-9, between approximately 1 and 2 hours after dosing from Day 10 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.

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.
- Animals fasted: Yes, iso-flurane
- 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):
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):
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 5-7.
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.
Female sacrificed in extremis during littering: Day 1 of lactation.

Several animals were necropsied later than after a maximum of 20 hours fasting, i.e. with a maximum of 1 hour and 12 minutes. The fasting period was only slightly longer and was considered not to have adversely affected the clinical laboratory, macroscopic or microscopic findings.

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, all Recovery males and the animal that was killed in extremis@: 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.

@: Recognizable fetuses were examined externally, sexed, euthanized by decapitation (if necessary) and preserved in 10% neutral-buffered formalin.
*: 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.

No thyroid was weighed from one animal (Group 1). This was a non-pregnant female, and hence organ weights of this animal cannot be used to compare with pregnant animals. Therefore, this deviation does not adversely affect the interpretation of the results.

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 adrenal glands from the selected 5 Main animals from Groups 2 and 3 and from Recovery animals 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.
- The preserved organs and tissues of the animal (Group 3) that was killed in extremis.
- The reproductive organs# of all Main animals that failed to conceive, or deliver healthy pups:
Group 1: One male and one female (failed to conceive)
Group 3: One male (one female was killed in extremis during parturition)
Group 4: One male and one female (failed to conceive)
- All gross lesions of all animals (all dose groups).

* Based on possible treatment-related histopathological changes in high dose Main animals.
# 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.
Postmortem examinations (offspring):
SACRIFICE
Pups were killed by decapitation on Day 5-7 of lactation.

Pups fromthe female that was killed in extremis were also killed by decapitation.

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. Any abnormal pup, organ or tissue, as well as pups from the female that was killed in extremis, were preserved in 10% buffered formalin for possible further examination.

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

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

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

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

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

Conception rate = Number of pregnant females/Number of females mated x 100

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

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

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

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

Viability index = Number of live pups on Day 4 lactation/Number of pups born alive x 100
Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY
No mortality occurred during the study period that was considered to be related to treatment with the test substance.
One female at 100 mg/kg/day was killed in extremis due to complications during parturition. No further mortality occurred.

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

Incidental findings included rales, salivation, diarrhoea and alopecia. The nature and/or incidence of these findings was considered to be within the normal range of biological variation for rats of this age and strain. No clinical signs were noted among control males.

BODY WEIGHT AND WEIGHT GAIN
No toxicologically significant changes in body weights and body weight gain were observed over the study period.

The statistically significant lower body weight gain of males at 300 mg/kg/day during the premating and Repro period was considered to be of no toxicological relevance since these variations were minor in nature, and body weight gain was similar to control values from Day 1 of the recovery phase onwards.

FOOD CONSUMPTION:
No toxicologically significant changes in food consumption before or after allowance for body weight were noted.

The statistically significant changes in (relative) food consumption among females at 300 mg/kg/day were incidentally noted during the premating and post-coitum phase and were slight in nature. Therefore, these variations were not considered to be of toxicological relevance.

HAEMATOLOGY
No toxicologically significant changes in haematology parameters were noted.

The statistically significant higher prothrombin time (PT) and mean corpuscular volume (MCV) in males at 300 mg/kg/day at the end of treatment and recovery phases respectively, were considered to be of no toxicological significance as they occurred in the absence of a treatment-related distribution, were absent at the end of the treatment phase and/or remained within the range considered normal for rats of this age and strain.

CLINICAL CHEMISTRY
The following statistically significant changes in clinical biochemistry parameters distinguished treated animals from control animals at the end of the treatment phase:
- Higher alanine aminotransferase activity (ALAT) and aspartate aminotransferase activity (ASAT) in males and females at 300 mg/kg/day at the end of treatment.
- Lower total protein and higher total bilirubin levels in males at 300 mg/kg/day at the end of treatment (within normal range for rats of this age and strain).
- Higher inorganic phosphate level in females at 300 mg/kg/day.
By the end of the treatment period these changes had returned to values comparable to controls.

Other statistically significant changes were considered to be of no toxicological significance as they occurred in the absence of a treatment-related distribution, remained within the range considered normal for rats of this age and strain and/or were absent at the end of the treatment phase. These changes included higher sodium and/or chloride levels in males at 30, 100 and 300 mg/kg/day at the end of treatment, lower glucose and calcium, and higher inorganic phosphate levels in males at 300 mg/kg/day at the end of recovery, and higher bile acid levels in females at 100 mg/kg/day.

NEUROBEHAVIOUR
Motor activity of females at 300 mg/kg/day appeared higher than controls, and achieved a level of statistical significance for low sensor counts.

No toxicologically significant changes in hearing ability, pupillary reflex, static righting reflex and grip strength were noted among the animals. Motor activity of males (all dose levels) and females (30 and 100 mg/kg/day) remained similar to control levels.

One animal at 30 mg/kg/day showed no grip reflex, and one male at 100 mg/kg/day did not show a hearing reflex. Given the incidental nature of these findings and absence of a treatment-related incidence, these were considered to be of no toxicological significance.

ORGAN WEIGHTS
A lower heart weight and heart to body weight ratio was recorded for males and females at 300 mg/kg/day at the end of treatment, achieving a level of statistical significance (except for relative heart weight of males). These changes were absent at the end of the recovery phase for males.

No further changes in organ weights were noted that were considered to be of toxicological significance. The statistically significant lower liver weight of males at 300 mg/kg/day at the end of the treatment phase was considered to be related to the lower terminal body weight (after correction for terminal body weight, liver weights were similar to those of control animals). The statistically significant lower epididymides weight, epididymides to body weight ratio and/or seminal vesicle to body weight ratio of males at 30, 100 and 300 mg/kg/day at the end of treatment did not show a dose-related trend. These changes were within the range considered normal for rats of this age and strain and were absent at the end of the recovery phase.

GROSS PATHOLOGY
Necropsy did not reveal any toxicologically relevant alterations.

Incidental necropsy findings at the end of the treatment period consisted of a nodule on the epididymides, pelvic dilation of the kidneys, red foci on the thymus or kidneys, reduced size of the thymus, a red-brown focus on the clitoral glands, enlarged clitoral glands or spleen, red discolouration of the mesenteric or mandibular lymph nodes, alopecia 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:
A morphological alteration in the adrenal gland consisting of a slight, not statistically significant, increase in incidence and severity to moderate, of multifocal vacuolation of the zona glomerulosa in animals of both sexes was noted at 300 mg/kg/day. Following a recovery period of at least two weeks for males this finding had reverted to a background incidence and severity of minimal.

No alterations in spermatogenesis staging were recorded.

There were no microscopic findings in any of the 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.

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.

DEVELOPMENTAL DATA:
No toxicologically significant effects on the number of females with live pups, gestation index, duration of gestation 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.
Dose descriptor:
NOAEL
Effect level:
>= 300 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: All parental findings observed at the end of treatment at 300 mg/kg/day were not considered to be adverse since these changes were generally slight and reversible in nature, and occurred in the absence of supportive histopathological lesions.
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
DEVELOPMENTAL DATA:
No toxicologically significant effects on early postnatal pup development (body weight, clinical signs, viability index and external macroscopy) were noted.

The female that was sacrificed due to complications during parturition delivered six live pups, one of which was cannibalized before necropsy. Four pups were not delivered. External macroscopic examination of the pups delivered by this female showed a small size, absence of milk in the stomach and cannibalism.
No further signs of difficult or prolonged parturition or signs of abortion or premature birth were noted among the pregnant females.

Incidental clinical symptoms consisted of a small size and absence of milk in the stomach. Incidental macroscopic findings consisted of a small size, enlargement of the right eye with blue discolouration and autolysis of pups found dead. No relationship with treatment was established for these observations and they were considered to be of no toxicological significance.

The incidence of postnatal loss at 100 and 300 mg/kg/day was slightly higher than would be expected based normal range values for rats of this age and strain, resulting in a slightly lower viability index at 100 mg/kg/day. However, the incidence did not show a dose-related trend and did not achieve a level of statistical significance.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 300 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No reproductive/developmental toxicity was observed at any dose level.
Reproductive effects observed:
not specified
Conclusions:
Overall, all parental findings observed at the end of treatment at 300 mg/kg/day were not considered to be adverse since these changes were generally slight and reversible in nature, and occurred in the absence of supportive histopathological lesions. Therefore, the parental, reproductive and developmental No Observed Adverse Effect Level (NOAEL) of 300 mg/kg/day was derived.
Executive summary:

Tall oil fatty acids, reaction products with polyethylenepolyamines was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 30, 100 and 300 mg/kg/day. The males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 28 days). A total of 5 animals each of Groups 1 and 4 were allowed a 2-week recovery period. The females were exposed for at least 42 days, i.e. 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, were homogeneous and were stable for at least 6 hours at room temperature.

Parental findings:

Treatment up to 300 mg/kg/day was well tolerated and did not result in toxicologically relevant clinical signs or changes in body weight and food intake. A higher motor activity was recorded for females at 300 mg/kg/day, although this was not accompanied by supportive clinical signs (e.g. hyperactivity) or changes in functional observation battery parameters. Therefore, this change was not considered to be of toxicological relevance.

Clinical biochemistry measurements at the end of treatment at 300 mg/kg/day showed notably higher alanine and aspartate aminotransferase activity in both sexes (but without corroborative histopathological findings, e.g. liver damage), along with lower total protein and higher total bilirubin levels in males (within normal range for rats of this age and strain) and higher inorganic phosphate level in females. These changes had resolved to values comparable to controls at the end of the recovery period for males. No (further) changes were noted in clinical biochemistry and haematology parameters at any dose level.

Macroscopic examination at the end of treatment revealed a lower absolute and relative heart weight for both sexes at 300 mg/kg/day. No histopathological correlate was found and no such change was apparent at the end of the recovery period for males. No (further) macroscopic abnormalities or organ weight changes were observed at any dose level.

Histopathology showed a slight, statistically non-significant, increase in incidence and severity of multifocal vacuolation of the zona glomerulosa of the adrenal glands in both sexes at 300 mg/kg/day. Following the recovery period this finding had reverted to background incidence and severity.

No signs of parental toxicity were observed at 30 and 100 mg/kg/day.

Reproductive/Developmental findings:

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

Overall, all parental findings observed at the end of treatment at 300 mg/kg/day were not considered to be adverse since these changes were generally slight and reversible in nature, and occurred in the absence of supportive histopathological lesions. Therefore, the parental, reproductive and developmental No Observed Adverse Effect Level (NOAEL) of 300 mg/kg/day was derived.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Consistent results from all studies within the whole group of Amidoamine/imidazolines (AAI), indicating a no concerns for reproduction toxicity. (See also document in support of category justification).
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

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

 

No reproductive/developmental toxicity was observed at any of the dose levels in an OECD 422 study with Fatty acid reaction product with pentaethylene-hexamine (AAI-PEHA), and thus a reproduction/developmental NOAEL of 300 mg/kg/day was determined. Similar OECD 422 studies have been performed on other substances from the group of AAI: on Fatty acid reaction product with diethylene-triamine (AAI-DETA) and on Fatty acid reaction product with tetraethylene-pentamine (AAI-TEPA). No indication of concern for reproductive or developmental toxicity was observed in these studies up to the highest dose tested. Also an OECD 414 developmental toxicity in rat on a similar substance showed no concern for developmental effects.

All already available data from the group of AAI substances, including 90-day studies in rats and dogs on a similar substance, suggest low toxicity and have shown no adverse effects on reproductive organs. (See also document in support of category justification).

Also the low likelihood of exposure can be considered as its use is limited to industrial and professional users where following its severe corrosive properties will provide for sufficient protection measures to prevent exposure. The likelihood of exposures via inhalation is low considering the high boiling point (> 300 °C) and very low vapour pressure (< 0.00017 mPa at 25°C) and use applications that do not involve the forming of aerosols, particles or droplets of an inhalable size.

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


Short description of key information:
NOAEL for reproduction/developmental of 300 mg/kg/day was established from a combined repeated dose/reproduction toxicity study with atty acids C18 unsat, reaction products with pentaethylenehexamine (AAI-PEHA).

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

Justification for selection of Effect on fertility via inhalation route:
Likelihood of exposures via inhalation is low considering the high boiling point (> 300 °C) and very low vapour pressure (< 0.00017 mPa at 25°C). The potential for inhalation is not significant to justify this study. Furthermore, as the substance is classified as corrosive, such testing should normally not be conducted.

Justification for selection of Effect on fertility via dermal route:
All substances from the group of Amidoamine/imidazolines (AAI) are corrosive to the skin and are not expected to easily pass the skin. The skin is therefore not a preferred route when studying reproductive toxicity.

Effects on developmental toxicity

Description of key information
No developmental toxicity was observed in an OECD 422 screening study with atty acids C18 unsat, reaction products with pentaethylenehexamine (AAI-PEHA); No developmental toxicity was observed in a developmental toxicity study (OECD 414) study on a similar substance.
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Limited reporting, method similar to OECD 414. US EPA evaluated: Reliable without restriction; guideline study.
Qualifier:
according to
Guideline:
EPA OPP 83-3 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
Timed-pregnant rats
Route of administration:
oral: gavage
Vehicle:
unchanged (no vehicle)
Analytical verification of doses or concentrations:
not specified
Details on mating procedure:
copulation plug-positive females
Duration of treatment / exposure:
gestation days (gd) 6 through 15.
Frequency of treatment:
Daily
Duration of test:
until gd 21
Remarks:
Doses / Concentrations:
o, 100, 300 and 1000 mg a.i./kg bw/day
Basis:
nominal conc.
No. of animals per sex per dose:
Twentyfive females per group
Control animals:
yes
Details on study design:
control group received Milli-Q water at a dose volume equivalent to that used in the high dose group.
Maternal examinations:
Clinical observations were made daily (twice daily during dosing), and maternal body weights were measured on gd 0, 6, 9, 12, 15, 18 and 21. At scheduled sacrifice on gd 21, the dams were evaluated for liver and gravid uterine weights.
Ovaries and uterine content:
gravid uterine weights, number of corpora lutea and number and status of implantation sites (including early and late resorptions, dead fetuses and live fetuses).
Fetal examinations:
Approximately one-half of the live fetuses in each litter were examined for visceral and craniofacial malformations and variations. The remaining one-half of the fetuses were stained with alizarin red S and were examined for skeletal malformations and variations.
Statistics:
The unit of comparison was the pregnant dam or the litter.
ANOVA, t-tests, Kruskal-Wallis Test, Mann-Whitney U Test and Fisher’s Exact Test were used where appropriate.
Details on maternal toxic effects:
Maternal toxic effects:no effects
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (nominal)
Based on:
act. ingr.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (nominal)
Based on:
act. ingr.
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
NOAEL (maternal and developmental) >1000 mg/kg-day (highest dose tested)
Executive summary:

The objective of this study was to evaluate the potential of the test substance to produce developmental toxicity when administered by a gavage to pregnant CD® rats during organogenesis. Maternal toxicity was also evaluated. Timed-pregnant rats were administered the test substance by gavage on gestation days (gd) 6 through 15. Twentyfive copulation plug-positive females per group were dosed with undiluted test substance at dose levels corresponding to 100, 300 and 1000 mg active ingredient/kg/day. An additional 25 females, assigned to the control group, received Milli-Q water at a dose volume equivalent to that used in the high dose group. Clinical observations were made daily (twice daily during dosing), and maternal body weights were measured on gd 0, 6, 9, 12, 15, 18 and 21. At scheduled sacrifice on gd 21, the dams were evaluated for liver and gravid uterine weights, number of corpora lutea and number and status of implantation sites (including early and late resorptions, dead fetuses and live fetuses). Approximately one-half of the live fetuses in each litter were examined for visceral and craniofacial malformations and variations. The remaining one-half of the fetuses were stained with alizarin red S and were examined for skeletal malformations and variations.

 

Maternal: The pregnancy rate was equivalent across groups and ranged from 88 - 100%. No females aborted or delivered early. At scheduled sacrifice, three females in the control group, two females in the 100 mg/kg/day group and one female in the 300 mg/kg/day group were found to be nonpregnant. One female from the control group and one female from the 300 mg/kg/day group contained no viable fetuses at scheduled sacrifice. Twenty-one to 25 live litters were available for evaluation from each group. One female in the 300 mg/kg/day treatment group became moribund and was sacrificed on gd 10. Two to three dams in the 300 and 1000 mg/kg/day treatment groups exhibited audible respiration during or subsequent to the treatment period. None of these observations were considered to be test substance related. There were no treatment-related effects on food consumption, gestational body weight and body weight gain, corrected body weight, corrected body weight gain, and gravid uterine weight. No treatmentrelated differences in gestational parameters including total number of implantations, number of viable implants, and number of nonviable implants, were observed in any dose group.

 

Fetal: Fetal body weights per litter were not affected by treatment.

No treatment-related malformations or variations were observed in this study.

 

Maternal toxicity NOEL: > 1000 mg/kg/day

Developmental toxicity NOEL: > 1000 mg/kg/day

 

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Consistent results from all studies within the whole group of Amidoamine/imidazolines (AAI), indicating a no concerns for developmental toxicity. (See also document in support of category justification).
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information
No developmental toxicity was observed in an OECD 422 screening study with Fatty acid reaction product with pentaethylene-hexamine (AAI-PEHA).

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

 

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

To strengthen the data for the group of AAI, a full prenatal developmental toxicity study in rats according to OECD 414 is therefore intended to perform on FA reaction product with DETA


Justification for selection of Effect on developmental toxicity: via oral route:
Only available guideline study

Justification for selection of Effect on developmental toxicity: via inhalation route:
Likelihood of exposures via inhalation is low considering the high boiling point (> 300 °C) and very low vapour pressure (< 0.00017 mPa at 25°C). The potential for inhalation is not significant to justify this study. Furthermore, as the substance is classified as corrosive, such testing should normally not be conducted.

Justification for selection of Effect on developmental toxicity: via dermal route:
All substances from the group of Amidoamine/imidazolines (AAI) are corrosive to the skin and are not expected to easily pass the skin. The skin is therefore not a preferred route when studying developmental toxicity.

Justification for classification or non-classification

The database of relevant studies available for the group of Amidoamine/imidazolines (AAI) include various OECD 422 studies and an OECD 414 study, that all show no concerns regarding reproduction or developmental toxicity. Also all already available data from the group of AAI substances, including a 90-day study in dogs on a similar substance, indicate low toxicity and no adverse effects on reproductive organs.