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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

An extended OECD 422 (10-week premating period) is available for di-(2-hydroxypropyl) C16-C18 (evennumbered), C18 unsaturated-alkyl amine CAS 1309955-79-0. Dose levels of 50, 150 and 300 mg/kg bw/day were examined. A NOAEL of 150 mg/kg/day was determined for reproductive toxicity (based on the lower number of corpora lutea and implantation sites).


An EOGRTS was performed on 2, 2’-(Octadec-9-enylimino) bisethanol CAS No 25307-17-9 which is used as read-across to di-(2-hydroxypropyl) C16-C18 (evennumbered), C18 unsaturated-alkyl amine CAS 1309955-79-0. Dose levels of 30, 70, and 150 mg/kg bw/day were examined. A NOAEL of 150 mg/kg bw/day was determined for reporductive toxicity.

Link to relevant study records

Referenceopen allclose all

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 27 Oct 2021 to 22 Sep 2022
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
At this time, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models that do not use live animals currently do not exist.
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: other guidance as listed under Principles of method if other than guideline
Principles of method if other than guideline:
The procedures described in this study plan essentially conform to the following
guidelines:
- EPA OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, 2000.
- EPA OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, 2000.
- OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
- EU Method B.26, Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents, 2008
- EPA OPPTS 870.3100, 90-Day Oral Toxicity in Rodents, 1998
GLP compliance:
yes
Limit test:
no
Justification for study design:
The total number of animals to be used in this study is considered to be the minimum required to properly characterize the effects of the test item. This study has been designed such that it does not require an unnecessary number of animals to accomplish its objectives.
At this time, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models which do not use live animals currently do not exist.
The oral route of administration was selected because this is a possible route of human exposure during manufacture, handling or use of the test item.
Species:
rat
Strain:
other: Crl: WI(Han)
Details on species / strain selection:
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 6-7 weeks
- Weight at study initiation: (P) Males: 181 – 215 g; Females: 107 – 144 g.
- Fasting period before study: No
- Housing: On arrival and during the pre-mating period, animals will be group housed (up to 5 animals of the same sex and same dosing group together) in Makrolon cages. During the mating phase, males and females will be cohabitated on a 1:1 basis in Makrolon plastic cages. During the post-mating phase, males will be housed in Makrolon cages with a maximum of 5 males/cage. Females will be individually housed in Makrolon cages. During the lactation phase, females will be housed in Makrolon plastic cages. Pups will be housed with the dam, except during locomotor activity monitoring of the dams. During locomotor activity monitoring, F0-animals will be housed individually in a Hi-temp polycarbonate cage without cage-enrichment, bedding material, food and water. Animals were socially housed for psychological/environmental enrichment and will be provided with items such as devices for hiding in, paper and/or objects for chewing, except when interrupted by study procedures/activities. It is considered that there are no known contaminants that would interfere with the objectives of the study.
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum. The feed was analyzed by the supplier for nutritional components and environmental contaminants.
- Water: tap water, ad libitum. During motor activity measurements, animals had no access to water for a maximum of 2 hours. Periodic analysis of the water was performed.
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24 (target) 21-23 (actual)
- Humidity (%): 40-70 (target) 38-56 (actual)
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From 09 Nov 2021 o 09 Mar 2022
Route of administration:
oral: gavage
Vehicle:
peanut oil
Details on exposure:
PREPARATION OF DOSING FORMULATIONS:
Test item dosing formulations were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements, prepared at least weekly and stored at 4°C. Test item was heated to a maximum temperature of 35±5°C to obtain a visually homogeneous solution before preparations of the formulations. The dosing formulations were removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing and dosed within 24 hours after removal from the refrigerator. Test item dosing formulations were kept at room temperature and continuously stirred (when practically possible) until dosing. Adjustments for specific gravity of the vehicle and test item were made. No correction factor to account for the purity/composition of the test item was used.

DOSE VOLUME: 4 mL/kg
Details on mating procedure:
After a minimum of 10 weeks of treatment, animals were cohabitated on a 1:1 basis within the same treatment group, avoiding sibling mating. 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. Once mating occurred, the males and females were separated.
A maximum of 14 days were allowed for mating, after which females who have not shown evidence of mating were separated from their males. In case less than 9 females per group shown evidence of mating, each non-mated female was re-mated once with a male for a maximum of 7 days (if possible). A male of the same group having previously shown evidence of mating was used for re-mating.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability analyses performed previously in conjunction with the method development and validation study demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.
Concentration and homogeneity analyses were performed by using a validated analytical procedure and UPLC-MS.
Duplicate sets of samples (approximately 500 mg) for each sampling time point were collected. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% for solutions of target concentration. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ≤ 10%.
Samples were taken in weeks 1, 6 and 12 for concentration determination (all groups) and homogeneity (low and high dose groups, the homogeneity results obtained from the top, middle and bottom for the low and high dose group preparations were averaged and utilized as the concentration results.
Duration of treatment / exposure:
Males: 92 days in total, or for a minimum of 13 weeks, up to and including the day before scheduled necropsy. This includes a minimum of 10 weeks prior to mating and during the mating period.
Females: 90 to119 days in total or for at least 10 weeks prior to mating, the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day before scheduled necropsy. Females were not dosed during littering.
Pups were not treated directly but could potentially be exposed to the test item in utero, via maternal milk, or from exposure to maternal urine/feces.
Frequency of treatment:
Daily
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
Low dose group
Dose / conc.:
150 mg/kg bw/day (nominal)
Remarks:
Mid dose group
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
High dose group
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
The dose levels were selected based on the results of a 14-day Dose Range Finder with Tallow amine propoxylate administered via oral gavage in male rats, a Prenatal Developmental Toxicity Study with Tallow amine propoxylate in pregnant female rats, and in an attempt to produce graded responses to the test item.
In the Dose Range Finding Study, three male rats per group were treated with 150, 200, 250, 300, 450 and 600 mg/kg bw/day. At 600 mg/kg bw/day, 3/3 males were sacrificed in extremis on Day 12 of treatment with signs of erected fur, hunched posture, decreased activity, pale tail and salivation. Body weight loss (up to 18%) and reduced food consumption were noted. Macroscopic examination showed decreased adipose tissue in 3/3 males and dark red discoloration of the adrenals in 1/3 males. At 450 mg/kg bw/day, no mortality occurred. Clinical signs that were noted included erected fur, hunched posture and salivation on several days. Reduced body weight gain and food consumption were noted during Day 1-14 of treatment. Absolute mean body weight was reduced by 22% and 15% compared to treatment at 150 and 300 mg/kg bw/day, respectively. Macroscopic examination revealed no abnormalities; relative kidney and liver weights were slightly decreased. At 300 mg/kg bw/day, 3/3 males were noted with erected fur, hunched posture and salivation on selective days only. Reduced body weight gain and food consumption was noted during Day 1-4 of treatment, which remained slightly lower between Days 4-14 of treatment. Macroscopic examination revealed no abnormalities; relative kidney and liver weights were slightly decreased.
In the Prenatal Developmental Toxicity Study, pregnant female rats were treated with 0, 100, 300 and 375 mg/kg bw/day from Days 6 to 20 post-coitum, inclusive. At 375 mg/kg bw/day, 13/22 females and at 300 mg/kg bw/day 3/22 females were sacrificed in extremis between post-coitum Days 12-19 of treatment with signs such as erected fur, hunched posture, decreased activity, orange/black feces, closed eyes and salivation. Body weight loss and reduced food consumption were noted. The high-dose level should produce some toxic effects, but not death nor obvious suffering. The mid-dose level is expected to produce minimal to moderate toxic effects. The low-dose level should produce no observable indications of toxicity.
Positive control:
No
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
Time schedule: Twice daily.
Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings.

DETAILED CLINICAL OBSERVATIONS: Yes
Time schedule: Once before the first administration of the test item and weekly during the Treatment Period.

BODY WEIGHT: Yes
Time schedule: On Day 1 of treatment (prior to dosing) and weekly thereafter. Mated females were weighted on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

FOOD CONSUMPTION: yes
Time schedule: Weekly, except for males and females which are housed together for mating and for females without evidence of mating. For mated females on Days 0, 4, 7, 11, 14, 17, and 20 postcoitum and during lactation on PND 1, 4, 7, and 13.

FOOD EFFICIENCY: No

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

OPHTHALMOLOGIC OBSERVATIONS: Yes.
All animals were examined once during the pretreatment period. During the dosing period, all animals from Groups 1 and 4 were examined during Week 13.

HEMATOLOGY: Yes.
Time schedule for collection of blood: on the day of scheduled necropsy.
- Anaesthetic used for blood collection: Yes (isoflurane).
- Animals fasted: Males (with a maximum of 24 hours). Females were not fasted.
- How many animals: 10 animals/sex/group.
- Parameters checked were: According to test guidelines.

CLINICAL CHEMISTRY: Yes.
Time schedule for collection of blood: on the day of scheduled necropsy.
- Animals fasted: Yes (with a maximum of 24 hours).
- How many animals: 10 animals/sex/group.
- Parameters checked were: According to test guidelines.
- Blood samples were processed for serum, and serum was analyzed for total Thyroxine (T4), Triiodothyronine (T3) and/or Thyroid-stimulating hormone (TSH).

URIANALYSIS: No.

NEUROBEHAVIOURAL EXAMINATION: Yes.
Time schedule for examinations: Five selected males per group were tested once during Week 13 of treatment and five selected females per group were tested once during the last week of lactation (i.e. PND 6-13). Tests were performed after completion of clinical observations .
- Battery of functions tested: According to test guidelines. Hearing ability, pupillary reflex, static right ing reflex , fore- and hindlimb grip strength (recorded as the mean of three measurements per animal) and locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system). Total movements and ambulations were reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or more fine movements like grooming, weaving or movements of the head.

GENERAL REPRODUCTION DATA: Yes.
From the mating period onwards, the following parameters were recorded for each female: male number paired with, mating date, confirmation of pregnancy and delivery day. Females were allowed to litter normally. Postnatal day (PND) 1 is defined as the day when a litter is found completed (i.e. membranes and placentas cleaned up, nest built and/or feeding of pups started). The day prior to PND 1 is considered to be the day when the female started to deliver and is defined as PND 0 and used for recording of delivery. Females that were littering were left undisturbed. Cage debris of pregnant females were examined for evidence of premature delivery and pregnant females were examined to detect signs of difficult or prolonged parturition or deficiencies in maternal care.
Oestrous cyclicity (parental animals):
Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage. Daily vaginal lavage was performed for all females beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period. On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrous. This was done for all females, except for females that had to be euthanized in extremis or died spontaneously.
Sperm parameters (parental animals):
For the testes of all males detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell- or stage-specificity of testicular findings were noted.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- On PND 4, the surplus pups (> 8 pups per litter) were euthanized by decapitation.

PARAMETERS EXAMINED
-Number and sex of pups, stillbirths, live births, postnatal mortality, clinical signs, weight gain, anogenital distance (AGD), presence of nipples/areolae in male pups

GROSS EXAMINATION OF DEAD PUPS: Yes
-For external and internal abnormalities. Pups that died before scheduled termination were examined externally and sexed (both externally and internally). The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.
Postmortem examinations (parental animals):
GROSS NECROPSY: yes
- All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs. The numbers of former implantation sites were recorded for all paired females. The number of corpora lutea will be recorded in all pregnant and formerly pregnant females (i.e. all females with implantation sites). Necropsy procedures were performed by qualified personnel with appropriate training and experience in animal anatomy and gross pathology.

ORGAN WEIGHTS: yes
-The organs detailed in table 1 under ''Any other information on materials and methods incl. tables'' were weighed at necropsy for all scheduled euthanasia animals and females with total litter loss. Organ weights will not be recorded for animals found dead or euthanized in poor condition or in extremis. Paired organs will be weighed together. Organ weights as a percent of body weight (using the terminal body weight) will be calculated.

HISTOPATHOLOGY: yes
- Representative samples of the tissues detailed in table 1 under ''Any other information on materials and methods incl. tables'' were collected from all animals and preserved. Additional tissue samples may be collected to elucidate abnormal findings. For females which fail to deliver a complete litter, uterine contents (i.e. any fetuses, placenta and implantation sites) were fixed (if applicable), but was not examined histopathologically in first instance. All tissues were examined by a board-certified toxicological pathologist with training and experience in laboratory animal pathology.
Postmortem examinations (offspring):
From two surplus pups per litter, blood was collected, if possible. All remaining pups were euthanized on PND 14-16. Sex was determined both externally and internally. Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development. No full histopathological examination was initially performed, however, if any abnormalities are observed during determination of sex, abnormalities may be collected and fixed in 10% buffered formalin. In addition, the thyroid was collected from two pups per litter (if possible, from one male and one female pup and preferably from the same pups as selected for (complete) blood collection) and preserved in 10% buffered formalin.
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and or 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Values may also be expressed as a percentage of predose or control values when deemed appropriate. Inferential statistics were performed according to the comparison matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations. The following pairwise comparisons were made: low dose group vs. control group; mid dose group vs. control group; high dose group vs. control group. Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test). For the motor activity data set (at least 3 groups) parametric (ANOVA) tests on group means were applied with Bonferroni correction for multiple testing. Mixed modelling techniques, comparing six different covariance structures, were used in order to select the best fitting statistical model. Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test). An overall Fisher’s exact test was used to compare all groups. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test was significant.
Reproductive indices:
For each group, the following calculations were performed:
- Mating index: (Number of females mated/Number of females paired) x 100
- Fertility index: (Number of pregnant females/Number of females paired) x 100
- Gestation index: (Number of females bearing live pups/Number of pregnant females) x 100
- Precoital time: Number of days between initiation of cohabitation and confirmation of mating
- Duration of gestation: Number of days between confirmation of mating and the beginning of parturition
Offspring viability indices:
- Post-implantation survival index (%): (Total number of offspring born/ Total number of uterine implantation sites) x 100
- Live birth index: (Number of live offspring on Day 1 after littering/ Total number of offspring born) x 100
- 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
- Viability index: (Number of live pups on Day 4 of lactation / Number of pups born alive) x 100
- Lactation index: (Number of live offspring on Day 13 after littering/ Number live offspring on Day 4 (after culling)) x 100
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
In males and females at 300 mg/kg bw/day, piloerection and/or hunched posture were frequently noted in multiple (to all) animals at 1-2 hours post-dose from Week 6 of treatment onwards. At the pre-dose observation, piloerection and hunched posture were only incidentally noted at 300 mg/kg bw/day from Week 11 and 13 onwards for males and females, respectively.
Also at 150 mg/kg bw/day, piloerection and/or hunched posture were incidentally observed in a few males and females from Week 11 onwards.
Salivation was seen after dosing among animals of the 150 and 300 mg/kg bw/day dose groups from Week 2 of the treatment period onwards. Taking into account the nature and minor severity of the effect and its time of occurrence (i.e., after dosing), this sign was considered to be a physiological response rather than a sign of systemic toxicity.
Any other clinical signs noted during the Treatment Period 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 to be unrelated to treatment with the test material.
Mortality:
mortality observed, treatment-related
Description (incidence):
Two females at 300 mg/kg bw/day were sacrificed in extremis during the Post-coitum Period.
One high-dose female was sacrificed in extremis on Day 18 post-coitum based on a deteriorating condition with clinical signs including hunched posture, piloerection, a pale and lean appearance and red staining of the snout. In addition, despite her pregnancy, this female displayed minimal weight gain up to slight weight loss over the post-coitum period. At necropsy, the uterus of this female contained 8 living fetuses and 1 early resorption. Furthermore, reddish foci were noted on the glandular mucosa of the stomach and yellowish discoloration of the mesenteric lymph nodes was observed.
Another high-dose female was sacrificed in extremis on Day 20 post-coitum based on a deteriorating condition with clinical signs including piloerection, hunched posture and a pale appearance. In addition, on the day of sacrifice, this female displayed lethargy and flat posture. Moreover, this female did not gain any weight between Days 17-20 post-coitum while she was pregnant. At necropsy, the uterus of this female contained 12 living fetuses. Furthermore, oily yellowish contents of the stomach, duodenum, jejunum and ileum were noted, a pale discoloration of the liver was observed, and the mesenteric lymph nodes were found with a yellowish discoloration.
Main microscopic findings related to the condition of these two animals were present in the non-glandular stomach (forestomach) (squamous cell hyperplasia slight-moderate, with lymphogranulocytic infiltrate and minimal erosion/ulcer or edema), small intestines and mesenteric lymph node (macrophage accumulation up to marked degree) and most likely secondary findings related to their poor health condition were noted in the thymus (minimal-slight increased apoptosis lymphocytes/decreased lymphoid cellularity) and bone marrow (slightly increased adipocytes). Macroscopic and microscopic findings of these organs were similar in scheduled sacrificed females at 300 mg/kg bw/day. Only the severity of the foamy macrophage accumulation in the jejunum and increased adipocytes in the bone marrow was slightly higher and the lesions of the small intestines were more extensive (included the ileum as well, instead of being present only in duodenum and/ or jejunum, as was the case for the scheduled sacrifices at 300 mg/kg bw/day).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
No changes in body weights and body weight gain were observed in males at 50 mg/kg bw/day and in females up to 150 mg/kg bw/day.
Progressively lower body weight gain was observed for males at 150 and 300 mg/kg bw/day from respectively Day 36 and 8 of treatment (Premating Period) onwards. In addition, occasional slight body weight loss was noted in most males at 300 mg/kg bw/day and in a few males at 150 mg/kg bw/day. This resulted in mean body weights of up to 13% and 27% lower than controls during the Mating Period for males at 150 and 300 mg/kg bw/day, respectively.
In females at 300 mg/kg bw/day, body weight gain was lower than control between Days 50-64 of the premating period, on Day 1 of mating (multiple females displayed slight body weight loss), and between Days 14-20 post-coitum (not statistically significant for all intervals). This resulted in lower mean body weights from Day 50 of treatment (Premating Period) onwards (up to 21% lower than control on Day 1 of lactation). From Day 7 of lactation, body weight gain in these females was higher than control, resulting in a slight recovery of the reduced body weights, with a mean body weight of 12% lower than control on Day 13 of lactation.
For females at 150 mg/kg bw/day, body weight gain was also higher than controls during the Lactation Period. As this did not affect the mean body weights and no effects on body weight gain was noted during the remainder of the treatment period, this was considered unrelated to treatment with the test material.
See also table 10 under ''any other information on results incl. tables''
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption before or after correction for body weight was similar to the control level in males at 50 mg/kg bw/day and in females up to 150 mg/kg bw/day.
In males at 300 mg/kg bw/day, absolute food consumption was lower throughout most of the treatment period (up to 23% lower than control, no statistics performed) and relative food consumption was lower between Days 1-36 of premating. The slightly increased relative food consumption from Day 57 of premating onwards could be attributed to the lower body weights of the males during this period.
In females at 300 mg/kg bw/day, absolute food consumption was lower than control between Days 43-71 of the premating period (up to 23% lower, no statistics performed) and from Day 17 post-coitum onwards through lactation (up to 36% lower). Relative food consumption was intermittently lower for these females (only reaching statistical significance over Days 1- 4 of lactation).
The slightly lower absolute food consumption for males at 150 mg/kg bw/day between Days 29- 71 of the premating period was considered unrelated to treatment with the test material, as the effect was only minimal and values normalized thereafter. The slightly higher relative food consumption during the mating period could be attributed to the lower body weight of the males during this period.
See also table 11 under ''any other information on results incl. tables''
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No ophthalmology findings were noted that were considered to be related to the test material.
The nature and incidence of ophthalmology findings noted during the Pretreatment Period and in Week 13 was similar among the groups, and occurred within the range considered normal for rats of this age and strain. These findings were therefore considered to be unrelated to the test material.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Hematological parameters of treated rats were considered unaffected by treatment with the test material in males and females at 50 mg/kg bw/day.
The following changes were found (relative changes in mean values as compared to the concurrent control group are indicated in parentheses):
- In males, lower mean corpuscular volume at 150 and 300 mg/kg bw/day (0.96 and 0.95x of control, respectively)
- In males, lower mean corpuscular hemoglobin at 150 and 300 mg/kg bw/day (0.95 and 0.94x of control, respectively)
- In males, higher platelet levels at 300 mg/kg bw/day (1.33x of control), mainly caused by three males with high values
- In females, higher red blood cell distribution width at 150 and 300 mg/kg bw/day (1.09 and 1.10x of control, respectively)
Any other changes in hematology parameters were considered to be unrelated to treatment with the test material as these occurred in the absence of a dose-related trend and/or due to general overlap of the individual data with the control group.

Coagulation parameters were considered not to have been affected by treatment with the test material in females. In males, a dose-dependent shorter activated partial thromboplastin time was noted at 50, 150 and 300 mg/kg bw/day (0.85, 0.76 and 0.74x of control, respectively.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Clinical chemistry parameters of treated rats were considered not to have been affected by treatment with the test material in males at 50 mg/kg bw/day and in females up to 150 g/kg bw/day.
The following changes were found (relative changes in mean values as compared to the concurrent control group are indicated in parentheses):
- In males, lower total protein levels at 300 mg/kg bw/day (0.93x of control)
- Higher total bilirubin levels in males at 150 and 300 mg/kg bw/day (1.28 and 1.25x of control, respectively) and in females at 300 mg/kg bw/day (1.22x of control)
- In males, higher urea levels at 300 mg/kg bw/day (1.27x of control)
- In males, higher low density lipoprotein levels at 300 mg/kg bw/day (1.58x of control)
- Higher phosphate levels in males at 300 mg/kg bw/day (1.12x of control) and lower phosphate levels in females at 300 mg/kg bw/day (0.37x of control)
Any other changes in clinical chemistry parameters were considered to be unrelated to treatment with the test material as these occurred in the absence of a dose-related trend and/or due to general overlap of the individual data with the control group.
Endocrine findings:
effects observed, treatment-related
Description (incidence and severity):
Serum levels of T3 and T4 in F0-males, and serum levels of T3, T4 and TSH in F0-females were considered unaffected by treatment with the test material up the highest dose level tested.
Serum levels of TSH were lower in males at 300 mg/kg bw/day (0.27x of control or 0.0423 mU/L). Mean values were within the historical control data range [ Historical control data (period 2015-2017): Thyroid Stimulating Hormone (mU/L) - mean: 0.1795, Central 95%: 0.0302-0.6685 (n=135)]
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Functional observation parameters were considered unaffected by treatment in males up to 150 mg/kg bw/day and in females at 50 mg/kg bw/day.
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals.
In males at 300 mg/kg bw/day, grip strength of both the front and hind legs was lower than control (28 and 33% lower, respectively).
In females, a dose-dependently lower grip strength of the front legs was observed at 150 and 300 mg/kg bw/day (10 and 22% lower than control, respectively, not statistically significant).
Motor activity was considered not to be affected by treatment with the test material. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the Test Period.
In females at 50 mg/kg bw/day, the higher number of total movements was considered unrelated to treatment with the test material in absence of a dose-dependent trend.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Microscopic findings were observed in the non-glandular stomach, small intestines, mesenteric lymph node (ln), kidneys, thymus and bone marrow of the 150 and/or 300 mg/kg bw/day group males and/or females and are summarized in Tables 2 - 5 under ''Any other information on results incl. tables''.
Squamous cell hyperplasia was noted in the non-glandular stomach (forestomach) in 1/10 males and 1/10 females of the 150 mg/kg bw/day group (minimal) and in 7/10 males and 4/10 females of the 300 mg/kg bw/day group (up to moderate).
Erosion/ulceration of the non-glandular mucosa was noted in 3/10 females of the 300 mg/kg bw/day group (up to slight).
Edema of the non-glandular mucosa was noted in 2/10 males and 2/10 females of the 300 mg/kg bw/day group (up to slight).
Lymphogranulocytic infiltrate of the non-glandular mucosa was noted in 1/10 males and 4/10 females of the 300 mg/kg bw/day group (up to slight).
Foamy macrophage accumulation of the duodenum was recorded in 4/9 males of the 150 mg/kg bw/day group (minimal) and in 9/10 males and 6/10 females of the 300 mg/kg bw/day group (up to slight).
Foamy macrophage accumulation of the jejunum was recorded in 10/10 males and 6/10 females of the 150 mg/kg bw/day group (up to moderate) and in 10/10 males and 10/10 females of the 300 mg/kg bw/day group (up to marked).
Foamy macrophage accumulation of the ileum was recorded in 2/10 females of the 300 mg/kg bw/day group (unscheduled sacrificed only, minimal).
The foamy macrophage accumulation of the small intestines was located in the lamina propria of the villi.
Intra-sinusoidal foamy macrophages of the mesenteric lymph node were recorded in 10/10 males and 10/10 females of the 150 mg/kg bw/day group (up to slight) and in 10/10 males and 10/10 females of the 300 mg/kg bw/day group (up to marked).
Foamy macrophage aggregates of the (para)cortex and/or medullary cords of the mesenteric lymph node were recorded in 3/10 males and 1/10 females of the 150 mg/kg bw/day group (up to slight) and in 5/10 males and 1/10 females of the 300 mg/kg bw/day group (minimal).
Lymphangiectasis of the mesenteric lymph node was recorded in 4/10 males of the 300 mg/kg bw/day group (up to slight).
An increased incidence and severity of hyaline droplet accumulation was noted in 10/10 males of the 300 mg/kg bw/day group (up to moderate).
An increased incidence and severity of decreased lymphoid cellularity was noted in 5/10 females of the 300 mg/kg bw/day group (up to moderate), compared to 0/10 Control females, 1/10 females of the 50 mg/kg bw/day group (minimal) and 0/10 females of the 150 mg/kg bw/day group. The single incidence at 50 mg/kg bw/day was considered to be within background pathology.
Increased apoptosis of lymphocytes was noted in 1/10 females at 150 mg/kg bw/day and in 9/10 females of the 300 mg/kg bw/day group (up to moderate). The single incidence at 150 mg/kg bw/day was considered to be within background pathology.
A higher incidence of increased adipocytes was noted in 5/10 females of the 300 mg/kg bw/day group (up to slight), compared to single incidences in the Control, 50 and 150 mg/kg bw/day group.
Furthermore, there were minor changes at 300 mg/kg bw/day, which were present at low incidence and severity. This included minimal tubular vacuolation of the kidneys in 3/10 females, which was regarded to be related to the moribundity and physiologic status of these animals.
The remainder of the recorded microscopic findings was within the range of background pathology encountered in rats of this age and strain. There was no test material-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.


For correlation of histopathology findings in reproductive organs with in-life reason for Males that failed to sire and females that failed to deliver healthy pups see Table 6 under ''any other information on results incl. tables''. Total litter loss occurred in a single female of the 150 mg/kg bw/day group on Lactation Day 6, and in 3/10 females of the 300 mg/kg bw/day group on Lactation Day 2 or Day 6. Furthermore, one 50 mg/kg bw/day couple, one 150 mg/kg bw/day female and one 300 mg/kg bw/day couple did not deliver offspring despite proof of mating. There were no morphological findings in the reproductive organs of either sex (including mammary gland in case of total litter loss), which could be attributed to the test material.
Histopathological findings: neoplastic:
not examined
Reproductive function: oestrous cycle:
effects observed, treatment-related
Description (incidence and severity):
Length and regularity of the estrous cycle were considered not to have been affected by treatment with the test material up to 150 mg/kg bw/day. All females up to 150 mg/kg bw/day had regular cycles of 4 days.
At 300 mg/kg bw/day, only one female had a regular estrous cycle of 4 days (female with no litter). Another two high-dose females were noted with an irregular cycle. Both females had a normal litter. For all remaining 7/10 females cycle regularity could not be determined as these females had only one complete estrous cycle (of 5 days) during the 14 days observation period. All these 7 females became pregnant and the fertility index at this dose level was unaffected.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Stage dependent qualitative evaluation of spermatogenesis in the testis was performed. The testes revealed normal progression of the spermatogenic cycle and the expected cell associations and proportions in the various stages of spermatogenesis were present.
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
Mating index was not affected by treatment with the test material. All females showed
evidence of mating.

Precoital time was considered not to be affected by treatment with the test material. Most females showed evidence of mating within 4 days, except for one female in the control group, one female at 150 mg/kg bw/day and one female at 300 mg/kg bw/day for which it took respectively 14, 14 and 6 days before mating could be confirmed. At the isolated incidences and in absence of a dose-related response, this was considered unrelated to treatment with the test material.

Number of corpora lutea and implantation sites were considered not to be affected by treatment with the test material up to 150 mg/kg bw/day. The number of corpora lutea and implantation sites was lower at 300 mg/kg bw/day (11.7 vs. 13.5 in control and 10.1 vs. 12.2 in control, respectively). The lower number of implantation sites at 150 mg/kg bw/day could be attributed to two females which only had 2 implantation sites while the number of corpora lutea was 11 and 12 in both females, respectively. After excluding these two females, a mean of 12.3 implantation sites was obtained which is comparable to the control group (i.e., 12.2). As this low number of implantation sites occurred in absence of a dose-response, it was considered not related to treatment with the test material.

Fertility index was considered not to be affected by treatment with the test material. The fertility indices were 100, 90, 100 and 90% for the control, 50, 150 and 300 mg/kg bw/day groups, respectively. One female at 50 mg/kg bw/day and one female at 300 mg/kg bw/day were not pregnant. In the absence of a dose-related incidence of non-pregnancy, this was considered unrelated to treatment with the test material.

See also Tables 7 and 8 under ''any other information on results incl. tables''
For males, “Repro period” represents the mating phase. For females, “Repro period” represents the mating, post-coitum and lactation phases.
As only 4 females at 300 mg/kg bw/day survived to scheduled sacrifice, clinical chemistry haematology and coagulation parameters were only available from these 4 females. The same applies to functional observations and motor activity.
As the macroscopic and microscopic findings of the high-dose females that were sacrificed in extremis were similar as in scheduled sacrificed females at 300 mg/kg bw/day, the findings from females sacrificed in extremis have been listed together with those from scheduled sacrificed females.
Key result
Dose descriptor:
NOAEL
Remarks:
Parental
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Clinical signs, lower body weights and food consumption, macroscopic and microscopic findings in the stomach, small intestine and mesenteric lymph nodes.
Key result
Dose descriptor:
NOAEL
Remarks:
Reproductive
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Lower number of corpora lutea and implantation sites
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
150
System:
gastrointestinal tract
Organ:
duodenum
jejunum
mesenteric lymph node
stomach
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No clinical signs occurred among pups surviving until scheduled necropsy that were considered to be related to treatment with the test material.
The nature and incidence of clinical signs noted remained within the range considered normal for pups of this age, and were therefore considered not to be test material-related.
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
Live birth index (number of live offspring on PND 1 as percentage of total number of
offspring born) was considered not to be affected by treatment with the test material. The live
birth indices were 100, 99, 100 and 100% for the control, 50, 150 and 300 mg/kg bw/day groups, respectively. The lower live birth index at 50 mg/kg bw/day was the result of one pup that was found dead at first litter check. In absence of a dose-response relation and as this mortality incidence remained within the range considered normal for pups of this age, this was considered unrelated to treatment with the test material.

Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was considered not to be affected by treatment with the test material up to 150 mg/kg bw/day. Viability indices were 99, 100 and 100% for the control, 50 and 150 mg/kg bw/day groups, respectively. At 300 mg/kg bw/day, the viability index was lower (69 vs. 99% in control). This lower viability index could mainly be attributed to two females with a total litter loss. All pups of these females were either found missing, found dead, or were sacrificed in extremis on PND 2. Consequently, both females were sent for necropsy with a total litter loss. For the pups of one of the high-dose females (with total litter loss) that were found dead, no milk was noted in their stomach at necropsy. In addition to these total litter losses, one pup of another high-dose litter was found missing on PND 3. The lower viability index in the control group could be attributed to one pup that went missing on PND 3. Missing pups were most likely cannibalized. As this occurred in the control group, this was not related to treatment with the test material.
See also table 9 under ''any other information on results incl. tables''
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weights of pups were considered not to be affected by treatment with the test material up to 50 mg/kg bw/day.
At 150 mg/kg bw/day, body weight of both male and female pups was lower on PND 4 and 7 (down to 12 and 9% lower than control for males and females, respectively, not statistically significant for females on PND 4 and for males on PND 7), but recovered on PND 13.
At 300 mg/kg bw/day, body weight of pups was lower from PND 1 onwards (down to 24 and 23% lower than control for males and females, respectively).
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Serum T4 levels in male and female PND 14-16 pups were considered not to be affected by treatment with the test material.
Anogenital distance (AGD):
effects observed, non-treatment-related
Description (incidence and severity):
Anogenital distance (absolute and corrected for body weight) in male and female pups was considered not to be affected by treatment with the test material. Shorter absolute anogential distance was noted for males and females at 300 mg/kg bw/day, which was considered related to the lower body weights on PND 1. After correcting the anogenital distance for body weight, the mean normalized anogenital distance was still lower, but individual values generally remained within the same range as control and therefore, this was considered not related to treatment with the test material.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
Treatment with the test material had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No macroscopic findings were noted among pups surviving until scheduled necropsy that were considered to be related to treatment with the test material.
The nature and incidence of macroscopic findings remained within the range considered normal for pups of this age, and were therefore considered not to be related to treatment with the test material.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Gestation index (females with living pups on Day 1 compared to the number of pregnant females) and duration of gestation were considered not to be affected by treatment with the test material. Except for one female at 150 mg/kg bw/day, all pregnant females had live offspring. The gestation indices were therefore 100, 100, 90 and 100 % for the control, 50, 150 and 300 mg/kg bw/day groups, respectively. The failed pregnancy of one female at 150 mg/kg bw/day that had two implantation sites only, was considered unrelated to treatment with the test material in absence of a dose-related trend.

No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed at 150 mg/kg bw/day. At 300 mg/kg bw/day, the pups of three high-dose litters, were found to be cold, dehydrated, with less or no milk in their stomach, and/or weighing less than 50% of other pups of a similar age on PND 2 and 6. Based on their poor clinical condition, the pups in these litters were sacrificed in extremis. At necropsy, no milk was noted in the stomachs of all pups of two of these three litters. No macroscopic abnormalities were noted for the pups of the other litter. These clinical signs and macroscopic findings were indicative of a deficiency in maternal care. At 150 mg/kg bw/day, the single pup of a mid-dose litter was sacrificed in extremis on PND 6. This pup was found to be dehydrated and weighed less than 50% of pups of a similar age from PND 4 onwards. These signs were indicative of a lack of maternal care. As this was observed in only one litter of this dose group, this was considered unrelated to treatment with the test material.

Post-implantation survival index (total number of offspring born as percentage of total
number of uterine implantation sites) was 93, 91, 97 and 97% for the control, 50, 150 and
300 mg/kg bw/day groups, respectively; and was not considered to be affected by treatment with the test material.

Litter size was considered not affected by treatment with the test material up to 150 mg/kg bw/day. Live litter sizes were 11.4, 11.8 and 11.0 living pups/litter for the control, 50 and 150 mg/kg bw/day groups, respectively. At 300 mg/kg bw/day, the live litter size was smaller than control (9.7 vs.11.4 pups/litter).

The lactation index [number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4 (after culling)] was considered not to be affected by treatment with the test material up to 150 mg/kg bw/day. The lactation indices were 100, 100 and 98% for the control, 50 and 150 mg/kg bw/day groups, respectively. At 300 mg/kg bw/day, the lower lactation index could be attributed to one female with a total litter loss. All pups of this female were sacrificed in extremis on PND 6 and, consequently, the female was sacrificed with a total litter loss. The lower lactation index at 150 mg/kg bw/day (98%) was the result a total litter loss of one female whose only pup was sacrificed in extremis on PND 6. As the incidence of pup mortality in this dose group remained in the range considered normal for pups of this age, this was considered unrelated to treatment with the test material.

Sex ratio was considered not to be affected by treatment with the test material.

See also table 9 under ''any other information on results incl. tables''
As females of the 1000 mg/kg dose group were euthanized in the post-coitum period, developmental data is available only for the 0, 100, and 300 mg/kg dose groups.

As at 300 mg/kg bw/day two females were sacrificed in extremis during the Post-coitum Period and one female was not pregnant, developmental data is available of only 7 females at this dose level up until PND 2. Between PND 2-6, data of only 5 litters was available, and from PND 6 onwards, data of only 4 litters was available as a results of high pup mortality resulting in 3 total litter losses. The developmental results at 300 mg/kg bw/day from PND 2 onwards should therefore be interpreted with caution.

Two females at 300 mg/kg bw/day were excluded from calculations of the gestation index and the post-implantation survival index as these females were sacrificed in extremis on Days 18 and 20 post-coitum, respectively.
Key result
Dose descriptor:
NOAEL
Remarks:
Developmental
Generation:
F1
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Small live litter sizes, high pup mortality and low pup body weights.
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects as a secondary non-specific consequence of other toxic effects
Dose response relationship:
yes
Relevant for humans:
yes

RESULTS OF THE DOSE FORMULATION ANALYISIS:


Accuracy
In the control group formulation prepared for the use in Week 1 and Week 12 of treatment, no test material was detected. Small responses at the retention time of the test item were observed in the chromatograms of the control group formulation prepared for use in Week 6 and Week 8. With a maximal contribution to the low-dose group samples of respectively 0.00037 and 0.3%, they were considered negligible. The concentrations analyzed in the formulations of treatment groups prepared for the use in Weeks 1, 8 and 12 were in agreement with target concentrations (i.e., mean sample concentration results were within or equal to 90-110% of target concentration). The concentrations analyzed in the low-dose group formulations prepared for the use in Week 6 were in agreement with target concentrations (i.e., mean sample concentration results were within or equal to 90-110% of target concentration). Initially analyzed concentrations in mid- and high-dose group formulations prepared for the use in Week 6 were above the criterion of 90- 110% (i.e., 113% and 118% respectively). In order to exclude a dilution error, samples were re-diluted and analysed the following day. After re-dilution, the mean recovery of mid-dose group formulations was within the criteria whereas the mean recovery of high-dose group formulations was slightly below the criteria (i.e., 88%). No analytical reason could be found for these out of specification results. As the formulations prepared for Week 1, 8 and 12 of treatment were all in agreement with target concentrations, the small deviation of the concentration of the Week 6 samples was considered not to have impacted the study integrity.


Homogeneity
Low- and high-dose group formulations were homogeneous (i.e., coefficient of variation ≤ 10%).


 


Table 2 - Summary Test Material-Related Microscopic Findings Non-Glandular Stomach – Both Sexes















































































































































































 

Males



Females



Dose level (mg/kg bw/day):



0



50



150



300



0



50



150



300



NON-GLANDULAR STOMACH



10



10



10



10



10



10



10



10



Hyperplasia, squamous cell



 



 



 



 



 



 



 



 



Minimal



-



-



1



4



-



-



1



1



Slight



-



-



-



2



-



-



-



3



Moderate



-



-



-



1



-



-



-



-



Erosion / Ulcer



 



 



 



 



 



 



 



 



Minimal



-



-



-



-



-



-



-



2



Slight



-



-



-



-



-



-



-



1



Edema



 



 



 



 



 



 



 



 



Minimal



-



-



-



1



-



-



-



-



Slight



-



-



-



1



-



-



-



2



Lymphogranulocytic infiltrate



 



 



 



 



 



 



 



 



Minimal



-



-



-



1



-



-



-



2



Slight



-



-



-



-



-



-



-



2



 


Table 3  - Summary Test Material-Related Microscopic Findings in Small Intestines and Mesenteric Lymph nodes – Both Sexes




















































































































































































































































































































 



Males



Females



Dose level (mg/kg bw/day):



0



50



150



300



0



50  



150



300



DUODENUMa



10



10



9



10



10



10



10



10



Foamy macrophage accumulation



 



 



 



 



 



 



 



 



Minimal



-



-



4



3



-



-     



-



3



Slight



-



-



-



6



-



-   



-



3



JEJUNUMa



10



10



10



10



10



10



10



10



Foamy macrophage accumulation



 



 



 



 



 



 



 



 



Minimal



-



-



-



-



-



-



4



1



Slight



-



-



5



-



-


-2

5



Moderate



-



-



5



7



-


--

3



Marked



-



-



-



3



-



-



-



1



ILEUMa



10



10



10



10



10



10



10



10



Macrophage accumulation



 



 



 



 



 



 



 



 



Minimal



-



-



-



-



-



-



-



2



MESENTERIC LNa



10



10



10



10



10



10



10



10



Foamy macrophage intra-sinusoidal



 



 



 



 



 



 



 



 



Minimal



-



-



3



-



-



-



4



-



Slight



-



-



7



-



-



-



6



2



Moderate



-



-



 



7



-



-



-



8



Marked



-



-



-



3



-



-



-



-



Foamy macrophage aggregates



 



 



 



 



 



 



 



 



Minimal



-



-



2



5



-



-



1



1



Slight



-



-



1



-



-



-



-



-



Moderate



-



-



-



-



-



-



-



-



Lymphangiectasis



 



 



 



 



 



 



 



 



Minimal



-



-



-



1



-



-



-



-



Slight



-



-



-



3



-



-



-



-



a = Number of tissues examined from each group.


 


Table 4 - Summary Test Material-Related Microscopic Findings Kidneys –Males




















































 



Males



Dose level (mg/kg bw/day):



0



50



150



300



KIDNEY



10



10



10



10



Hyaline droplet accumulation



 



 



 



 



Minimal



7



3



4



1



Slight



1



3



5



2



Moderate



-



-



-



7



 


Table 5 - Summary Test Material-Related Microscopic Findings Thymus and Bone Marrow –Females


 












































































































 



Females



Dose level (mg/kg bw/day):



0



50



150



300



THYMUS



10



10



10



10



Decreased cellularity, lymphoid



 



 



 



 



Minimal



-



1



-



2



Slight



-



-



-



2



Moderate



-



-



-



1



Increased apoptosis



 



 



 



 



Minimal



-



-



1



6



Slight



-



-



-



2



Moderate



-



-



-



1



BONE MARROW



10



10



10



10



Increased adipocytes



 



 



 



 



Minimal



1



-



1



3



Slight



-



-



1



2



a = Number of tissues examined from each group.


 


Table 6 - Correlation of Histopathology Findings of Reproductive Organs with In-Life Reason for Males that Failed to Sire and Females that Failed to Deliver Healthy Pups.




































Group



Dose level mg/kg bw/day



In-Life Reason



Histopathology



1



0



n.a.



 



2



50



Not pregnant



n.a.d



3



150



Total litter loss, LD6


Implantation sites only



n.a.d


n.a.d



4



300



Total litter loss, LD2


Total litter loss, LD6


Not pregnant


Total litter loss, LD2



n.a.d.


n.a.d.


n.a.d.


n.a.d.



n.a.d. = no abnormalities detected, histology in line with normal cycle/lactation day.


n.a = not applicable; LD = Lactation Day; PC = Post coitum.


 


Table 7 - Reproduction Data Summary


















































































 

GROUP 1 - CONTROL



GROUP 2 - 50 MG/KG BW/DAY



GROUP 3 - 150 MG/KG BW/DAY



GROUP 4 - 300 MG/KG BW/DAY



Females paired



10



10



10



10



Females mated



10



10



10



10



Pregnant females



10



9



10



9



Females with implantations only



0



0



1



0



Females dead on post coitum Day 18



0



0



0



1



Females dead on post coitum Day 20



0



0



0



1



Females with living pups on Day 1



10



9



9



7



Mating index (%)


(Females mated / Females paired) * 100



100



100



100



100



Fertility index (%)


(Pregnant females / Females mated) * 100



100



90



100



90



Gestation index (%)


(Females with living pups on Day 1 / Pregnant females) * 100



100



100



90



100 !



!: Two females at 300 mg/kg bw/day were excluded from calculation of the gestation index since these females were sacrificed in extremis on post-coitum Day 18 and 20, respectively


 


Table 8 - Corpora Lutea and Implantation Sites Summary Females 





























































 



 


GROUP 1 CONTROL



 


GROUP 2 - 50 MG/KG BW/DAY



 


GROUP 3 - 150 MG/KG BW/DAY



 


GROUP 4 - 300 MG/KG BW/DAY



 


AT NECROPSY


Corpora Lutea



 


MEAN



 


13.5



 


13.6



 


12.4



 


11.7 +



 



ST.DEV



1.4



1.9



1.0



1.1



 



N



10



9



10



9



Implantations



MEAN



12.2



13.0



10.2



10.1 ++



 



ST.DEV



1.0



2.1



4.5



1.2



 



N



10



9



10



9



+/++ Steel-test significant at 5% (+) or 1% (++) level


 


Table 9- Developmental Data




























































































































































































































































































































































 



GROUP 1 CONTROL



GROUP 2 - 50 MG/KGBW/DAY



GROUP 3 - 150 MG/KGBW/DAY



GROUP 4 - 300 MG/KGBW/DAY



LITTERS TOTAL



 


10



 


9



 


9



 


7



DURATION OF GESTATION MEAN (+)



 


21.3



 


21.3



 


21.2



 


21.0



ST.DEV.



0.5



0.5



0.4



0.0



N



10



9



9



7



DEAD PUPS AT FIRST LITTER CHECK



LITTERS AFFECTED (#)



0



1



0



0



TOTAL



0



1



0



0



MEAN (+)



0.0



0.1



0.0



0.0



ST.DEV.



0.0



0.3



0.0



0.0



N



10



9



9



7



LIVING PUPS AT FIRST LITTER CHECK



% OF MALES / FEMALES (#)



44 / 56



48 / 52



49 / 51



47 / 53



TOTAL



114



106



99



68



MEAN (+)



11.4



11.8



11.0



9.7 +



ST.DEV.



1.4



2.1



3.9



1.0



N



10



9



9



7



POSTNATAL LOSS


% OF LIVING PUPS



 


0.9



 


0.0



 


0.0



 


30.9



LITTERS AFFECTED (#)



1



0



0



3



TOTAL (#)



1



0



0



21 ##



MEAN (+)



0.1



0.0



0.0



3.0



ST.DEV.



0.3



0.0



0.0



4.8



N



10



9



9



7



CULLED PUPS TOTAL



33



34



34



7



LIVING PUPS DAY 4 P.P. TOTAL



80



72



65



40



MEAN (+)



8.0



8.0



7.2



5.7



ST.DEV.



0.0



0.0



2.3



3.9



N



10



9



9



7



BREEDING LOSS DAYS 5 - 13 P.P.


% OF LIVING PUPS AT DAY 4 P.P.



 


0.0



 


0.0



 


1.5



 


20.0



LITTERS AFFECTED (#)



0



0



1



1



TOTAL (#)



0



0



1



8 ##



MEAN (+)



0.0



0.0



0.1



1.1



ST.DEV.



0.0



0.0



0.3



3.0



N



10



9



9



7



LIVING PUPS DAY 13 P.P.


% OF MALES / FEMALES (#)



 


50 / 50



 


51 / 49



 


52 / 48



 


50 / 50



TOTAL



80



72



64



32



MEAN (+)



8.0



8.0



7.1



4.6



ST.DEV.



0.0



0.0



2.7



4.3



N



10



9



9



7



 



 



 



 



 



Total number of offspring born



114



107



99



68



Total number of uterine implantation sites



122



117



102



91



Number of live offspring on Day 1 after littering



114



106



99



68



Number of live offspring on Day 4 (before culling)



113



106



99



47



Number of live offspring on Day 4 (after culling)



80



72



65



40



Number of live offspring on Day 13 after littering



80



72



64



32



 Post-implantation survival index (%)


(Total number of offspring born/Total number of uterine implantation sites) * 100



 


93



 


91



 


97



 


97 !



Live birth index (%)


(Number of live offspring on Day 1 after littering/Total number of offspring born) * 100



100



99



100



100



Viability index (%)


(Number of live offspring on Day 4 (before culling)/Number of live offspring on Day 1 after littering)*100



99



100



100



69



Lactation index (%)


(Number of live offspring on Day 13 after littering/Number of live offspring on Day 4 (after culling)) * 100



100



100



98



80



+/++ Steel-test significant at 5% (+) or 1% (++) level


# / ## Fisher's Exact test significant at 5% (#) or 1% (##) level


!: Two females at 300 mg/kg bw/day were excluded from calculation of the post-implantation survival index since these females were sacrificed in extremis on post-coitum Day 18 and 20, respectively


 


Table 10 – Body Weights (gram) summary.






































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































 



 



 


GROUP 1 CONTROL



 


GROUP 2


50 MG/KG/DAY



 


GROUP 3


150


MG/KG/DAY



 


GROUP 4


300


MG/KG/DAY



 


MALES PRE MATING



 


 



 


 



 


 



 


 



 


 



DAY 1



MEAN



200



196



197



197



WEEK 1



ST.DEV



9.5



9.1



9.0



5.0



 



N



10



10



10



10



DAY 8



MEAN



245



240



239



232 *



WEEK 2



ST.DEV



13.2



10.2



9.9



5.5



 



N



10



10



10



10



DAY 15



MEAN



284



277



277



258 **



WEEK 3



ST.DEV



14.4



11.7



12.8



6.9



 



N



10



10



10



10



DAY 22



MEAN



315



305



303



277 **



WEEK 4



ST.DEV



16.2



14.3



17.7



10.5



 



N



10



10



10



10



DAY 29



MEAN



341



329



325



289 **



WEEK 5



ST.DEV



18.1



16.0



20.9



16.6



 



N



10



10



10



10



DAY 36



MEAN



363



349



334 *



295 **



WEEK 6



ST.DEV



20.4



19.4



25.2



18.7



 



N



10



10



10



10



DAY 43



MEAN



376



365



346 *



303 **



WEEK 7



ST.DEV



24.1



21.2



27.7



24.0



 



N



10



10



10



10



DAY 50



MEAN



393



377



353 **



305 **



WEEK 8



ST.DEV



24.5



21.8



30.1



22.3



 



N



10



10



10



10



DAY 57



MEAN



405



389



360 **



307 **



WEEK 9



ST.DEV



25.9



21.6



33.3



24.9



 



N



10



10



10



10



DAY 64



MEAN



419



402



366 **



317 **



WEEK 10



ST.DEV



28.2



21.1



33.9



25.4



 



N



10



10



10



10



MALES - MATING PERIOD



 


 



 


 



 


 



 



 


 



DAY 1



MEAN



426



408



372 **



322 **



WEEK 1



ST.DEV



28.3



21.9



35.8



24.8



 



N



10



10



10



10



DAY 8



MEAN



432



414



375 **



317 **



WEEK 2



ST.DEV



30.0



22.8



33.7



23.3



 



N



10



10



10



10



DAY 15



MEAN



437



420



382 **



323 **



WEEK 3



ST.DEV



32.3



22.0



37.3



23.7



 



N



10



10



10



10



DAY 22



MEAN



443



428



389 **



326 **



WEEK 4



ST.DEV



32.5



23.4



40.1



23.0



 



N



10



10



10



10



FEMALES PRE MATING



 


 



 


 



 


 



 


 



 


 



DAY 1



MEAN



126



122



118



122



WEEK 1



ST.DEV



11.1



7.2



5.8



7.8



 



N



10



10



10



10



DAY 8



MEAN



145



142



138



142



WEEK 2



ST.DEV



9.3



7.8



6.8



8.1



 



N



10



10



10



10



DAY 15



MEAN



166



161



159



161



WEEK 3



ST.DEV



14.6



7.8



10.6



9.2



 



N



10



10



10



10



DAY 22



MEAN



184



181



176



177



WEEK 4



ST.DEV



13.9



9.7



10.7



11.1



 



N



10



10



10



10



DAY 29



MEAN



196



191



185



187



WEEK 5



ST.DEV



16.9



12.6



12.1



11.9



 



N



10



10



10



10



DAY 36



MEAN



206



204



194



195



WEEK 6



ST.DEV



14.5



12.7



11.1



13.9



 



N



10



10



10



10



DAY 43



MEAN



215



210



203



203



WEEK 7



ST.DEV



19.4



11.6



14.4



13.4



 



N



10



10



10



10



DAY 50



MEAN



223



218



209



203 *



WEEK 8



ST.DEV



19.8



13.2



13.1



15.2



 



N



10



10



10



10



DAY 57



MEAN



228



222



212



208 *



WEEK 9



ST.DEV



21.0



13.3



13.8



15.2



 



N



10



10



10



10



DAY 64



MEAN



229



227



214



213 *



WEEK 10



ST.DEV



17.3



13.9



12.6



15.4



 



N



10



10



10



10



FEMALES MATING PERIOD



 



 



 



 



 



DAY 1



MEAN



234



229



218



210 **



WEEK 1



ST.DEV



20.0



12.1



15.0



12.7



 



N



10



10



10



10



DAY 8



MEAN



261



--



208



 



WEEK 2



ST.DEV



--



--



--



 



 



N



1 x



0 x



1



 



DAY 15



MEAN



--



--



 



 



WEEK 3



ST.DEV



--



--



 



 



 



N



0 x



0 x



 



 



DAY 22



MEAN



--



--



 



 



WEEK 4



ST.DEV



--



--



 



 



 



N



0 x



0 x



 



 



FEMALES - POST COITUM



 


 



 


 



 


 



 


 



 


 



DAY 0



MEAN



236



233



220



212**



 



ST.DEV.



20.5



15.3



10.8



15.3



 



N



9



7



10



9



DAY 4



MEAN



246



246



232



220 **



 



ST.DEV.



21.3



18.0



12.5



17.6



 



N



9



7



10



9



DAY 7



MEAN



254



252



238



224 **



 



ST.DEV.



23.4



15.7



13.3



17.6



 



N



9



7



10



9



DAY 11



MEAN



266



267



253



236 **



 



ST.DEV.



23.3



17.3



14.9



19.7



 



N



9



7



10



9



DAY 14



MEAN



275



275



259



239 **



 



ST.DEV.



23.2



16.7



16.4



22.2



 



N



9



7



10



9



DAY 17



MEAN



298



300



283



255 **



 



ST.DEV.



25.8



20.5



24.0



28.3



 



N



9



7



10



9



DAY 20



MEAN



337



340



315



277 **



 



ST.DEV.



28.9



22.5



32.7



11.0



 



N



9



7



10



8



FEMALES - LACTATION



 



 



 



 



 



DAY 1



MEAN



265



260



247



210 **



 



ST.DEV.



26.2



12.7



11.3



12.4



 



N



10



9



9



7



DAY 4



MEAN



274



268



263



225 **



 



ST.DEV.



24.9



13.2



15.9



11.7



 



N



10



9



9



5



DAY 7



MEAN



283



277



272



238 **



 



ST.DEV.



22.5



13.9



16.4



6.7



 



N



10



9



8



4



DAY 13



MEAN



292



283



285



257 *



 



ST.DEV.



24.5



14.2



22.6



13.6



 



N



10



9



8



4



*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level


x Explanations for excluded data are listed in the study report (tables of the individual values)


 


Table 11 – Food Consumption (g/animal/day)






































































































































































































































































































































































































































































































































































































































































































































































































































































































































 



 



GROUP 1 - CONTROL



GROUP 2 –


50 MG/KGBW/DAY



GROUP 3 –


150 MG/KGBW/DAY



GROUP 4 –


300 MG/KGBW/DAY



PRE MATING - MALES



 



 



 



 



 



DAYS 1-8



MEAN



22



21



20



19



WEEKS 1-2



ST.DEV



0.5



0.2



0.6



0.9



 



N (CAGE)



2



2



2



2



DAYS 8-15                            



MEAN



22



22



22



19



WEEKS 2-3                          



ST.DEV



0.1



1.4



1.0



1.2



 



N (CAGE)



2



2



2



2



DAYS 15-23                          



MEAN



22



21



21



19



WEEKS 3-4                          



ST.DEV



0.1



1.0



0.3



1.0



 



N (CAGE)



2



2



2



2



DAYS 23-29



MEAN



22



21



21



17



WEEKS 4-5



ST.DEV



0.9



1.2



1.6



0.7



 



N (CAGE)



2



2



2



2



DAYS 29-36



MEAN



22



21



19



17



WEEKS 5-6



ST.DEV



0.7



1.2



0.7



2.0



 



N (CAGE)



2



2



2



2



DAYS 36-43



MEAN



21



21



19



17



WEEKS 6-7



ST.DEV



1.4



1.3



0.1



1.7



 



N (CAGE)



2



2



2



2



DAYS 43-50



MEAN



21



20



18



15



WEEKS 7-8



ST.DEV



---



1.7



0.0



3.1



 



N (CAGE)



1 x



2



2



2



DAYS 50-57



MEAN



19



19



17



15



WEEKS 8-9



ST.DEV



0.4



1.6



0.3



2.9



 



N (CAGE)



2



2



2



2



DAYS 57-64



MEAN



19



19



17



17



WEEKS 9-10



ST.DEV



0.6



0.8



0.3



0.9



 



N (CAGE)



2



2



2



2



DAYS 64-71



MEAN



19



18



17



17



WEEKS 10-11



ST.DEV



0.6



0.5



0.1



1.2



 



N (CAGE)



2



2



2



2



MEAN OF MEANS OVER PRE MATING



MEAN



21



20



19



17



MATING PERIOD - MALES 



 



 



 



 



 



DAYS 1-8



MEAN



20



19



18



16



WEEKS 1-2



ST.DEV



0.2



0.1



0.8



0.9



 



N (CAGE)



2



2



2



2



DAYS 8-15



MEAN



18



18



18



16



WEEKS 2-3



ST.DEV



0.5



0.2



0.6



0.5



 



N (CAGE)



2



2



2



2



DAYS 15-22



MEAN



18



18



18



16



WEEKS 3-4



ST.DEV



0.3



0.1



0.0



0.1



 



N (CAGE)



2



2



2



2



DAYS 22-29



MEAN



---



---



---



---



WEEKS 4-5



ST.DEV



---



---



---



---



 



N (CAGE)



0



0



0



0



PRE MATING - FEMALES



 



 



 



 



 



DAYS 1-8



MEAN



14



13



12



13



WEEKS 1-2



ST.DEV



0.1



0.1



0.5



0.2



 



N (CAGE)



2



2



2



2



DAYS 8-15



MEAN



14



14



13



14



WEEKS 2-3



ST.DEV



0.0



0.1



0.7



0.1



 



N (CAGE)



2



2



2



2



DAYS 15-23



MEAN



14



15



14



14



WEEKS 3-4



ST.DEV



0.3



0.1



0.3



0.3



 



N (CAGE)



2



2



2



2



DAYS 23-29



MEAN



15



15



14



14



WEEKS 4-5



ST.DEV



0.4



0.4



0.4



0.2



 



N (CAGE)



2



2



2



2



DAYS 29-36



MEAN



15



15



14



13



WEEKS 5-6



ST.DEV



0.4



0.5



0.2



0.1



 



N (CAGE)



2



2



2



2



DAYS 36-43



MEAN



15



15



14



14



WEEKS 6-7



ST.DEV



0.1



0.1



0.8



0.6



 



N (CAGE)



2



2



2



2



DAYS 43-50



MEAN



14



15



13



12



WEEKS 7-8



ST.DEV



0.1



0.2



0.6



0.6



 



N (CAGE)



2



2



2



2



DAYS 50-57



MEAN



14



14



13



11



WEEKS 8-9



ST.DEV



0.4



0.1



0.6



0.3



 



N (CAGE)



2



2



2



2



DAYS 57-64



MEAN



13



14



12



12



WEEKS 9-10



ST.DEV



0.4



0.6



0.7



0.7



 



N (CAGE)



2



2



2



2



DAYS 64-71



MEAN



13



13



13



10



WEEKS 10-11



ST.DEV



0.1



0.7



0.6



0.5



 



N (CAGE)



2



2



2



2



MEAN OF MEANS OVER PRE MATING



MEAN



14



14



13



13



MATING PERIOD - FEMALES


DAYS 1-8



MEAN



---


 



---


 



---


 



---


 



WEEKS 1-2



ST.DEV



---



---



---



---



 



N (CAGE)



0



0



0



0



POST COITUM - FEMALES



 



 



 



 



 



DAYS 0-4



MEAN



15



17



15



14



 



ST.DEV.



1.8



0.9



2.8



2.2



 



N



8



7



10



9



DAYS 4-7



MEAN



16



18



16



15



 



ST.DEV.



1.4



1.2



1.9



4.3



 



N



8



7



9



9



DAYS 7-11



MEAN



18



19



18



19



 



ST.DEV.



2.6



1.6



2.5



5.3



 



N



9



7



10



8



DAYS 11-14



MEAN



18



18



18



17



 



ST.DEV.



2.3



1.0



2.7



3.9



 



N



9



7



10



9



DAYS 14-17



MEAN



18



19



18



18



 



ST.DEV.



3.6



2.0



2.2



5.9



 



N



9



7



10



9



DAYS 17-20



MEAN



22



21



21



16 **



 



ST.DEV.



2.3



1.6



2.3



3.6



 



N



9



7



10



8



 


MEAN OF MEANS



 



 


18



 


19



 


18



 


16



LACTATION - FEMALES



 



 



 



 



 



DAYS 1-4



MEAN



28



27



26



18**



 



ST.DEV.



3.3



3.0



5.5



3.9



 



N



10



9



9



5



DAYS 4-7



MEAN



39



37



37



31 **



 



ST.DEV.



2.1



3.0



4.1



1.4



 



N



10



9



8



4



DAYS 7-13



MEAN



51



49



49



41 **



 



ST.DEV.



2.6



4.3



4.6



3.9



 



N



10



9



8



4



MEAN OF MEANS



 



39



38



38



30



*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level


x Explanations for excluded data are listed in the study report (tables of the individual values)

Conclusions:
In conclusion, based on the results of this combined 90-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the following No Observed Adverse Effect Levels (NOAELs) of Tallow amine propoxylate were established:
Parental NOAEL: 50 mg/kg bw/day (based on clinical signs, lower body weights and food consumption, macroscopic and microscopic findings in the stomach, small intestine and mesenteric lymph nodes)
Reproduction NOAEL: 150 mg/kg bw/day (based on the lower number of corpora lutea and implantation sites)
Developmental NOAEL: 150 mg/kg bw/day (based on the small live litter sizes, high pup mortality and low pup body weights)
Executive summary:

The objectives of this study were to determine the potential toxic effects of Tallow amine propoxylate when given orally by gavage for a minimum of 90 days to Wistar Han rats, and to evaluate the potential to affect male and female reproductive performance such as gonadal function, mating behavior, conception, parturition and early postnatal development.
In addition, parental, reproduction (up to and including implantation) and developmental (from implantation onwards) No Observed Adverse Effect Levels (NOAELs) were evaluated.
The dose levels in this study were selected to be 0, 50, 150, 300 mg/kg bw/day, based on the results of a Dose Range Finder and a Prenatal Developmental Toxicity Study.


The study design was as follows:




















































 


Group No.



 


 


Test Item Id.



 


Dose Level (mg/kg bw/day)



Dose Volume


(mL/kg bw)



 


Dose Concentration (mg/mL)



Number of Animals



Males



Females



1



-



0 (Vehicle)



4



0



10



10



2



Tallow amine propoxylate



50



4



12.5



10



10



3



150



4



37.5



10



10



4



300



4



75



10



10



Id.= identification.


Chemical analyses of formulations were conducted in Weeks 1, 6, 8 and 12 of the study and confirmed that formulations in peanut oil were prepared accurately and homogeneously in Weeks 1, 8 and 12. The concentrations of the formulations of Groups 3 and 4 prepared for Week 6 of treatment were above the acceptance criterion. No analytical reason could be found for these out of specification result. As the formulations prepared for Weeks 1, 8 and
12 of treatment were all in agreement with target concentrations, the small deviation of the concentration of the Week 6 samples was considered not to impact the study integrity.
The following parameters and end points were evaluated in this study: mortality/moribundity, clinical signs, body weight and food consumption, ophthalmoscopy, functional observations, clinical pathology (hematology, coagulation, clinical chemistry, and measurement of thyroid hormones (total triiodothyronine (T3), thyroxine (T4) and thyroidstimulating hormone (TSH) in F0-males and females), gross necropsy findings, organ weights
and histopathologic examinations.
In addition, the following reproduction/developmental parameters were determined: estrous cycle, mating and fertility indices, precoital time, number of corpora lutea and implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy, measurement of thyroid hormone T4 (PND 14-16
pups)).
Parental results
Two premature sacrifices occurred in females at 300 mg/kg bw/day. One female was sacrificed in extremis on Day 18 post-coitum based on a deteriorating condition with clinical signs including hunched posture, piloerection, a pale and lean appearance and red staining of the snout. In addition, despite her pregnancy, this female displayed minimal weight gain up to slight weight loss over the post-coitum period. At necropsy, the uterus of this female contained 8 living fetuses and 1 early resorption. Furthermore, reddish foci were noted on the glandular mucosa of the stomach and yellowish discoloration of the mesenteric lymph nodes was observed.
Another female was sacrificed in extremis on Day 20 post-coitum based on a deteriorating condition with clinical signs including piloerection, hunched posture and a pale appearance.
In addition, on the day of sacrifice, this female displayed lethargy and flat posture. Moreover, this female did not gain any weight between Days 17-20 post-coitum while she was pregnant. At necropsy, the uterus of this female contained 12 living fetuses. Furthermore, oily yellowish contents of the stomach, duodenum, jejunum and ileum were noted, a pale discoloration of the liver was observed, and the mesenteric lymph nodes were found with a yellowish discoloration.
Main microscopic findings related to the condition of these two animals were present in the non-glandular stomach (forestomach) (squamous cell hyperplasia slight-moderate, with lymphogranulocytic infiltrate and minimal erosion/ulcer or edema), small intestines and mesenteric lymph node (macrophage accumulation up to marked degree) and most likely secondary findings related to their poor health condition were noted in the thymus (minimalslight increased apoptosis lymphocytes/decreased lymphoid cellularity) and bone marrow
(slight increased adipocytes). Macroscopic and microscopic findings of these organs were similar as in scheduled sacrificed females at 300 mg/kg bw/day. Only the severity of the foamy macrophage accumulation in the jejunum and increased adipocytes in the bone marrow was slightly higher and the lesions of the small intestines were more extensive (included the ileum as well, instead of being present only in duodenum and/or jejunum, as was the case for the scheduled sacrifices at 300 mg/kg bw/day).
At 300 mg/kg bw/day, piloerection and hunched posture were frequently noted in both males and females. These signs were also incidentally observed in males and females at 150 mg/kg bw/day. Dose-dependent progressively lower body weight gain and body weights were noted for males at 150 and 300, together with a lower food consumption at 300 mg/kg bw/day. In addition, occasional slight body weight loss was observed for males at 300 mg/kg bw/day. In females at 300 mg/kg bw/day, lower body weight gain and body weights with occasional lower food intake
were also noted from the end of premating onwards. From Day 7 of lactation, body weight gain increased, resulting in a slight recovery of the decreased body weights. The lower body weight (gain) in males at 150 mg/kg bw/day was considered adverse. Furthermore, the combination of clinical signs, magnitude of the lower body weight (gains) and/or lower food consumption was considered adverse for males and females at 300 mg/kg bw/day.
Grip strength of both the front and hindlegs was lower in males at 300 mg/kg bw/day, and in females, a dose-dependent lower grip strength of the front legs was noted at 150 and 300 mg/kg bw/day. These results could be a secondary consequence of the general poor condition of these animals (including low body weights). The lower front- and hindleg grip strength of males at 300 mg/kg bw/day and the lower hindleg grip strength of females at 300 mg/kg bw/day were considered adverse.
Non-adverse changes noted in hematology parameters in males included: lower mean corpuscular volume and mean corpuscular hemoglobin at 150 and 300 mg/kg bw/day, and higher platelet counts at 300 mg/kg bw/day. In females, higher red blood cell distribution width at 300 mg/kg bw/day was also considered non-adverse.
A dose-dependent shorter activated partial thromboplastin time in males at 50, 150 and 300 mg/kg bw/day was considered non-adverse.
At 300 mg/kg bw/day lower total protein (males), higher total bilirubin (males and females, also at 150 mg/kg bw/day for males), higher urea (males) and higher low density lipoprotein (males) levels were noted. In addition, phosphate levels were higher in males and lower in females at 300 mg/kg bw/day. Moreover, TSH levels were lower in males at 300 mg/kg bw/day. All theseclinical chemistry changes were considered non-adverse.
Adverse morphological alterations were observed in males starting at 150 mg/kg bw/day in the small intestines and mesenteric lymph node in the form of macrophage accumulation, and in both sexes at 300 mg/kg bw/day in the non-glandular stomach in the form of squamous cell hyperplasia, erosion/ulcer, edema and/or lymphogranulocytic infiltrate and small intestines and mesenteric lymph node in the form of the presence of foamy macrophage accumulation.
Non-adverse test material-related alterations were present at 150 mg/kg bw/day in the nonglandular stomach of both sexes, thymus of a single male and small intestines and mesenteric lymph node of females and at 300 mg/kg bw/day in the thymus of both sexes, bone marrow of females and kidneys of males.
No test material-related changes were noted in any of the remaining parameters investigated in this study (i.e., ophthalmic examination parameters and hearing ability, pupillary reflex and static righting reflex).
Reproductive results
At 300 mg/kg bw/day, non-adverse effects were seen on estrous cycle regularity in 9/10 females which displayed an irregular estrous cycle or in which the estrous cycle could not be determined as only one estrous cycle was completed during the observation period. All females at this dose level, except the one female with a regular cycle, became pregnant. The lower number of corpora lutea and implantation sites was considered adverse. This latter effect is potentially related to the general poor condition of the females, but a direct effect of the test material could not be excluded.
No test material-related changes were noted in any of the remaining reproductive parameters investigated in this study (i.e., mating and fertility indices, precoital time, spermatogenic profiling, and histopathological examination of reproductive organs).
Developmental results
At 150 mg/kg bw/day, the lower pup body weights on PND 4 and 7 were considered non-adverse as the pup body weights recovered on PND 13. At 300 mg/kg bw/day, the lower live litter sizes and low pup body weights were considered adverse and could be a secondary effect of maternal toxicity although a direct effect of the test material cannot be excluded. In addition, adverse lower viability and lactation indices were noted that could be attributed to the lack of maternal care of three females resulting from a poor condition of these females leading up to and following parturition.
No test material-related changes were noted in any of the other developmental parameters investigated in this study (i.e., gestation index, duration of gestation, parturition, postimplantation survival index, live birth index, sex ratio, and some early postnatal pup development parameters consisting of anogenital distance, areola/nipple retention, T4 thyroid hormone levels and macroscopic examination).
In conclusion, based on the results of this combined 90-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the following No Observed Adverse Effect Levels (NOAELs) of Tallow amine propoxylate were established:Parental NOAEL: 50 mg/kg bw/day (based on clinical signs, lower body weights and food consumption, macroscopic and microscopic findings in the stomach, small intestine and mesenteric lymph nodes)


Reproduction NOAEL: 150 mg/kg bw/day (based on the lower number of corpora lutea and implantation sites)


Developmental NOAEL: 150 mg/kg bw/day (based on the small live litter sizes, high pup mortality and low pup body weights)


 

Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
Experimental start date (Animal arrival) 25 March 2020 Experimental completion date (Anatomical Pathology Report ) 23 February 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
See section 13.2 for the read-across justification.
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Justification for study design:
Purpose
The purpose of this study was to assess the influence of 2,2' (Octadec 9 enylimino)bisethanol on reproductive performance when administered by oral gavage administration to Han Wistar rats. Cohorts of F1 animals were used to assess the potential for systemic toxicity, and potential effects on sexual maturation and estrous cycles.


Route of Administration
The oral gavage route of administration was chosen to simulate the conditions of possible human exposure.

Rationale for Dose Level Selection
Dose levels of 30, 70 and 150 mg/kg/day were selected in conjunction with the Sponsor based on the findings from a preliminary reproductive study conducted at this laboratory (Covance study no XG05HV).

In the preliminary study oral administration of 2,2'-(Octadec-9-enylimino)bisethanol at dose levels of 30, 70 or 150 mg/kg/day had no adverse effect on general clinical condition (F0/F1), mating performance and fertility (F0), organ weights (F0) or macropathology (F0/F1). However at 150 mg/kg/day significant effects were apparent on both F0 and F1 body weight performance and two out of the eight litters showed high mortality.

Based on the findings in the preliminary study there was nothing to preclude the use of 150 mg/kg/day as the high dose for use on this OECD 443 study, with low and intermediate dose levels of 30 and 70 mg/kg/day, to provide approximately 2-fold dose intervals.
Specific details on test material used for the study:
Test item: 2,2'-(Octadec-9-enylimino)bisethanol
Test item identity (including alternative names): ETHOMEEN O/12 (Trade name)
Bis (2-hydroxyethyl) oleyl amine (Chemical name)
CAS number: 25307-17-9
Appearance: Yellow liquid
Storage conditions: Room temperature (15 to 25°C), under nitrogen in the dark.
Supplier: Sponsor
Batch number: 1853056
Expiry date: 19 May 2022
Purity: 99.7%
Supplier’s responsibilities: Characterization of the test item and the documentation of the methods of synthesis, fabrication or derivation and stability.
Archive sample: A 0.5 mL representative sample was taken, placed in a well closed glass container and stored in the archives under the same conditions as the bulk material.
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Animal Model
The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The HanWistar (RccHan™;WIST) strain was used because of the historical control data available at this laboratory.
Sex:
male/female
Details on test animals or test system and environmental conditions:
Strain/Species RccHan™;WIST rat.

Supplier Envigo RMS Limited.

Number of animals ordered 106 males and 106 females; males unrelated to females.
Spare animals were removed from the study room after treatment commenced.

Duration of acclimatization Six days before commencement of treatment.

Age of the F0 animals at the start of the treatment 28 to 34 days old.

Weight range of the F0 animals at the start of the treatment Males 72 to 102 g.
Females 65 to 95 g.

Animal Care and Husbandry
Environmental Control
Animal facility Limited access - to minimize entry of external biological and chemical agents and to minimize the transference of such agents between rooms.

Air supply Filtered fresh air which was passed to atmosphere and not recirculated.

Temperature and relative humidity Monitored and maintained within the range of 20-24°C and 40-70%.

There were no deviations from these ranges.

Lighting Artificial lighting, 12 hours light: 12 hours dark.

Electricity supply Public supply with automatic stand-by generators.

Animal Accommodation
Cages Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals.

Solid (polycarbonate) bottom cages were used throughout the study except during pairing.

Grid bottomed cages were used during pairing. These were suspended above absorbent paper which was changed daily.

Cage distribution The cages were distributed on the racking to equalize, as far as possible, environmental influences amongst the groups.

Bedding Solid bottom cages contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week.

Environmental Enrichment
Aspen wood based product A soft white untreated wood based products; provided to each cage throughout the study (except for F0 females during lactation and except when F1 Cohort 1A animals were separated into single housing overnight during urine collection) and replaced when necessary.

Plastic shelter Provided to each cage throughout the study (except during pairing and during lactation and except when F1 Cohort 1A animals were separated into single housing overnight during urine collection) and replaced at the same time as the cages.

Paper shavings From Day 20 after mating and throughout lactation, approximately two handfuls of paper shavings were provided to each cage as nesting material; this nesting material was changed at the same frequency as the cage bedding.

Diet Supply
Diet SDS VRF1 Certified pelleted diet.

The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.

Availability Non-restricted (except when diet was removed overnight before blood sampling for hematology or blood chemistry and during urine collection).

Water Supply
Supply Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.

Availability Non-restricted (except during urine collection).

Supplier Certificates of Analysis
Certificates of analysis for the diet were scrutinized and approved before any batch of diet was released for use. Certificates of analysis were routinely provided by the water supplier.

Certificates of analysis were also received from the suppliers of the softwood based bark-free fiber bedding and Aspen wood based product.

No specific contaminants were known that may have interfered with or prejudiced the outcome of the study and therefore no special assays were performed.

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
Test Item Preparation and Analysis
Method of preparation The required amount of test item was weighed and approximately 50% of the required volume of vehicle was added. It was magnetically stirred until uniformly mixed and then made up to the required volume with vehicle and mixed again with a magnetic stirrer until homogenous.

A series of formulations at the required concentrations were prepared by dilution of individual weighings of the test item.

Frequency of preparation Weekly.

Storage of formulation Refrigerated (2 to 8°C).

Test item accounting
Detailed records of compound usage were maintained. The amount of test item necessary to prepare the formulations and the amount actually used were determined on each occasion. The difference between these amounts was checked before the formulations were dispensed.

Formulation Analysis
Stability and homogeneity Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations at 0.5 to 50 mg/mL were analyzed to assess the stability and homogeneity of the test item in the liquid matrix (Covance Study Number NQ85KJ).

• Ambient temperature (15 to 25℃) for 24 hours.
• Refrigerated temperature (2 to 8℃) for eight days.

Achieved concentration Samples of each dose preparation l for Week 1 (F0 and F1 generation) and last week (F1 generation) were analyzed for achieved concentration of the test item.


Formulation A daily record of the usage of formulation was maintained based on weights. This balance was compared with the expected usage as a check of correct administration. No significant discrepancy was found.
Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.
Details on mating procedure:
F0 Generation
F0 pairing commenced After ten weeks of treatment.

Male/female ratio 1:1 from within the same treatment groups (sibling pairing was not permitted).

Duration of pairing Up to two weeks.

Daily checks for evidence of mating Ejected copulation plugs in cage tray and sperm in the vaginal smear.

Day 0 of gestation When positive evidence of mating was detected.

Male/female separation Day when mating evidence was detected.
Pre-coital interval Calculated for each female as the time between first pairing and evidence of mating.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical method involved extraction in acetone and dilution in acetonitrile/water/formic
acid 50/50/0.1 v/v/v followed by liquid chromatographic analysis with mass spectrometric
detection (LC-MS/MS). Sample concentrations were determined with reference to single
bracketing standards. Procedural recovery samples were prepared concurrently with samples
and results were corrected for the mean recovery value at each level at analysis.
Duration of treatment / exposure:
F0 animals For ten weeks before pairing until termination after litters were weaned.

F1 animals From weaning until termination of respective cohort; although direct exposure starts at weaning on Day 21 of age, all offspring had potential indirect exposure in utero and through the milk during lactation.

Cohort 1A : General toxicity and pathology of the tissues of the male and female reproductive systems - treated from weaning to 13 weeks of age.

Cohort 1B : Spare Cohort - treated from weaning to 14 weeks of age.


Frequency of treatment:
Once daily at approximately the same time each day. Animals were not dosed if parturition was in progress at the scheduled time of administration.
Details on study schedule:
Selection of Offspring to Form F1 Generation
Selection
Nominally Day 21 of age (direct dose administration from Day 21 of age).

Formal start of F1 generation Nominally Day 28 of age

Method Where possible, two male and two female were selected from each selected litter and were allocated to each of the two cohorts. If more were required, up to three males and three females were selected from each selected litter.

Selected animals were microchipped on Day 18 to 21 of age and separated from littermates on Day 21 of age.

Up to two male and two female offspring per group were retained as spares, to provide potential replacement in the event of any mortality. These spares had body weights and clinical signs recorded weekly and were terminated after commencement of the F1 generation.

Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Group 1 (control): F0 generation, F1 generation cohorts 1A and 1B
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Remarks:
Group 2: F0 generation, F1 generation cohorts 1A and 1B
Dose / conc.:
70 mg/kg bw/day (actual dose received)
Remarks:
Group 3: F0 generation, F1 generation cohorts 1A and 1B
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Remarks:
Group 4: F0 generation, F1 generation cohorts 1A and 1B
No. of animals per sex per dose:
F0 generation: three groups of 25 male and 25 female rats

F1 generation
Cohorts 1A and 1B: 20 male and 20 female progeny
Control animals:
yes, concurrent vehicle
Details on study design:
Rationale for Dose Level Selection
Dose levels of 30, 70 and 150 mg/kg/day were selected in conjunction with the Sponsor based on the findings from a preliminary reproductive study conducted at this laboratory (Covance study no XG05HV).

In the preliminary study oral administration of 2,2'-(Octadec-9-enylimino)bisethanol at dose levels of 30, 70 or 150 mg/kg/day had no adverse effect on general clinical condition (F0/F1), mating performance and fertility (F0), organ weights (F0) or macropathology (F0/F1). However at 150 mg/kg/day significant effects were apparent on both F0 and F1 body weight performance and two out of the eight litters showed high mortality.

Based on the findings in the preliminary study there was nothing to preclude the use of 150 mg/kg/day as the high dose for use on this OECD 443 study, with low and intermediate dose levels of 30 and 70 mg/kg/day, to provide approximately 2-fold dose intervals.


Parental animals: Observations and examinations:
Clinical Observations - F0 generation
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.

During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

Signs Associated with Dosing
Detailed observations were performed to establish and confirm a pattern of signs in association with dosing according to the following schedule:

F0 generation Week 1 - Daily.
Weeks 2 to 4 - twice weekly (middle and end of the week)
Week 5 onward - once each week (Days 0, 7, 14 and 20 after mating and Days 1, 7, 14 and 20 of lactation for F0 females).

Detailed observations were recorded at the following times in relation to dose administration:
• Prior to dosing.
• One to two hours after completion of dosing.
• As late as possible in the working day.

Clinical Signs
A detailed physical examination was performed on each animal to monitor general health according to the following schedule:

Physical examination Once each week

After mating of F0 females: Days 0, 5, 12, 18 and 20 after mating and Days 1, 7, 14 and 21 of lactation.

Particular attention was paid to possible signs of neurotoxicity such as convulsions, tremor and abnormalities of gait or behavior.

Mortality
A viability check was performed near the start and end of each working day. Animals were killed for reasons of animal welfare where necessary.

A complete necropsy was performed in all cases

Body weight
F0 males Day that treatment commenced.
Each week.
Before necropsy.
F0 females Day that treatment commenced.
Each week until mating detected.
Days 0, 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 after mating.
Days 1, 4, 7, 14, 21 and 28 of lactation.
Before necropsy

Food Consumption
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded as follows:

F0 males and females Weekly, until paired for mating.

For females after mating food consumption was performed to match the body weight recording:
Days 0-1, 2-3, 4-5, 6-7, 8-9, 10-11, 12-13, 14-15, 16-17 and 18 19 after mating
Days 1-3, 4-6, 7-13, and 14-20 of lactation.

From these records the mean weekly or daily consumption per animal (g/animal/week or g/animal/day) was calculated for each relevant phase.

Parturition Observations and Gestation Length - F0 Generation
Duration of gestation Time that elapsed between mating and commencement of parturition.

Parturition observations From Day 20 after mating animals were checked three times daily for evidence of parturition. The progress and completion of parturition was monitored; numbers of live and dead offspring were recorded and any difficulties observed were noted.

Records Made During Littering Phase - F0 Generation
The records maintained were as follows:

Clinical observations Observed approximately 24 hours after birth (Day 1 of age) and then daily for evidence of ill-health or reaction to treatment.

On Day 1 of age, all offspring received a qualitative assessment of body temperature, state of activity and reaction to handling.

Hematology, Peripheral Blood - F0 Generation
Blood samples were collected after overnight withdrawal of food. Sampling for Cohort 1A was performed on the morning after overnight collection of urine. These animals were, therefore, also deprived of water overnight but had access to water for a minimum period of one hour prior to the commencement of blood sampling procedures. Samples were collected at the following occasions:

Occasion Generation Animals
Termination F0 Adults Ten male and ten female animals per group

Animals were held under light general anesthesia induced by isoflurane. Blood samples (nominally 0.5 mL) were withdrawn from the sublingual vein, collected into tubes containing EDTA anticoagulant and examined for the following characteristics using a Bayer Advia 120 analyzer:
• Hematocrit (Hct)*
• Hemoglobin concentration (Hb)
• Erythrocyte count (RBC)
• Absolute reticulocyte count (Retic)
• Mean cell hemoglobin (MCH)*
• Mean cell hemoglobin concentration (MCHC)*
• Mean cell volume (MCV)
• Total leucocyte count (WBC)
• Differential leucocyte count:
• Neutrophils (N)
• Lymphocytes (L)
• Eosinophils (E)
• Basophils (B)
• Monocytes (M)
• Large unstained cells (LUC)
• Platelet count (Plt)

* Derived values calculated in ClinAxys

Blood film (prepared for all samples) - Romanowsky stain, examined for abnormalities by light microscopy, in the case of flags from the Advia 120 analyzer. Confirmation or a written description from the blood film was made where appropriate. A manual count of the differential white blood cell parameters was performed where necessary.
Additional blood samples (nominally 0.5 mL) were taken into tubes containing citrate anticoagulant and examined using a Stago STA Compact Max analyzer and appropriate reagent in respect of:
• Prothrombin time (PT) - using IL PT Fibrinogen reagent.
• Activated partial thromboplastin time (APTT) - using IL APTT reagent.

Blood Chemistry - F0 Generation
Blood samples were collected after overnight withdrawal of food. Sampling for Cohort 1A was performed on the morning after overnight collection of urine. These animals were, therefore, also deprived of water overnight but had access to water for a minimum period of one hour prior to the commencement of blood sampling procedures. Samples were collected at the following occasions:
Occasion Generation Animals
Termination F0 Adults Ten male and ten female animals per group
F1 Cohort 1A Ten male and ten female animals per group

Animals were held under light general anesthesia induced by isoflurane. Blood samples (nominally 0.7 mL) were withdrawn from the sublingual vein and collected into tubes containing lithium heparin as anticoagulant. After separation, the plasma was examined using a Roche Cobas 6000 Analyzer in respect of:
• Alkaline phosphatase (ALP)
• Alanine aminotransferase (ALT)
• Aspartate aminotransferase (AST)
• Gamma-glutamyl transpeptidase (gGT)
• Total bilirubin (Bili)
• Bile acid (Bi Ac)
• Urea
• Creatinine (Creat)
• Glucose (Gluc)
• Total cholesterol (Chol)
• Sodium (Na)
• Potassium (K)
• Chloride (Cl)
• Calcium (Ca)
• Inorganic phosphorus (Phos)
• Total protein (Total Prot)
• Albumin (Alb)

Albumin/globulin ratio (A/G Ratio) was calculated from total protein concentration and analyzed albumin concentration.


Thyroid Hormone Analysis - TSH and T4

Blood samples were collected at the following occasions:
Occasion Generation Animals
Termination F0 Adults Ten male and ten female animals per group

Ten male and ten female animals per group


Conditions Adults: Following overnight deprivation of food.
Blood sample site Adults: Sublingual vein.
Anesthetic Adults and Offspring on Day 22 of age: Isoflurane
Anticoagulant None.
Tubes Greiner Minicollect - with clot activator.
Blood volume 1 mL
Treatment of samples Samples were kept at ambient temperature (15 to 25°C) for a minimum of 30 minutes prior to centrifugation.
Centrifugation conditions At 2000g for ten minutes at 4°C.
Number of aliquots Adults and offspring on Day 22 of age: Two per animal. Aliquot 1: 0.2 mL serum for T4
Aliquot 2: residual serum for TSH
Final storage conditions Deep frozen (approximately -60°C to -90°C).
Fate of samples Aliquot 1 (T4): dispatched to the Department of Bioanalysis, Covance.
Aliquot 2 (TSH): dispatched to the Department of Immunology & Immunotoxicology, Covance.
T4 Performed by the Department of LC-MS/MS Bioanalysis, Covance.
TSH Performed by the Department of Immunology & Immunotoxicology, Covance.
Oestrous cyclicity (parental animals):
Estrous Cycle Monitoring - F0 Generation
Dry and wet smears were taken as follows:

Dry smears For 15 days before pairing, using cotton swabs.
Wet smears After pairing until evidence of mating confirmed.

For four days before scheduled termination (nominally Days 25 to 28 post partum). Females that failed to litter or mate were retained and smeared for four days starting on the day on which the first batch of ‘true’ Day 25 post partum females started smearing, and were then killed with that first batch of females.
Sperm parameters (parental animals):
Sperm Analysis - F0 Generation
Immediately after scheduled sacrifice of each F0 male and the left vas deferens, epididymis and testis were removed and the epididymis and testis were weighed.

The following tests were performed:
Sperm motility: all groups
A sample of sperm was expressed from the left vas deferens into prewarmed (target 37°C) medium M199, which contained 0.5% w/v bovine serum albumin (BSA Fraction V). A sample for assessment was taken into a 100 µm depth cannula by capillary action and, at least 200 sperm per animal analyzed using the Hamilton Thorne IVOS II Computer Assisted Sperm Analyzer (CASA).

F0 Group 1 Males 9 & 19 had insufficient sperm to assess 200.

Sperm morphology
Groups 1 and 4 A 200 µL aliquot of the sperm/medium mixture (described above) was diluted with 800 µL of 10% neutral buffered formalin. After staining with nigrosine and eosin an air-dried smear was prepared. Slides were examined by light microscopy for the assessment of sperm morphology. At least 200 sperm were assessed for each male, where possible.

F0 Group 1 Males 9 & 19 and Group 4 Male 91 & 98 had insufficient sperm to assess 200.

Sperm morphology: Groups 2 and 3 Fixed samples retained for possible future assessment.

Sperm count: Groups 1 and 4 The left cauda epididymis of each male was weighed and then the tunica was removed, then homogenized for at least 30 seconds in 10 mL of a mixture of 0.9% saline and 0.01% merthiolate (SM). An aliquot of this mixture was added to a pre-prepared IDENT stain tube before being assessed for sperm count using CASA.

Sperm count: Groups 2 and 3 Samples frozen for possible future assessment.

Homogenization-resistant spermatid counts: Groups 1 and 4 After removal of the tunica, the left testis of each male then homogenized for at least 30 seconds in 25 mL of SM. An aliquot of this mixture was added to a pre-prepared IDENT stain tube before being assessed for homogenization resistant spermatid count using CASA.

Homogenization-resistant spermatid counts: Groups 2 and 3 Samples frozen for possible future assessment.


Light Microscopy
Tissues preserved for examination were examined as follows:
Right testis (except F1 Group 4 male 480, the left tissues were used) A detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell- or stage-specificity of testicular findings was noted.

Vagina The stage of vaginal estrus was evaluated based on vaginal epithelial morphology (and appearance of the uterus and endometrial glands).
All other findings were either reported as "present" or assigned a severity grade. In the latter case one of the following five grades was used - minimal, slight, moderate, marked or severe. A reviewing pathologist undertook a peer review of the microscopic findings.
Litter observations:
Litter size
Daily records were maintained of mortality and consequent changes in litter size from Days 1-21 of age.
On Day 4 of age, litters containing more than eight offspring were reduced to eight by random culling, leaving, whenever possible, four male and four female offspring in each litter.

Sex ratio of each litter
Recorded on Days 1, 4 (before and after culling) and on Day 21 of age.

Individual offspring body weights
Recorded on Days 1, 4 (before culling), 7, 14 and 21 of age.
Selected F1 generation: Days 23, 25, 27* and 29* of age.
* - Only applicable before formal commencement of the F1 generation at nominal four weeks of age (Day 28 of age ± 2 days).

Unselected F1 offspring: Day 22 of age

Weaning of offspring The dam was removed from the litter cage and offspring were weaned on Day 21 of age.

Ano-genital distance Day 1 of age - all offspring.

Nipple/areolae count Day 13 of age - male offspring.


Sexual Maturation - F1 Generation
Males Sexual maturation was assessed by daily examination from Day 38 of age until balano-preputial separation occurred. Body weight was recorded on the day of completion of separation.
Females Sexual maturation was assessed by daily examination from Day 25 of age until vaginal opening occurred. Body weight was recorded on the day of vaginal opening.
For Cohort 1A: a wet smear was taken daily from the day of vaginal opening until first estrus was detected.

Mortality - F1 Generation
A viability check was performed near the start and end of each working day. Animals were killed for reasons of animal welfare where necessary.

A complete necropsy was performed in all cases

Body Weight - F1 Generation
The weight of animals was recorded as follows:
F1 selected animals From nominal Week 4 of age, twice during Week 1 of the F1 generation and weekly thereafter.

Before necropsy.

Food consumption - F1 Generation
F1 selected animals From nominal Week 4 of age, twice during Week 1 of the F1 generation and weekly thereafter.
From these records the mean weekly or daily consumption per animal (g/animal/week or g/animal/day) was calculated for each relevant phase.

Haematology, Peripheral Blood - F1 Cohort 1A Generation
Blood samples were collected after overnight withdrawal of food. Sampling for Cohort 1A was performed on the morning after overnight collection of urine. These animals were, therefore, also deprived of water overnight but had access to water for a minimum period of one hour prior to the commencement of blood sampling procedures. Samples were collected at the following occasions:
Occasion Generation Animals
Termination F1 Cohort 1A Ten male and ten female animals per group

Animals were held under light general anesthesia induced by isoflurane. Blood samples (nominally 0.5 mL) were withdrawn from the sublingual vein, collected into tubes containing EDTA anticoagulant and examined for the following characteristics using a Bayer Advia 120 analyzer:
• Hematocrit (Hct)*
• Hemoglobin concentration (Hb)
• Erythrocyte count (RBC)
• Absolute reticulocyte count (Retic)
• Mean cell hemoglobin (MCH)*
• Mean cell hemoglobin concentration (MCHC)*
• Mean cell volume (MCV)
• Total leucocyte count (WBC)
• Differential leucocyte count:
• Neutrophils (N)
• Lymphocytes (L)
• Eosinophils (E)
• Basophils (B)
• Monocytes (M)
• Large unstained cells (LUC)
• Platelet count (Plt)

* Derived values calculated in ClinAxys

Blood film (prepared for all samples) - Romanowsky stain, examined for abnormalities by light microscopy, in the case of flags from the Advia 120 analyzer. Confirmation or a written description from the blood film was made where appropriate. A manual count of the differential white blood cell parameters was performed where necessary.
Additional blood samples (nominally 0.5 mL) were taken into tubes containing citrate anticoagulant and examined using a Stago STA Compact Max analyzer and appropriate reagent in respect of:
• Prothrombin time (PT) - using IL PT Fibrinogen reagent.
• Activated partial thromboplastin time (APTT) - using IL APTT reagent.


Blood Chemistry - F1 Cohort 1A Generation
Blood samples were collected after overnight withdrawal of food. Sampling for Cohort 1A was performed on the morning after overnight collection of urine. These animals were, therefore, also deprived of water overnight but had access to water for a minimum period of one hour prior to the commencement of blood sampling procedures. Samples were collected at the following occasions:
Occasion Generation Animals
Termination F0 Adults Ten male and ten female animals per group
F1 Cohort 1A Ten male and ten female animals per group

Animals were held under light general anesthesia induced by isoflurane. Blood samples (nominally 0.7 mL) were withdrawn from the sublingual vein and collected into tubes containing lithium heparin as anticoagulant. After separation, the plasma was examined using a Roche Cobas 6000 Analyzer in respect of:
• Alkaline phosphatase (ALP)
• Alanine aminotransferase (ALT)
• Aspartate aminotransferase (AST)
• Gamma-glutamyl transpeptidase (gGT)
• Total bilirubin (Bili)
• Bile acid (Bi Ac)
• Urea
• Creatinine (Creat)
• Glucose (Gluc)
• Total cholesterol (Chol)
• Sodium (Na)
• Potassium (K)
• Chloride (Cl)
• Calcium (Ca)
• Inorganic phosphorus (Phos)
• Total protein (Total Prot)
• Albumin (Alb)

Albumin/globulin ratio (A/G Ratio) was calculated from total protein concentration and analyzed albumin concentration.

Urinalysis - F1 Cohort 1A Generation
Urine samples were collected after overnight withdrawal of food and water at the following occasion:
Occasion Generation Animals
Termination F1 Cohort 1A Ten males and ten females animals per group

The individual samples were examined for the following characteristics:
• Using manual methods:
• Clarity and Color (App) - by visual assessment
• Volume (Vol) - using a measuring cylinder
• pH - using a pH meter
• Specific gravity (SG) - by direct refractometry using a SG meter

• Using Multistix reagent strips interpreted using the Clinitek®500 instrument:
• Glucose (Gluc)
• Ketones (Keto)
• Bile pigments (Bili)
• Blood pigments (UBld)

• Using a Cobas 6000 Analyzer:
• Protein - total (T-Prot) and concentration (Prot)
• Sodium - total (T-Na) and concentration (U-Na)
• Potassium - total (T-K) and concentration (U-K)
• Chloride - total (T-Cl) and concentration (U-Cl)

A microscopic examination of the urine sediment was performed. An aliquot of the urine sample was centrifuged, stained with Kova stain and the resulting deposit spread on a microscope slide. The number of elements seen in nine high or low power fields (HPF or LPF) was recorded in the raw data and entered onto the database and the number seen /HPF or /LPF was derived from these data as described below.
• Epithelial cells (Epi)
• Leucocytes (WBC)
• Erythrocytes (RBC)
• Casts
• Other abnormal components (A)

The slide was also examined for abnormalities in spermatozoa and crystals


Thyroid Hormone Analysis - TSH and T4
Blood samples were collected at the following occasions:
Occasion Generation Animals
Termination F1 Offspring Ten litters per group on Day 4 of age
(pooled litter sample for T4 only and retained pending possible analysis)
Ten male and ten female animals per group on Day 22 of age
(from as many litters as possible)

F1 Adults - Cohort 1A Ten male and ten female animals per group

Animal numbers are documented in Attachment 13.3 and Attachment 13.4.

Conditions Adults: Following overnight deprivation of food.

Offspring: No overnight deprivation of food.
Offspring Day 4 of age: Decapitation
Offspring Day 22 of age: Orbital sinus
Anesthetic Offspring on Day 22 of age: Isoflurane
Offspring Day 4 of age: Not required
Anticoagulant None.
Tubes Greiner Minicollect - with clot activator.
Blood volume Offspring Day 22 of age: 1 mL
Offspring Day 4 of age: Maximum possible.
Treatment of samples Samples were kept at ambient temperature (15 to 25°C) for a minimum of 30 minutes prior to centrifugation.
Centrifugation conditions At 2000g for ten minutes at 4°C.
Number of aliquots Offspring on Day 22 of age: Two per animal. Aliquot 1: 0.2 mL serum for T4
Aliquot 2: residual serum for TSH
Offspring on Day 4 of age: single aliquot for T4 analysis, all available collected.
Final storage conditions Deep frozen (approximately -60°C to -90C).
Fate of samples Aliquot 1 (T4): dispatched to the Department of Bioanalysis, Covance.
Aliquot 2 (TSH): dispatched to the Department of Immunology & Immunotoxicology, Covance.
T4 Performed by the Department of LC-MS/MS Bioanalysis, Covance.
TSH Performed by the Department of Immunology & Immunotoxicology, Covance.

Cohort Specific in Life Investigations - F1 Generation
Estrous Cycle Monitoring - Cohort 1A
Dry and wet smears were taken as follows:

Wet smears (using pipette lavage) Following onset of vaginal patency until first cornified (estrus) smear was recorded.

For at least three days prior to the start of the necropsy phase and on the day of termination.
Dry smears (using cotton swabs) For two weeks from approximately Day 75 of age.

Estrous Cycle Monitoring - Cohort 1B
Wet smears were taken as follows:

Wet smears (using pipette lavage) For at least three days prior to the start of the necropsy phase and on the day of termination.

Postmortem examinations (parental animals):
Any abnormality in the appearance or size of any organ and tissue was recorded and the required tissue samples preserved in appropriate fixative.

The organs weighed, tissue samples fixed and sections examined microscopically (if applicable) are detailed as follows:

Tissue and regions examined

Abnormalities
Adrenals
Brain (cerebellum, cerebrum, midbrain)
Cecum
Colon
Duodenum
Epididymides
Esophagus
Eyes
Femurs - (longitudinal section through joint)
Heart (including auricular and ventricular regions)
Ileum
Jejunum
Kidneys
Liver (section from two lobes)
Lungs (section from two major lobes including bronchi)
Optic nerves
Ovaries with oviduct
Pancreas
Pituitary
Prostate - dorsolateral and ventral combined
Rectum
Sciatic nerves
Seminal vesicles (with coagulating gland)
Skeletal muscle
Skin with mammary glands (inguinal area)
Spinal cord (transverse and longitudinal sections at the cervical, thoracic and lumbar levels)
Spleen
Sternum - bone marrow
Stomach
Testes
Thymus
Thyroid with parathyroids
Trachea
Urinary bladder
Uterus with cervix
Vagina
Vas Deferens


Histology
F0 animals and Cohort 1A
Wet tissues Wet tissues were dispatched to the Test Site (Covance Harrogate, UK) for processing.

Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.

Full List All adult animals killed or dying prematurely.

All terminal adult animals of Groups 1 and 4 killed at a scheduled interval.

Processing - reproductive organs and pituitary The reproductive organs were examined from F0 animals in Groups 2 and 3 that showed reduced fertility. This included males that failed to sire a pregnancy and females that were not pregnant, failed to litter or females with a litter death and abnormal estrous cycles.

Processing - stomach and jejunum All F0 and Cohort 1A males and females from Groups 2 and 3.
Processing - duodenum F0: All males from Groups 2 and 3.
Cohort 1A: All males and females from Groups 2 and 3.

Processing - ileum Cohort 1A: All males and females from Groups 2 and 3.

Processing - abnormalities only All F0/Cohort 1A terminal adult animals of Groups 2 and 3 killed at a scheduled interval.

Routine staining Sections were stained with hematoxylin and eosin.
Postmortem examinations (offspring):
Any abnormality in the appearance or size of any organ and tissue was recorded and the required tissue samples preserved in appropriate fixative.

For females of Cohort 1A, counts were performed for the number of ovarian follicles and corpora lutea.

Pathology procedures - Cohort 1A
Tissue and regions examined

Abnormalities
Adrenals
Brain (cerebellum, cerebrum, midbrain)
Cecum
Colon
Duodenum
Epididymides
Esophagus
Eyes
Femurs - (longitudinal section through joint)
Heart (including auricular and ventricular regions)
Ileum
Jejunum
Kidneys
Liver (section from two lobes)
Lungs (section from two major lobes including bronchi)
Lymph nodes - mesenteric
- left axillary
Optic nerves
Ovaries with oviduct
Pancreas
Pituitary
Prostate - dorsolateral and ventral combined
Rectum
Sciatic nerves
Seminal vesicles (with coagulating gland)
Skeletal muscle
Skin with mammary glands (inguinal area)
Spinal cord (transverse and longitudinal sections at the cervical, thoracic and lumbar levels)
Spleen
Sternum- bone marrow
Stomach
Testes
Thymus
Thyroid with parathyroids
Trachea
Urinary bladder
Uterus with cervix
Vagina
Vas Deferens


Pathology procedures - Cohort 1B
Tissue and regions examined

Abnormalities
Adrenals
Brain (cerebellum, cerebrum, midbrain)
Cecum
Colon
Duodenum
Epididymides
Esophagus
Eyes
Femurs - (longitudinal section through joint)
Heart (including auricular and ventricular regions)
Ileum
Jejunum
Kidneys
Liver (section from two lobes)
Lungs (section from two major lobes including bronchi)
Lymph nodes - mesenteric
- left axillary
Optic nerves
Ovaries with oviduct
Pancreas
Pituitary
Prostate - dorsolateral and ventral combined
Rectum
Sciatic nerves
Seminal vesicles (with coagulation gland)
Skeletal muscle
Skin with mammary glands (inguinal area)
Spinal cord (transverse and longitudinal sections at the cervical, thoracic and lumbar levels)
Spleen
Sternum - bone marrow
Stomach
Testes
Thymus
Thyroid with parathyroids
Trachea
Urinary bladder
Uterus with cervix
Vagina
Vas Deferens

Pathology procedures - Unselected F1 offspring on Day 22 of age
Ten male and ten females per group; one male or one female from each litter to ensure that all litters are represented.
Tissue and regions examined

Abnormalities
Brain (cerebellum, cerebrum, midbrain)
Epididymides
Ovaries
Pituitary
Prostate
Seminal vesicles
Skin with mammary glands (inguinal area)
Spleen
Testes
Thymus
Uterus with cervix and oviducts
Vagina


Sperm Analysis - F1 Cohort 1A Generation
Immediately after scheduled sacrifice of each F1 Cohort 1A male and the left vas deferens, epididymis and testis were removed and the epididymis and testis (except F1 Group 4 male 480, the right tissues were used) were weighed.

The following tests were performed:
Sperm motility: all groups A sample of sperm was expressed from the left vas deferens into prewarmed (target 37°C) medium M199, which contained 0.5% w/v bovine serum albumin (BSA Fraction V). A sample for assessment was taken into a 100 µm depth cannula by capillary action and, at least 200 sperm per animal analyzed using the Hamilton Thorne IVOS II ComputerAssisted Sperm Analyzer (CASA).


F1 Group 1 male 412 unable to assess 200 sperm. 20 samples were incorrectly analyzed using the wrong volume, reanalysis of the saved images resulted in 200 sperm not being analyzed for six samples).

Sperm morphology: Groups 1 and 4 A 200 µL aliquot of the sperm/medium mixture (described above) was diluted with 800 µL of 10% neutral buffered formalin. After staining with nigrosine and eosin an air-dried smear was prepared. Slides were examined by light microscopy for the assessment of sperm morphology. At least 200 sperm were assessed for each male, where possible.


F1 Group 1 male 413 unable to assess 200 sperm and Group 4 male 472 less than 200 sperm were analyzed in error.

Sperm morphology: Groups 2 and 3 Fixed samples retained for possible future assessment.

Sperm count: Groups 1 and 4 The left cauda epididymis of each male was weighed and then the tunica was removed, then homogenized for at least 30 seconds in 10 mL of a mixture of 0.9% saline and 0.01% merthiolate (SM). An aliquot of this mixture was added to a pre-prepared IDENT stain tube before being assessed for sperm count using CASA.

Sperm count: Groups 2 and 3 Samples frozen for possible future assessment.

Homogenization-resistant spermatid counts: Groups 1 and 4 After removal of the tunica, the left testis of each male then homogenized for at least 30 seconds in 25 mL of SM. An aliquot of this mixture was added to a pre-prepared IDENT stain tube before being assessed for homogenization resistant spermatid count using CASA.

Homogenization-resistant spermatid counts: Groups 2 and 3 Samples frozen for possible future assessment.


Histology
F0 animals and Cohort 1A
Wet tissues Wet tissues were dispatched to the Test Site (Covance Harrogate, UK) for processing.

Processing Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.

Full List All adult animals killed or dying prematurely.

All terminal adult animals of Groups 1 and 4 killed at a scheduled interval.

Processing - reproductive organs and pituitary The reproductive organs were examined from F0 animals in Groups 2 and 3 that showed reduced fertility. This included males that failed to sire a pregnancy and females that were not pregnant, failed to litter or females with a litter death and abnormal estrous cycles.

Processing - stomach and jejunum All F0 and Cohort 1A males and females from Groups 2 and 3.

Processing - duodenum F0: All males from Groups 2 and 3.

Cohort 1A: All males and females from Groups 2 and 3.

Processing - ileum Cohort 1A: All males and females from Groups 2 and 3.

Processing - abnormalities only All F0/Cohort 1A terminal adult animals of Groups 2 and 3 killed at a scheduled interval.

Routine staining Sections were stained with hematoxylin and eosin.
Cohort 1B

Processing Tissue samples were dehydrated and embedded in paraffin wax.
Full List All adult animals killed or dying prematurely.
Processing - abnormalities only All animals.
Processing to block - reproductive organs Al animals (see Section 4).

On completion of the study phase the Histology slides were transferred back to the Test Facility with any necessary supporting documentation. The wet tissues, blocks and raw paper data were returned to the Test Facility.


Immunophenotyping of Spleen Leucocytes - Cohort 1A
Ten males and ten females per group from Cohort 1A were selected for immunophenotyping.
Where possible, one male or one female was assigned from each selected litter.

The whole spleen was weighed. After weighing, a 3-5 mm mid transverse section was removed and retained for histopathological evaluation. The remaining portions of the spleen was then weighed, placed in to a vial of chilled Hank’s Balanced Salt Solution (HBSS) and held in wet ice until processing for analysis.


Light Microscopy
Tissues preserved for examination were examined as follows:
Right testis (except F1 Group 4 male 480, the left tissues were used) A detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell- or stage-specificity of testicular findings was noted.
Ovaries F0 and Cohort 1A - Qualitative evaluation of one section from each ovary
Cohort 1A - Quantitative evaluation of five sections from the middle third from each ovary for the assessment of primordial follicle and small growing follicle population as well as evaluation of corpora lutea numbers in one section from each ovary.
Vagina The stage of vaginal estrus was evaluated based on vaginal epithelial morphology (and appearance of the uterus and endometrial glands).

All other findings were either reported as "present" or assigned a severity grade. In the latter case one of the following five grades was used - minimal, slight, moderate, marked or severe. A reviewing pathologist undertook a peer review of the microscopic findings
Statistics:
Please refer to "Any other information on materials and methods"
Reproductive indices:
Mating Performance and Fertility - F0 Generation
Individual data was tabulated. Group values were calculated for males and females separately for the following:

Percentage mating (%) = (Number of animals mating /Animals paired) x 100

Conception rate (%) = (Number of animals achieving pregnancy / Animals mated) x 100

Fertility index (%) = (Number of animals achieving pregnancy / Animals paired) x 100

Gestation Length and Gestation Index - F0 Generation
Gestation length was calculated as the number of gestation days up to and including the day on which offspring were first observed, with Day 1 = day of mating for calculation purposes. Where parturition had started overnight, this value was adjusted by subtracting half of one day.

Gestation index was calculated for each group as:

Gestation index (%) = (Number of live litters born /Animals paired) x 100
Offspring viability indices:
Sexual Maturation - F1 Generation
Individual values were tabulated for age and body weight at completion. Group mean values were calculated from individual values presented.

Survival Indices - F0 Generation
The following were calculated for each litter:
Post implantation survival index (%) = Total number of offspring born x 100
Total number of uterine implantation sites

Post-implantation survival index was expressed as 100% where the number of offspring exceeded the number of implantation sites recorded.
Live birth index (%) = (Number of offspring on Day 1 after littering / Total number of offspring born) x 100

Viability index (%) = (Number of live offspring on Day 4 before culling / Number of live offspring on Day 1 after littering) x 100

Lactation index (%) = (Number of live offspring on Day 21 after littering / Number of live offspring on Day 4 (after culling) ) x 100

Group mean values were calculated from individual litter values.

Sex Ratio - F0 Generation

The percentage of male offspring in each litter was calculated at Day 1, and for live offspring on Days 1, 4 (before and after culling) and 21 of age.

Percentage males = Number of males in litter x 100
Total number of offspring in litter

Group mean values were calculated from individual litter values.

Offspring Examinations - F0 Generation

Ano-genital distance were presented both as absolute/unadjusted and adjusted for body weight, using the weight recorded on Day 1 of age.

A check was performed to assess for the presence or absence of nipple/areolae for the male offspring on Day 13 of age. As no nipples were present, no data is presented.

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
A few animals receiving 150 mg/kg/day were observed with increased salivation in association with dose administration; this sign is often seen in association with oral gavage administration and is attributed to the taste of the formulation rather than an effect of treatment.

No further signs were observed at either routine physical examination or in association with dose administration that were considered to relate to administration of the test item.
Mortality:
mortality observed, treatment-related
Description (incidence):
A total of four animals (one male and three females) were euthanized for welfare reasons.
Group 1(Control) male no. 18 was euthanized on Day 43 of treatment. Terminal signs included abnormal breathing and partially closed eyelids. Macroscopic findings at necropsy were limited to an enlarged mandibular lymph node. However, no histopathological lesions accounting for the poor clinical condition were observed.

Three females (animal nos. 278, 279 and 280 that received 150 mg/kg/day) were sacrificed following the day parturition completed, Day 2 of lactation and Day 21 of gestation, respectively.
• Terminal signs for female no 278 included thin build, labored breathing, piloerection, pallor and a hunched posture. Macroscopic examination revealed a distended stomach with abnormal gastrointestinal tract content, small spleen and thick/dark red fluid in the vagina.
Histopathological evaluation revealed treatment related findings such as slight accumulation of the foamy macrophages within the jejunum and minimal epithelial hyperplasia of the non-glandular gastric region, decreased cellularity of the splenic red pulp, minimal degranulation of the pancreas, moderate involution/atrophy of the thymus, one uterine polyp and moderate intraluminal haemorrhage the vagina and uterus.
• Terminal signs for female no. 279 included thin build, piloerection, pallor and a hunched posture. Macroscopic examination revealed thickened non-glandular mucosa of the stomach, abnormal caecal and rectal content, pale areas on the lungs and generally pale organs.
Histopathological evaluation revealed treatment related findings such as slight accumulation of the foamy macrophages within the jejunum and moderate oedema with slight hyperplasia of the gastric non-glandular region, minimal focal accumulation of the macrophages within the lung, moderate degranulation of the pancreas, moderate atrophy/involution of the thymus, moderate ulceration of the urinary bladder and moderate diffuse hyperplasia of the uterus with slight fibrinoid necrosis of the vascular media.

• Terminal signs for female no. 280 included decreased activity, piloerection, pale eyes and partially closed eyelids. Macroscopic examination revealed thickened non-glandular mucosa of the stomach, abnormal gastrointestinal tract content, abnormal nasal turbinate content and a small spleen. Histopathological evaluation revealed treatment related findings such as slight accumulation of the foamy macrophages within the jejunum and slight oedema, minimal mixed inflammation, moderate ulceration and moderate epithelial hyperplasia of the non-glandular gastric region, decreased cellularity of the splenic white pulp, slight degranulation of the pancreas and moderate diffuse hyperplasia of the uterus.

The premature deaths at 150 mg/kg/day were considered to be related to treatment.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males receiving 2,2'-(Octadec-9-enylimino)bisethanol had low body weight gain resulting in low mean absolute body weights from Day 8 of study up to termination; a dose response was apparent. The overall bodyweight gain from Day 1 to 120 was significantly low for male receiving 70 or 150 mg/kg/day (p<0.01).

During the 10-week pre-pairing treatment period body weight gain for females during Week 4 of treatment was significantly low at all dose levels; however, the overall gain for females before pairing showed no adverse effects of treatment.

During gestation body weight gain was slightly low during Days 18-20 for females receiving 150 mg/kg/day (p<0.01); however, the overall weight gain GD0-20 showed no adverse effect of treatment.

On Day 1 of lactation mean body weight for females receiving 150 mg/kg/day was low when compared with Controls (p<0.01), however overall the bodyweight gain at 70 or 150 mg/kg/day from Day 1-21 of lactation was high when compared with Control values (p<0.01); these differences were attributed to high weight gain during the first two weeks of lactation.

PLEASE REFER TO THE ATTACHED TABLES; "BODY WEIGHT AND BODY WEIGHT CHANGE - GROP MEAN VALUES FOR MALES AND FEMALES BEFORE PAITING (F0)
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Males receiving 70 or 150 mg/kg/day showed low food consumption from Week 1 of treatment (p<0.05/0.01); a dose response was apparent. Males at 30 mg/kg/day also showed occasions of low consumption during Week 2 (p<0.05), Week 5(p<0.01) and Week 9 (p<0.05) of treatment.
Before pairing females at 150 mg/kg/day showed low food consumption during Weeks 4, 5, 7, 8 and 9 (p<0.05/0.01); food consumption for females receiving 30 or 70 mg/kg/day was unaffected by treatment.

During gestation females receiving 150 g/kg/day showed slightly low food consumption, with statistical significance achieved on Days 2-6 and 12-20 (p<0.05/0.01); food consumption at 30 or 70 mg/kg/day was similar to Controls.

Food consumption during lactation was unaffected by administration of 2,2'-(Octadec-9-enylimino)bisethanol at dose levels up to and including 150 mg/kg/day.

PLEASE REFER TO THE ATTACHED TABLE: "FOOD CONSUMPTION - GROUP MEAN VALUES BEFORE PAIRING (F0)
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Males and females receiving 70 or 150 mg/kg/day and males receiving 30 mg/kg/day showed slightly but statistically significantly shorter prothrombin clotting times when compared with Controls.

At 150 mg/kg/day the mean neutrophil count for males and females was high when compared with Controls, the difference attaining statistical significance for the male animals (p<0.01). Females at 150 mg/kg/day also showed a high mean eosinophil count when compared with Controls (p<0.05).

Males at 150 mg/kg/day also showed:
• low mean haemoglobin (p<0.01)
• low mean cell haemoglobin (p<0.01)
• low mean cell volume (p<0.05)
• high platelet count (p<0.01)

Contrary to male animals, females at all dose levels showed high mean cell haemoglobin (p<0.05) and a longer activated partial thromboplastin time at 150 mg/kg/day (p<0.01) when compared with Controls.

The effects on erythrocyte parameters in males are possibly treatment related but are only of a minor nature. The percentage of reticulocytes is not affected. Females are much less affected, actually showing a non dose related small increase of MCH which in the absence of anemia has no biological meaning.

PLEASE REFER TO THE ATTACHED TABLE: "HAEMATOLOGY - GROUP MEAN VALUES AT SCHEDULED TERMINATION (F0)"
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Alanine transaminase activity for males and females receiving 150 mg/kg/day was elevated when compared with Controls (p<0.05). Males at 150 mg/kg/day also showed low total protein and albumin (p<0.01), with males showing a high A/G ratio at 70 mg/kg/day (p<0.05) and 150 mg/kg/day (p<0.01).

Other statistically significant differences were as follows:
• gamma-glutamyl transpeptidase activity at 150 mg/kg/day increased for male animals only (p<0.01)
• bile acid concentration at 150 mg/kg/day was high for males (p<0.01) but low for females (p<0.05)
• urea concentration at 70 and 150 mg/kg/day were maginally high for males only (p<0.05); however, there was no dose response and most individual values were within the concurrent control range.

• cholesterol levels at all dose levels were slightly low for females only (p<0.05)
• sodium concentrations at 150mg/kg/day were high for males (p<0.05) and low for females (p<0.05).

As observed differences to Control were of relative minor clinical significance, and mostly showed no consistency between males and females, these effects are not considered to represent adverse treatment related effects.

PLEASE REFER TO THE ATTACHED TABLE: "BLOOD CHEMISTRY - GROUP MEAN VALUES AT SCHEDULED TERMINATION (F0)"
Endocrine findings:
effects observed, non-treatment-related
Description (incidence and severity):
Thyroid Hormone Analysis
Thyroid stimulating hormone (TSH)

Administration of 2,2'-(Octadec-9-enylimino)bisethanol by oral gavage led to no significant differences in serum TSH levels in F0 adult male animals, F1 male offspring or F1 adult female animals at dose levels up to and including 150 mg/kg/day when compared with Controls.

However, serum TSH levels were high when compared with Controls for the following:
• F0 adult females at 150 mg/kg/day
• F1 female offspring at 150 mg/kg/day
• F1 adult males at 70 or 150 mg/kg/day

These differences lacked consistency and showed no correlation with thyroxine levels and were therefore considered to be unrelated to administration of the test item.

Thyroxine (T4)

The mean serum T4 concentrations in samples obtained from F1 offspring on Day 22 of age, F0 adult females and F1 adult males were slightly variable across treatment Groups 2 to 4, but within ±20% of the mean T4 values in the respective Control group.

F0 adult male animals and F1 adult female animals that received 150 mg/kg/day showed mean serum T4 levels that were low when compared with their Control group (at 77% and 75% of Controls, respectively). As the observed differences to control are relatively minor, and show no consistent pattern between ages and sexes, they are not considered to represent adverse effects from treatment with the test substance.

PLEASE REFER TO THE TABLES IN ANY OTHER INFORMATION ON RESULTS: "GROUP MEAN SERUM TSH CONCENTRATIONS IN MALE RATS" AND "FEMALE RATS"
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
effects observed, non-treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
In unscheduled and terminal sacrifice animals, 2,2’-(Octadec-9-enylimino) bisethanol-related microscopic findings were observed in the stomach (both sexes administered 30, 70 or 150 mg/kg/day), jejunum (both sexes administered 70 or 150 mg/kg/day) and duodenum (males administered 150 mg/kg/day).

The predominant microscopic finding in the stomach was characterized as diffuse minimal to moderate epithelial hyperplasia of the non-glandular region associated with erosion/vesicle or ulceration in a few animals. Edema and mixed inflammation were considered secondary to the erosion/vesicle or ulceration. This finding correlated with the 2,2’-(Octadec-9-enylimino) bisethanol related macroscopic observation of edema and thickening of the non-glandular mucosa.
Foamy cell accumulation observed within the jejunum (both sexes) and duodenum (males only) correlated with the 2,2’-(Octadec-9-enylimino) bisethanol related macroscopic observation of abnormal color and thickening.

PLEASE REFER TO THE TABLE IN ANY OTHER INFORMATION ON RESULTS: "MICROSCOPIC FINDINGS - TERMINAL SACRIFICE F0 GENERATION"
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
With the exception of one female at 150 mg/kg/day all females showed regular 4/5-day estrous cycles.

Stage of Estrous Cycle at Termination
The majority of females showed an estrus smear prior to scheduled termination.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
No adverse effects were observed following treatment with 2,2'-(Octadec-9-enylimino) bisethanol at dose levels up to and including 150 mg/kg/day.
Reproductive performance:
no effects observed
Description (incidence and severity):
Pre-Coital Interval
Pre-coital interval was unaffected by treatment with the vast majority of animals showing positive evidence for mating within four days of pairing, with the exception of one Control female (13-14 days) and one female at 70 mg/kg/day (5-8 days)

Mating Performance and Fertility
Mating performance and fertility did not show any adverse effects of treatment.

Gestation Length and Gestation Index
The duration of gestation was unaffected by treatment at dose levels up to and including 150 mg/kg/day; however, at 150 mg/kg/day the gestation index was slightly low when compared with Controls (91% vs 100%). This was related to the sacrifice for welfare reasons of one female on GD21 and another following completion of parturition.

Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
30 mg/kg bw/day (actual dose received)
System:
gastrointestinal tract
Organ:
stomach
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At 150 mg/kg/day there was a slight increase in the number of offspring (litters) with clinical signs; three litters with offspring cold to touch and two litters scattered in the cage. The general condition of offspring at 30 or 70 mg/kg/day was similar to Controls

No signs were observed at either routine physical examination or in association with dose administration that could be associated with administration of the test item.
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
A total of eight animals died prematurely; death for five of these animals is related to accidental trauma rather than administration of the test item. From the remaining three deaths two are also unrelated to administration of the test item: Group 1 female no. 616 (no vagina) and in Group 2 male no. 512 (multiple pathology). The death of Group 4 male no. 466 is probably related to the observed effects in GI-system and is possibly test substance treatment related.

• Group 2 male no. 512 (30 mg/kg/day) was killed for reasons of animal welfare on Day 47 of the F1 generation. Terminal signs included thin build and piloerection. Macroscopic examination dark areas on the liver, small epididymides, small testes, small thymus and a distended urinary bladder. Histopathological examination revealed signs of sexual immaturity with absence of sperm, distension of the urinary bladder, thymic atrophy/involution and depletion of the pancreatic acini.

• Group 4 male no 466 (150 mg/kg/day) was killed for welfare reasons on Day 64 of the F1 generation following significant body weight loss. Macroscopic examination revealed that the stomach had an irregular surface and depressions on the non-glandular mucosa. Histopathological evaluation revealed treatment related findings such as slight accumulation of the foamy macrophages within the jejunum, ileum and duodenum, diffuse hypertrophy of the villi within the ileum and duodenum, and oedema, inflammation, erosion/vesicle and hyperplasia of the gastric non-glandular region and involution/atrophy of the thymus.

• Group 4 male no. 550 (150 mg/kg/day) was killed for welfare reasons on Day 25 of the F1 generation. Terminal signs and macroscopic findings were related to trauma of the left tibia; death was not attributed to treatment. Histopathological findings included a callus formation suggesting the bone trauma on the tibia.

• Group 4 male no. 559 (150 mg/kg/day) was found dead on Day 10 of the F1 generation. No clinical signs were observed up to Day 9. Macroscopic examination revealed findings including a perforated esophagus and adhesions in the thoracic cavity. Histopathological examination revealed minimal to moderate diffuse mixed inflammation within the thoracic cavity including heart, lungs and thymus. This death was attributed to dosing trauma and is not attributed to administration of the test item.

• Group 4 male no. 560 (150 mg/kg/day) was found dead and cannibalised on Day 74 of the F1 generation. Macroscopic examination revealed fluid in the thoracic cavity. Histopathological examination revealed minimal to moderate diffuse mixed inflammation within the thoracic cavity including heart, lungs and thymus. This death was attributed to dosing trauma and is not attributed to administration of the test item.

• Group 1 Control female no. 616 was despatched to necropsy on Day 55 of age having failed to achieve vaginal opening; macroscopic examination revealed that the vulva was imperforate. Histopathological evaluation revealed the absence of vagina and imperforate vulva.

• Group 3 female no 650 (70 mg/kg/day) was killed for welfare reasons on Day 60 of the F1 generation. Terminal signs include decreased activity, noisy breathing and pale ears. Macroscopic examination revealed a perforated esophagus, thick clear fluid in the thoracic cavity and the heart had a thickened pericardium; Histopathological examination revealed minimal diffuse mixed inflammation within the thoracic cavity including oesophagus, heart and lungs and involution/atrophy of the thymus; this death is attributed to doing trauma and is not attributed to administration of the test item.

• Group 3 female no 655 (70 mg/kg/day) was killed for welfare reasons on Day 11 of the F1 generation. Terminal signs included decreased activity, laboured breathing, thin build and piloerection. Macroscopic examination revealed findings of a perforated esophagus with fluid and adhesions in the thoracic cavity. Histopathological examination revealed treatment related findings such as slight accumulation of foamy cells within the sinuses of the mesenteric lymph node, slight to moderate mixed inflammation within the thoracic cavity, lungs and thymus, minimal hepatic necrosis and minimal splenic extramedullary hematopoiesis; this death is attributed to doing trauma and is not attributed to administration of the test item.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
From Day 1 of age mean body weights for both male and female offspring at 150 mg/kg/day were significantly low when compared with Controls (p<0.01). Body weight gain at 150 mg/kg/day was also low from Day 1 up to Day 7 for males (p<0.01) and from Day 1 up to Day 14 for females (p<0.05/0.01); the overall body weight gain for both male and female offspring from Day 1 up to weaning on Day 21 of age was low at 150 mg/kg/day when compared with Controls (p<0.01).

Offspring body weight for offspring at 30 and 70 mg/kg/day showed no adverse effects of parental treatment.


At weaning on Day 21 of age selected F1 males at all dose levels and females at 30 mg/kg/day showed slight differences in mean bodyweight that were not dose related. However, both males and females at 70 or 150 mg/kg/day showed low bodyweight gain up to Day 25 of age (p<0.01) and a dose response was apparent.
At the formal commencement of the F1 generation on nominal Day 28 (±2) of age, the mean bodyweight for selected F1 animals at 150 mg/kg/day was low when compared with Controls (p<0.01). Subsequent body weight gain from Day 1 to Day 64 of the F1 generation was low for males receiving 70 or 150 mg/kg/day (p<0.01) and for females at 150 mg/kg/day (p<0.05).

PLEASE REFER TO THE ATTACHED TABLES: "BODY WEIGHT AND BODY WEIGHT CHANGE - GROUP MEAN VALUES FOR OFFSPRING (F1)"
and
"BODY WEIGHT AND BODY WEIGHT CHANGE - GROUP MEAN VALUES FOR MALES AND FEMALES (F1)"
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
From Day 1 of the F1 generation males at 150 mg/kg/day showed slightly but significantly low food consumption when compared with Controls (p<0.05/0.01) and at 70 mg/kg/day low food consumption was recorded for male animals from Day 15 of the F1 generation low food consumption (p<0.05/0.01).

Females at 150 mg/kg/day showed slightly low food consumption from Day 8 of the F1 generation (p<0.05/0.01).

Food consumption for females at 70 mg/kg/day and for males and females at 30 mg/kg/day was essentially similar to Controls and considered unaffected by treatment.

PLEASE REFER TO THE ATTACHED TABLE: "FOOD CONSUMPTION - GROUP MEAN VALUES FOR MALES AND FEMALES (F1)"
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Males and females that received the test item had statistically significantly shorter prothrombin clotting times when compared with Controls (p<0.05/0.01); similar to the results for F0 animals.
Males at all dose levels showed a low haematocrit (p< 0.05/0.01) and males at 150 mg/kg/day had low haemoglobin (p<0.01) when compared with Controls. The biological relevance of this finding is limited considering the lack of changes in eyrthrocytes, with no shifts in mean cell hemaglobin, mean cell hemaglobin conentrtaion and mean cell volume.

Females that received 70 or 150 mg/kg/day had low haemoglobin (p<0.01) and mean cell haemoglobin concentration (p<0.05/0.01). Females at all dose levels had low mean cell haemoglobin (p<0.05/0.01) and females at 150 mg/kg/day also had low mean cell volume (p<0.05)and a high platelet count (p<0.01). The majority of these differences were not evident in the male animals with the exception of low haemoglobin for males that received 150 mg/kg/day. The biological relevance of the low hemagolobin is limited in view of no changes in hematocrit, RBC and reticulocytes, and only minor shifts in mean cell hemaglobin, mean cell hemaglobin conentrtaion and mean cell volume.

White blood cell counts were high for males at 150 mg/kg/day and for females at all dose levels.

• Leucocyte counts high for males at 150 mg/kg/day (p<0.05) and for females high at all dose levels(p<0.05)

• Neutrophil and monocyte counts high for males at 70 of 150 mg/kg/day(p<0.01)

• Large unstained cell count high for males at 150 mg/kg/day(p<0.05)

PLEASE REFER TO THE ATATCHED TABLE: "HAEMATOLOGY - GROUP MEAN VALUES AT SCHEDULED TERMINATION (F1 COHORT 1A)"
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Males that received 70 or 150 mg/kg/day showed low Alkaline phosphatase activity (p<0.01), which is of no biological significance, and males at 150 mg/kg/day had slightly high Alanine amino-transferase activity (p<0.01). This was not evident in female animals, with females at 150 mg/kg/day showing high Gamma glutamyl transpeptidase activity (p<0.01).

Both males and females at 150 mg/kg/day had low cholesterol levels when compared with Controls (male p<0.05, females p<0.01)

Glucose and potassium levels were low for males at dose levels up to and including 150 mg/kg/day when compared with Controls (p<0.01).

Creatinine levels for females at all dose levels were low when compared with Controls (p<0.05/0.01); this decrease is oif noi biological/toxicological significance.

When compared with Controls males at 150 mg/kg/day also had marginally high plasma urea (p<0.05), with low total protein (p<0.05) and a high A/G ratio (p<0.01).

PLEASE REFER TO THE ATTACHED TABLE: "BLOOD CHEMISTRY - GROUP MEAN VALUES AT SCHEDULED TERMINATION (F1 COHORT 1A)"
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Urinary protein levels were significantly high for both male and females that received the test item at dose levels up to and including 150 mg/kg/day (p<0.05/0.01), but total urinary protein levels were unaffected by treatment. Other differences included:

• Low urinary output for males at 70 and 150 mg/kg/day (p<0.05)

• Low total potassium levels for males at 70 and 150 mg/kg/day (p<0.05/0.01)

• Low pH for females at all dose levels (p<0.05/0.01)

PLEASE REFER TO THE ATTACHED TABLE: "URINALYSIS - GROUP MEAN VALUES AT SCHEDULED TERMINATION (F1 COHORT 1A)"
Sexual maturation:
effects observed, non-treatment-related
Description (incidence and severity):
At 150 mg/kg/day completion of sexual maturation for both males and females was approximately three days later than the concurrent Controls; however, as the mean body weight at completion was similar to Controls, these delays are considered to be related to a general delay in physical development rather than a specific effect on sexual maturation.

PLEASE REFER TO THE ATTACHED TABLE: "SEXUAL MATURATION - GROUP MEAN AGE AND BODY WEIGHT AT COMPLETION (F1)"
Anogenital distance (AGD):
effects observed, treatment-related
Description (incidence and severity):
Ano-genital distance of male offspring on Day 1 of age was unaffected by parental treatment at dose levels up to and including 150 mg/kg/day.

The ano-genital distance for female offspring at 150 mg/kg/day was significantly smaller than Controls (p<0.05), but as this is a decrease rather than an increase in distance it is of no biological/toxicological significance; values at 30 or 70 mg/kg/day were similar to Controls

PLEASE REFER TO THE ATTACHED TABLE: "ANO-GENITAL DISTANCE - GROUP MEAN AND ADJUSTED VALUES FOR OFFSPRING (F1)"
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
No nipples were seen on examination of male offspring on Day 13 of age
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Male offspring derived from the group that received 150 mg/kg/day showed slightly but statistically significantly low absolute mean brain and thymus weight when compared with Controls; in the absence of any similar observation in the female offspring at the same dose level, and since body weight relative brain and thymus weights were similar to Controls, these differences were considered to reflect the lower terminal body weights and not a specific effect of treatment on these organs.

* PLEASE REFER TO THE TABLE IN ANY OTHER INFORMATION ON RESULTS: "ORGAN WEIGHT PARAMETERS - UNSELECTED OFFPSRING ON DAY 22 OF AGE (F1)"

F1 Generation (Cohort 1A)
Body weight relative kidney weights were statistically significantly high for both males and females at all dose levels (p<0.05/0.01). Females at all dose levels and males at 70 or 150 mg/kg/day had significantly high body weight relative spleen weight (p<0.05/0.01).

2,2’-(Octadec-9-enylimino) bisethanol-related low absolute and body weight relative thymus weights were noted at the terminal sacrifice of males that received 150 mg/kg/day (p<0.01 and p<0.05 respectively). Males at 70 or 150 mg/kg/day also had high body weight relative heart weight(p<0.05) and males at 150 mg/kg/day had high bodyweight relative adrenal weight. (p<0.01).

High bodyweight relative mesenteric lymph nodes weight and liver weight were recorded for females that received 150 mg/kg/day (p<0.01 and p<0.05 respectively). The high mesenteric lymph node weights may be associated with the test item related microscopic changes identified in this tissue.

No test item related microscopic changes were detected in any of the other organs and thus no definite or adverse effect of treatment is inferred. Differences in relative brain weight and reproductive organ relative weights were considered to be secondary to the effect on body weight.

PLEASE REFER TO THE TABLE IN ANY OTHER INFORMATION ON RESULTS:"ORGAN WEIGHT PARAMETERS - TERMINAL SACRIFICE F1 GENERATION (COHORT 1A)"

Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Macroscopic examination of offspring that died or were killed prior to scheduled termination or were culled on Day 4 of age did not reveal any findings that could be attributed to parental treatment.

Macroscopic examination of unselected F1 offspring on Day 22 of age did not reveal any abnormality and as such no data is presented.

F1 Generation (Cohort 1A)
At terminal sacrifice, thickening was noted for the stomach of some animals administered 70 or 150 mg/kg/day. This finding was inconsistently associated with depression, in both sexes, and correlated with the 2,2’-(Octadec-9-enylimino) bisethanol related microscopic observation of diffuse hyperplasia of the non-glandular mucosa, erosion/vesicles, ulceration, edema and mixed inflammation.

PLEASE REFER TO THE TABLE IN ANY OTHER INFORMATION ON RESULTS: "MACROSCOPIC FINDINGS - TERMINAL SACRIFICE F1 GENERATION (COHORT 1A)"

F1 Generation (Cohort 1B)
At terminal sacrifice, thickening was noted for the stomach of occasional animals administered 70 or 150 mg/kg/day. This finding was inconsistently accompanied with depression, in both sexes, and correlated with the 2,2’-(Octadec-9-enylimino) bisethanol related microscopic observation of diffuse hyperplasia of the non-glandular mucosa, erosion/vesicles, ulceration, edema and mixed inflammation.

PLEASE REFER TO THE TABLE IN ANY OTHER INFORMATION ON RESULTS: "MACROPSCOPIC FINSINGS - TERMINAL SACRIFICE F1 GENERATION (COHORT 1B)"

F1 Generation (Cohort 1B)
Low absolute thymus weights were noted at terminal sacrifice in males that received 150 mg/kg/day.

Other variations in organ weights consisted of decreases/increases of absolute or relative weights of pituitary glands (both sexes administered 150 mg/kg/day), testes, epididymides and seminal vesicles and coagulating glands (males administered 70 or 150 mg/kg/day) and ovaries (females administered 150 mg/kg/day).

The effects of 2,2’-(Octadec-9-enylimino) bisethanol administration on organ weights and organ weights relative to body weight are mostly characterised as consequential to changes observed in body weight and not indicative of specific organ toxicity. The lower absolute and relative thymus weight in males at 70 and at 150 mg/kg/day is indicative for a secondary thymus involution.

PLEASE REFER TO THE TABLE IN ANY OTHER INFORMATION ON RESULTS: "ORGAN WEIGHT PARAMETERS - TERMINAL SACRIFICE F1 GENERATION (COHORT 1B)"
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
In unscheduled and terminal sacrifice animals, 2,2’-(Octadec-9-enylimino) bisethanol-related microscopic findings were observed in the stomach (males administered 70 or 150 mg/kg/day and females administered 30 or 150 mg/kg/day), jejunum (both sexes administered 30, 70 or 150 mg/kg/day), duodenum (both sexes administered 150 mg/kg/day), ileum (both sexes administered 70 or 150 mg/kg/day), mesenteric lymph nodes (males and females administered 70 or 150 mg/kg/day) and thymus (males administered 70 or 150 mg/kg/day).

Findings in the stomach included diffuse hyperplasia of the non-glandular mucosa, erosion/vesicles, ulceration, edema and mixed inflammation and correlated with macroscopic observations of thickening and/or depression. Findings in the jejunum, duodenum and ileum included diffuse accumulation of foamy cells within the lamina propria and lymphangiectasis accompanied by hypertrophic villi within the duodenum and ileum. Changes observed within the mesenteric lymph nodes consisted of foamy cell accumulation within the sinuses with sinus dilation and reflect the lymph nodes drainage function of the intestinal lesions. Changes observed within the thymus consisted of atrophy/involution and considered as probably due to body weight loss.


F1 Generation (Cohort 1B)
In unscheduled and terminal sacrifice animals, 2,2’-(Octadec-9-enylimino) bisethanol-related microscopic findings were observed in the stomach (males administered 70 or 150 mg/kg/day and females administered 150 mg/kg/day) and jejunum (males administered 150 mg/kg/day); histopathological examination was limited to those with macroscopic abnormality.

Findings in the stomach included diffuse hyperplasia of the non-glandular mucosa, erosion/vesicles, ulceration, edema and mixed inflammation and correlated with macroscopic observations of thickening and/or depression. Findings in the jejunum included diffuse accumulation of foamy cells within the lamina propria.

PLEASE REFER TO THE TABLES IN ANY OTHER INFORMATION ON RESULTS:
a) "MICROSCOPIC FINDINGS - TERMINAL SACRIFICE F1 GENERATION (COHORT 1A)"
b) "MICROSCOPIC FINDINGS - TERMINAL SACRIFICE F1 GENERATION (COHORT 1B)"
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Litter Size, Survival Indices and Sex Ratio
The number of implantation sites, post-implantation survival, live birth index and the number of offspring on Day 1 (total and live Day 1) were unaffected by parental treatment at dose levels up to and including 150 mg/kg/day.

However, at 150 mg/kg/day the viability index on Day 4 of age was slightly low when compared with Controls at 94.6% compared with 100% in the Control group; this was attributed to litter no 289 with a viability index of 18% and no relationship to treatment was inferred.

Litter survival following standardization of the litter size on Day 4 of age up to weaning on Day 21 of age was unaffected by treatment at all dose levels.

Sex ratio showed no effects of treatment.

Estrous cycles following Vaginal Opening - Cohort 1A
The interval between completion of vaginal opening and the first estrus smear showed no adverse effects of treatment.

Estrous Cycles from nominal Day 75 of age - Cohort 1A
Assessment of estrous cycles from nominal Day 75 of age showed a slight but statistically significant shift at 150 mg/kg/day from a 4-day cycle to 4/5 day or 5-day cycles when compared with Controls (p<0.05). At 70 mg/kg/day the incidence of 4/5 day cycles was also high when compared with Controls, but this difference did not attain statistical significance. The biological significance of these findings is minimal since the normal cycle duration is 4-5 days.

Estrous cycles at 30 mg/kg/day were similar to Controls

Stage of Estrous Cycle at Termination - Cohort 1A and 1B
The majority of females showed an estrus smear prior to scheduled termination.

Ovarian Follicle Counts and Corpora Lutea - Cohort 1A
Assessment of the follicle and corpora lutea counts in the Control and high dose did not show any adverse effects of treatment.

Sperm Assessment - Cohort 1A
Following treatment at 150mg/kg/day, there was a slight but statistically significant decrease in cauda epididymis total (million), this was within the historical control range. Motility, motion, testis and morphology parameters were unaffected.

There were positive statistically significant increases in some motion parameters (STR, LIN) and decreases in morphological parameters (tail abnormal and head misshapen).
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
30 mg/kg bw/day (actual dose received)
System:
gastrointestinal tract
Organ:
stomach
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Key result
Reproductive effects observed:
no
Lowest effective dose / conc.:
150 mg/kg bw/day (actual dose received)
Treatment related:
no

 

Group Mean Serum TSH Concentration (pg/mL) in Male Rats

Group

Treatment

Dose (mg/kg/day)

 

F0 Adults
Day 28

F1 Offspring
Day 22

F1 Adults
Cohort 1A
Day 91

 
 

1

Control

0

Mean

1260

287

542

 

SD

591

NA

304

 

%CV

46.8

NA

56.1

 

N

10

10

9

 

2

2,2'-(Octadec-9-enylimino)
bisethanol

30

Mean

2170

374

799

 

SD

1240

145

473

 

%CV

56.9

38.8

59.2

 

N

10

10

10

 

3

2,2'-(Octadec-9-enylimino)
bisethanol

70

Mean

1280

315

1140

 

SD

729

NA

617

 

%CV

56.7

NA

54.3

 

N

10

10

10

 

4

2,2'-(Octadec-9-enylimino)
bisethanol

150

Mean

1550

409

1090

 

SD

984

202

527

 

%CV

63.6

49.2

48.3

 

N

10

10

10

 

Where fewer than 66.7% of the individual values were BLQ, the mean was calculated using 0.5 x LLOQ. SD and %CV were not calculated

 

 

Group Mean Serum TSH Concentration (pg/mL) in Female Rats

Group

Treatment

Dose (mg/kg/day)

 

F0 Adults
Day 28

F1 Offspring
Day 22

F1 Adults
Cohort 1A
Day 91

 
 

1

Control

0

Mean

441

238

585

 

SD

267

NA

226

 

%CV

60.7

NA

38.6

 

N

10

10

10

 

2

2,2'-(Octadec-9-enylimino)
bisethanol

30

Mean

607

421

738

 

SD

324

NA

501

 

%CV

53.4

NA

67.9

 

N

10

10

10

 

3

2,2'-(Octadec-9-enylimino)
bisethanol

70

Mean

543

305

627

 

SD

631

NA

224

 

%CV

116.1

NA

35.7

 

N

9

10

10

 

4

2,2'-(Octadec-9-enylimino)
bisethanol

150

Mean

904

998

579

 

SD

393

876

NA

 

%CV

43.5

87.8

NA

 

N

10

10

10

 

Where fewer than 66.7% of the individual values were BLQ, the mean was calculated using 0.5 x LLOQ. SD and %CV were not calculated

 

 

 

 

Organ Weight Parameters – Terminal Sacrifice F0 Generation

Sex

2,2’- (Octadec-9-enylimino) bisethanol

Males

Females

Dose (mg/kg/day)

0

30

70

150

0

30

70

150

 

 

 

 

 

 

 

 

 

Kidney

 

 

 

 

 

 

 

 

Absolute Weight (g)

2.484

97

99

97

1.685

101

113

115

Relative Mean Organ Weight (%)

0.545

101

110**

120**

0.693

103

110**

119**

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Liver

 

 

 

 

 

 

 

 

Absolute Weight (g)

13.267

98

94

96

9.222

101

107

113

Relative Mean Organ Weight (%)

2.90

102

105

118**

3.77

103

105

117**

 

 

 

 

 

 

 

 

 

Spleen

 

 

 

 

 

 

 

 

Absolute Weight (g)

0.670

99

104

100

0.536

99

103

104

Relative Mean Organ Weight (%)

0.147

103

116**

124**

0.221

100

101

107

 

 

 

 

 

 

 

 

 

Adrenals

 

 

 

 

 

 

 

 

Absolute Weight (g)

0.059

105

98

100

0.073

104

107

107

Relative Mean Organ Weight (%)

0.0130

110

108

123**

0.0303

104

104

110*

 

 

 

 

 

 

 

 

 

Brain

 

 

 

 

 

 

 

 

Absolute Weight (g)

2.121

100

100

99

1.908

100

100

100

Relative Mean Organ Weight (%)

0.466

105

111**

122**

0.790

101

98

102

 

 

 

 

 

 

 

 

 

Epididymides

 

 

 

 

 

 

 

 

Absolute Weight (g)

1.462

99

99

96

 

 

 

 

Relative Mean Organ Weight (%)

0.323

103

110*

118**

 

 

 

 

Sex

2,2’- (Octadec-9-enylimino) bisethanol

Males

Females

Dose (mg/kg/day)

0

30

70

150

0

30

70

150

 

 

 

 

 

 

 

 

 

Testes

 

 

 

 

 

 

 

 

Absolute Weight (g)

3.929

100

101

102

 

 

 

 

Relative Mean Organ Weight (%)

0.862

104

113**

126**

 

 

 

 

 

 

 

 

 

 

 

 

 

Ovaries and oviducts

 

 

 

 

 

 

 

 

Absolute Weight (g)

 

 

 

 

0.133

110

113

113

Relative Mean Organ Weight (%)

 

 

 

 

0.0548

112*

110*

117**

* = p<0.05; ** = p<0.01 statistically significant difference (absolute or relative) compared with respective control mean value.

 

 

 

Macroscopic Findings – Terminal Sacrifice F0 generation

Sex

2,2’-(Octadec-9-enylimino) bisethanol

Males

Females

Dose Level (mg/kg/day)

0

30

70

150

0

30

70

150

Stomach

 

 

 

 

 

 

 

 

Number Examined

24

25

25

25

24

24

22

20

  Thickened

0

8

9

21

0

3

11

9

  Edematous

0

0

0

0

0

2

2

1

 

 

 

 

 

 

 

 

 

Jejunum

 

 

 

 

 

 

 

 

Number examined

24

25

25

25

24

24

22

20

  Abnormal color

0

0

2

5

0

0

0

3

  Thickened

0

0

0

9

0

0

0

1

 

 

 

 

 

 

 

 

 

 

Microscopic Findings – Terminal Sacrifice F0 Generation

Sex

2,2’-(Octadec-9-enylimino) bisethanol

Males

Females

Dose Level (mg/kg/day)

0

30

70

150

0

30

70

150

Stomach

 

 

 

 

 

 

 

 

Number Examined

25

25

25

25

25

25

25

25

 

 

 

 

 

 

 

 

 

  Hyperplasia, Epithelial, Non-Glandular Region

 

 

 

 

 

 

 

 

Minimal

0

8

6

4

0

6

6

5

Slight

0

3

9

13

0

2

12

13

Moderate

0

0

3

6

0

1

2

7

  Erosion/Vesicle, Non-Glandular Region

 

 

 

 

 

 

 

 

Minimal

0

0

0

0

0

0

0

0

Slight

0

0

1

0

0

0

0

2

Moderate

0

0

0

0

0

0

2

0

  Ulceration, Non-Glandular Region

 

 

 

 

 

 

 

 

Minimal

0

0

2

1

0

0

0

0

Slight

0

1

0

0

0

1

0

1

Moderate

0

0

1

0

0

0

0

2

  Edema, Non-Glandular Region

 

 

 

 

 

 

 

 

Minimal

0

1

1

0

0

3

1

2

Slight

0

1

2

1

0

0

4

5

Moderate

0

0

0

0

0

0

1

5

  Inflammation, Non-Glandular Region

 

 

 

 

 

 

 

 

Minimal

0

1

2

2

0

4

6

7

Slight

0

0

1

2

0

0

0

2

 

 

 

 

 

 

 

 

 

Jejunum

 

 

 

 

 

 

 

 

Number Examined

25

25

25

25

24

25

25

25

Accumulation, Foamy Cells

 

 

 

 

 

 

 

 

Minimal

0

0

3

0

0

0

16

2

Slight

0

0

12

3

0

0

3

11

Moderate

0

0

7

22

0

0

0

12

 

 

 

 

 

 

 

 

 

Duodenum

 

 

 

 

 

 

 

 

Number Examined

25

25

25

25

25

0

0

25

Accumulation, Foamy Cells

 

 

 

 

 

 

 

 

Minimal

0

0

0

3

0

-

-

0

Slight

0

0

0

6

0

-

-

0

Moderate

0

0

0

0

0

-

-

0

 

 

 

 

 

 

 

 

 

 

Formulation Analysis

The mean achieved concentrations of formulation samples taken during the course of the study were within 6% of the nominal concentration, confirming the accuracy of formulation. The difference from mean remained within 2%, confirming precise analysis.

For First Week (F0 generation), procedural recoveries were outside of the range established during the validation. The standard and sample responses remained consistent throughout, and therefore the procedural recoveries were considered to be prepared incorrectly and were not used to correct results. All system suitability criteria for this run was met except standard comparison. At most the standards were made with an error of 10%, and the worse possible outcome is that the results are marginally outside of the acceptance criteria stated in the study plan. The results are reported for information only but considered meaningful.

For Week 1 (F1 generation) Group 1 due to a response at the same retention time as the test item, re-dilution of original samples took place and contingency samples were analyzed. Both the re-dilution of original samples and contingency samples confirmed the original results, however the response is less than the quantifiable range for the method. There are four results reported for this group.

For Last week (F1 generation), Group 2 had a difference from mean outside of specified limits. Re-dilutions of original samples provided variable results and therefore were deemed unreliable. The contingency samples were analyzed, and confirmed the original results, therefore four results are reported for this group and the co-efficient of variation was 10.17%.

For Last week (F1 generation) contingency analysis, procedural recoveries were outside of the range established during the validation. No results were deemed outliers and therefore were used to correct results.

For all other occasions, procedural recoveries remained within the range established during the validation, confirming the continued accuracy of the analytical procedure.

 

Organ Weight Parameters – Unselected offspring on Day 22 of age (F1)

Sex

2,2’- (Octadec-9-enylimino) bisethanol

Males

Females

Dose (mg/kg/day)

0

30

70

150

0

30

70

150

 

 

 

 

 

 

 

 

 

Body weight (g)

49.2

99

100

91*

54.1

107

106

95

 

 

 

 

 

 

 

 

 

Thymus

 

 

 

 

 

 

 

 

Absolute Weight (g)

0.196

97

99

88*

0.192

106

109

91

Relative Mean Organ Weight (%)

0.398

98

99

97

0.426

99

103

96

 

 

 

 

 

 

 

 

 

Spleen

 

 

 

 

 

 

 

 

Absolute Weight (g)

0.212

109

97

88

0.192

116

118

104

Relative Mean Organ Weight (%)

0.431

110

96

97

0.426

108

111

109

 

 

 

 

 

 

 

 

 

Brain

 

 

 

 

 

 

 

 

Absolute Weight (g)

1.461

97

96

94*

1.367

103

102

97

Relative Mean Organ Weight (%)

2.970

98

96

104

3.031

96

97

101

* = p<0.05; ** = p<0.01 statistically significant difference (absolute or relative) compared with respective control mean value.

 

 

Organ Weight Parameters – Terminal Sacrifice F1 Generation (Cohort 1A)

Sex

2,2’- (Octadec-9-enylimino) bisethanol

Males

Females

Dose (mg/kg/day)

0

30

70

150

0

30

70

150

 

 

 

 

 

 

 

 

 

Thymus

 

 

 

 

 

 

 

 

Absolute Weight (g)

0.411

93.4

81.5**

66.4**

0.323

106.5

97.5

96.9

Relative Mean Organ Weight (%)

0.110

94.5

90.9

84.5*

0.145

108.9

101.3

106.2

 

Mesenteric lymph nodes

 

 

 

 

 

 

 

 

Absolute Weight (g)

0.018

100

100

133.3

0.015

153.3

126.6

173.3*

Relative Mean Organ Weight (%)

0.0049

104

112.2

173.4

0.0070

150

131.4

181.4*

 

 

 

 

 

 

 

 

 

Kidney

 

 

 

 

 

 

 

 

Absolute Weight (g)

2.297

107

105

90*

1.448

104

105

105

Relative Mean Organ Weight (%)

0.619

107*

117**

113**

0.648

107**

110**

115**

 

 

 

 

 

 

 

 

 

Heart

 

 

 

 

 

 

 

 

Absolute Weight (g)

1.029

101

94

84**

0.744

103

97

92

Relative Mean Organ Weight (%)

0.278

101

105*

107*

0.333

105

101

101

 

 

 

 

 

 

 

 

 

Sex

2,2’- (Octadec-9-enylimino) bisethanol

Males

Females

Dose (mg/kg/day)

0

30

70

150

0

30

70

150

Liver

 

 

 

 

 

 

 

 

Absolute Weight (g)

12.689

103

93

84*

7.520

103

104

109

Relative Mean Organ Weight (%)

3.40

102

104

105

3.36

105

108

120**

 

 

 

 

 

 

 

 

 

Spleen

 

 

 

 

 

 

 

 

Absolute Weight (g)

0.634

107

98

93

0.443

110

105

107

Relative Mean Organ Weight (%)

0.171

106

109*

117**

0.198

113*

109*

117**

 

 

 

 

 

 

 

 

 

Adrenals

 

 

 

 

 

 

 

 

Absolute Weight (g)

0.067

97

98

92

0.074

100

96

96

Relative Mean Organ Weight (%)

0.0181

97

110

117**

0.330

102

101

106

 

 

 

 

 

 

 

 

 

Brain

 

 

 

 

 

 

 

 

Absolute Weight (g)

2.038

102

97

93**

1.887

100

97*

96**

Relative Mean Organ Weight (%)

0.553

102

109*

118**

0.848

103

101

106

 

 

 

 

 

 

 

 

 

Epididymides

 

 

 

 

 

 

 

 

Absolute Weight (g)

1.274

100

95

87**

 

 

 

 

Relative Mean Organ Weight (%)

0.345

100

107

110**

 

 

 

 

 

 

 

 

 

 

 

 

 

Testes

 

 

 

 

 

 

 

 

Absolute Weight (g)

3.637

102

100

99

 

 

 

 

Relative Mean Organ Weight (%)

0.98

103

113**

125**

 

 

 

 

 

 

 

 

 

 

 

 

 

Ovaries and oviducts

 

 

 

 

 

 

 

 

Absolute Weight (g)

 

 

 

 

0.121

109

101

110

Relative Mean Organ Weight (%)

 

 

 

 

0.540

112

106

121**

 

 

 

 

 

 

 

 

 

* = p<0.05; ** = p<0.01 statistically significant difference (absolute or relative) compared with respective control mean value.

 

 

 

Organ Weight Parameters – Terminal Sacrifice F1 Generation (Cohort 1B)

Sex

2,2’- (Octadec-9-enylimino) bisethanol

Males

Females

Dose (mg/kg/day)

0

30

70

150

0

30

70

150

 

 

 

 

 

 

 

 

 

Pituitary

 

 

 

 

 

 

 

 

Absolute Weight (g)

0.009

100

100

89**

0.013

92

100

85**

Relative Mean Organ Weight (g)

0.0023

100

104

113*

0.0057

95

96

88**

 

 

 

 

 

 

 

 

 

Thymus

 

 

 

 

 

 

 

 

Absolute Weight (g)

0.372

110

98

73**

0.325

99

102

91

Relative Mean Organ Weight (g)

0.0956

111

103

88

0.141

103

102

98

 

 

 

 

 

 

 

 

 

Sex

2,2’- (Octadec-9-enylimino) bisethanol

Males

Females

Dose (mg/kg/day)

0

30

70

150

0

30

70

150

Testes

 

 

 

 

 

 

 

 

Absolute Weight (g)

3.743

101

103

102

 

 

 

 

Relative Mean Organ Weight (g)

0.96

101

109**

125**

 

 

 

 

 

 

 

 

 

 

 

 

 

Seminal vesicles and coagulating glands

 

 

 

 

 

 

 

 

Absolute Weight (g)

1.045

105

120*

97

 

 

 

 

Relative Mean Organ Weight (g)

0.267

105

128**

119**

 

 

 

 

 

 

 

 

 

 

 

 

 

Epididymides

 

 

 

 

 

 

 

 

Absolute Weight (g)

1.280

104

103

94

 

 

 

 

Relative Mean Organ Weight (g)

0.329

104

109*

115**

 

 

 

 

 

 

 

 

 

 

 

 

 

Ovaries and oviducts

 

 

 

 

 

 

 

 

Absolute Weight (g)

N/A

N/A

N/A

N/A

0.124

104

106

110**

Relative Mean Organ Weight (g)

N/A

N/A

N/A

N/A

0.0540

108

107

119**

 

 

 

 

 

 

 

 

 

* = p<0.05; ** = p<0.01 statistically significant difference (absolute or relative) compared with respective control mean value.

 

 

Macroscopic Findings – Terminal Sacrifice F1 Generation (Cohort 1A)

Sex

2,2’-(Octadec-9-enylimino) bisethanol

Males

Females

Dose Level (mg/kg/day)

0

30

70

150

0

30

70

150

Stomach

 

 

 

 

 

 

 

 

Number Examined

20

20

20

20

20

20

20

20

  Thickened

1

1

5

7

0

0

3

4

  Depression

0

0

0

2

0

0

1

1

 

 

 

 

 

 

 

 

 

 

Macroscopic Findings – Terminal Sacrifice F1 Generation (Cohort 1B)

Sex

2,2’-(Octadec-9-enylimino) bisethanol

Males

Females

Dose Level (mg/kg/day)

0

30

70

150

0

30

70

150

Stomach

 

 

 

 

 

 

 

 

Number Examined

20

20

20

20

20

20

20

20

  Thickened

0

0

3

2

0

0

0

1

  Depression

0

0

3

4

0

0

0

2

 

 

 

 

 

 

 

 

 

 

 

 

Microscopic Findings – Terminal Sacrifice F1 Generation (Cohort 1A)

Sex

2,2’-(Octadec-9-enylimino) bisethanol

Males

Females

Dose Level (mg/kg/day)

0

30

70

150

0

30

70

150

Stomach

 

 

 

 

 

 

 

 

Number Examined

20

20

20

20

20

20

20

20

 

 

 

 

 

 

 

 

 

  Hyperplasia, Epithelial, Non-Glandular Region

 

 

 

 

 

 

 

 

Minimal

0

0

6

7

0

0

0

6

Slight

0

0

1

7

0

0

0

7

Moderate

0

0

0

3

0

0

0

1

  Erosion/Vesicle, Non-Glandular Region

 

 

 

 

 

 

 

 

Minimal

0

0

0

0

0

1

0

0

Slight

0

0

2

3

0

0

0

3

Moderate

0

0

0

1

0

0

0

1

  Ulceration, Non-Glandular Region

 

 

 

 

 

 

 

 

Minimal

0

0

0

0

0

0

0

0

Slight

0

0

0

2

0

0

0

0

Moderate

0

0

0

2

0

1

0

1

  Edema, Non-Glandular Region

 

 

 

 

 

 

 

 

Minimal

0

0

0

4

0

1

0

5

Slight

0

0

4

3

0

0

0

1

Moderate

0

0

0

3

0

0

0

0

 

 

 

 

 

 

 

 

 

 

Sex

2,2’-(Octadec-9-enylimino) bisethanol

Males

Females

Dose Level (mg/kg/day)

0

30

70

150

0

30

70

150

Stomach

 

 

 

 

 

 

 

 

  Inflammation, Non-Glandular Region

 

 

 

 

 

 

 

 

Minimal

0

0

3

5

0

0

0

3

Slight

0

0

2

4

0

1

0

2

Jejunum

 

 

 

 

 

 

 

 

Number Examined

20

20

20

20

20

20

20

20

  Accumulation, Foamy Cells

 

 

 

 

 

 

 

 

Minimal

0

1

15

1

0

2

8

0

Slight

0

0

0

7

0

0

0

9

Moderate

0

0

0

10

0

0

0

11

Marked

0

0

0

2

0

0

0

0

  Lymphangiectasis

 

 

 

 

 

 

 

 

Minimal

0

0

0

2

0

0

0

0

Duodenum

 

 

 

 

 

 

 

 

Number Examined

20

20

20

20

20

20

20

20

  Accumulation, Foamy Cells

 

 

 

 

 

 

 

 

Minimal

0

0

0

10

0

0

0

2

Slight

0

0

0

2

0

0

0

1

  Hypertrophy, Villous

 

 

 

 

 

 

 

 

Minimal

0

0

0

12

0

0

0

1

Slight

0

0

0

2

0

0

0

0

  Lymphangiectasis

 

 

 

 

 

 

 

 

Minimal

0

0

0

3

0

0

0

0

Ileum

 

 

 

 

 

 

 

 

Number Examined

20

20

20

20

19

20

19

20

  Accumulation, Foamy Cells

 

 

 

 

 

 

 

 

Minimal

0

0

4

12

0

0

3

6

Slight

0

0

0

0

0

0

0

0

Moderate

0

0

0

0

0

0

0

0

  Hypertrophy, Villous

 

 

 

 

 

 

 

 

Minimal

0

0

0

8

0

0

0

1

Slight

0

0

0

0

0

0

0

0

  Lymphangiectasis

 

 

 

 

 

 

 

 

Minimal

0

0

0

1

0

0

0

0

 

 

 

 

 

 

 

 

 

Mesenteric Lymph Node

 

 

 

 

 

 

 

 

Number Examined

20

20

20

20

19

20

20

20

  Accumulation, Foamy Cells

 

 

 

 

 

 

 

 

Minimal

0

0

8

0

0

0

7

0

Slight

0

0

11

3

0

0

11

5

Moderate

0

0

1

14

0

0

1

13

Marked

0

0

0

2

0

0

0

2

  Dilated/Cystic, Sinuses

 

 

 

 

 

 

 

 

Minimal

0

2

3

0

0

1

1

2

Slight

0

0

1

5

0

0

0

11

Moderate

0

0

0

14

0

0

0

6

Thymus

 

 

 

 

 

 

 

 

Number Examined

20

18

18

20

20

0

1

20

  Involution/Atrophy

 

 

 

 

 

 

 

 

Minimal

0

0

2

13

0

0

1

1

Slight

0

1

0

0

0

0

0

0

 

 

 

 

 

 

 

 

 

 

 

Microscopic Findings – Terminal Sacrifice F1 Generation (Cohort 1B)

Sex

2,2’-(Octadec-9-enylimino) bisethanol

Males

Females

Dose Level (mg/kg/day)

0

30

70

150

0

30

70

150

Stomach

 

 

 

 

 

 

 

 

Number Examined

0

1

5

8

0

0

0

3

 

 

 

 

 

 

 

 

 

  Hyperplasia, Epithelial, Non-Glandular Region

 

 

 

 

 

 

 

 

Slight

0

0

2

1

0

-

-

2

Moderate

0

0

2

5

0

-

-

1

  Erosion/Vesicle, Non-Glandular Region

 

 

 

 

 

 

 

 

Minimal

0

0

0

1

0

-

-

0

Slight

0

0

0

1

0

-

-

0

  Ulceration, Non-Glandular Region

 

 

 

 

 

 

 

 

Slight

0

0

0

0

0

-

-

1

Moderate

0

0

0

1

0

-

-

0

  Edema, Non-Glandular Region

 

 

 

 

 

 

 

 

Minimal

0

0

2

1

0

-

-

2

Slight

0

0

0

2

0

-

-

0

  Inflammation, Non-Glandular Region

 

 

 

 

 

 

 

 

Minimal

0

0

2

2

0

-

-

1

Slight

0

0

0

2

0

-

-

1

 

 

 

 

 

 

 

 

 

Jejunum

 

 

 

 

 

 

 

 

Number Examined

0

1

0

1

0

-

-

0

Accumulation, Foamy Cells

 

 

 

 

 

 

 

 

Minimal

0

0

0

1

0

-

-

0

 

 

 

 

 

 

 

 

 

 

 

Conclusions:
Based on the results of this study, it is concluded that the No Observed Adverse Effect Level (NOAEL) for both systemic and reproductive toxicity in the Han Wistar rat is 150 mg/kg/day and the NOAEL for the local gastrointestinal irritant effect was not established and lies below 30 mg/kg/day.
Executive summary:

The purpose of this study was to assess the influence of 2,2'‑(Octadec‑9‑enylimino)bisethanol on reproductive performance when administered  by oral gavage administration to Han Wistar rats. Cohorts of F1 animals were used to assess the potential for systemic toxicity, and potential effects on sexual maturation and estrous cycles.

Dose levels for the EOGRTS were selected after evaluation in a RF in which four groups of eight male and eight female rats received 2,2’‑(Octadec-9-enylimino)bisethanol at doses of 0, 30, 70 or 150 mg/kg/day by oral gavage. Males were treated daily for 15 days before pairing and until termination. Females were treated daily for 15 days before pairing, throughout pairing, gestation and lactation. The F1 generation comprised of 10 male and 10 female progeny from each group, and they continued to receive the relevant dose, as per the F0 generation, throughout the study until termination following sexual maturation at approximately eight weeks of age. The oral administration at dose levels of 30, 70 or 150 mg/kg/day had no adverse effect on general clinical condition (F0/F1), mating performance and fertility (F0), organ weights (F0) or macropathology (F0/F1). However, at 150 mg/kg/day clear effects were apparent on both F0 and F1 body weight performance and two out of the eight litters at this dose level were compromised resulting in high mortality/litter death. It was not clear whether this is indicative for a too severe perinatal toxicity or mostly accidental as suggested based on results on similar structures. Based on the findings in this preliminary study it was decided to use of 150 mg/kg/day as the high dose in the subsequent OECD 443 study.

In the F0 generation of the EOGRTS, three groups of 25 male and 25 female rats received 2,2'‑(Octadec‑9‑enylimino)bisethanol at dose levels of 30, 70 or 150 mg/kg/day at a volume dose of 4 mL/kg/day. Males were treated for ten weeks before pairing, up to necropsy after litters were weaned. Females were treated for ten weeks before pairing, throughout pairing up to necropsy on Day 28 of lactation.

For the F0 generation data were recorded on clinical condition, body weight, food consumption, estrous cycles, mating performance and fertility, gestation length and parturition observations and reproductive performance.  Clinical pathology (hematology, blood chemistry and thyroid-related hormones), sperm assessment, organ weight, macroscopic pathology and microscopic pathology investigations were performed. 

For F1 offspring, clinical condition, litter size and survival, sex ratio, body weight, ano‑genital distance, organ weights and macropathology were assessed.  Nipple counts were performed on male offspring on Day 13 of age.  Serum samples that were collected from selected offspring on Day 22 of age were analysed for thyroid-related hormones.

The F1 generation comprised of two cohorts:

Cohort 1A: 20 male and 20 female progeny were selected from each dose group and continued to receive 2,2'‑(Octadec‑9‑enylimino)bisethanol at doses of 0 (Control), 30, 70 or 150 mg/kg/day from weaning until scheduled sacrifice at approximately Week 13 of age. 

Cohort 1B: 20 male and 20 female progeny were selected from each dose group and continued to receive 2,2'‑(Octadec‑9‑enylimino)bisethanol at doses of 0 (Control), 30, 70 or 150 mg/kg/day from weaning until scheduled sacrifice at approximately Week 14 of age. 

For F1 Cohort 1A, data were recorded on clinical condition, body weight, food consumption, sexual maturation and estrous cycles. Clinical pathology (hematology, blood chemistry, urinalysis and biomarkers), sperm assessment, ovarian follicle and corpora lutea counts, organ weight, macroscopic pathology, full microscopic pathology and immunophenotyping investigations were performed. 

For F1 Cohort 1B, data was recorded on clinical condition, body weight, food consumption, sexual maturation, organ weight and macroscopic pathology investigations were performed.

 

Results

Oral administration of 2,2’-(Octadec-9-enylimino) bisethanol to Han Wistar rats at dose levels of 30, 70 and 150 mg/kg/day showed no treatment-related effects on general condition (F0/F1), estrous cycles (F0/F1), thyroid hormones(F0/F1), sperm parameters (F0/F1), mating performance (F0), fertility (F0), gestation length (F0), ano genital distance (F1), spleen cell immunophenotyping (F1) or sexual maturation (F1).

There was a total of four premature deaths at 150 mg/kg/day that were considered to relate to treatment; three F0 females and one F1 male.  Macroscopic and histopathological examination of these animals revealed findings related to the stomach and small intestine. 

Histopathological examination of F0 and F1 animals at scheduled termination revealed findings in the non-glandular portion of the stomach that consisted of epithelial hyperplasia associated with erosion/vesicle or ulceration, edema and mixed inflammation and correlated with macroscopic observations of thickened stomach and depressions. The combination of these findings was considered adverse in this study at all dose levels. The higher neutrophil counts for F0 males and females at 150 mg/kg/day, the higher eosinophil counts in F0 females at 150 mg/kg/day, and the higher white blood cell counts in F1 males at 150 mg/kg/day and F1 females at all dose levels were considered likely to reflect the mixed inflammation in the stomach.

In the jejunum (F0/F1), duodenum (F0/F1) and ileum (F1) showed histopathological changes at scheduled termination.  

  • In the F0 adults the jejunum and duodenum showed foamy cell accumulation within the lamina propria, probably related to the absorption of the test item and/or metabolites and correlated with the macroscopic observations of thickened and abnormal color. The jejunum was more severely affected than the duodenum. At the severity observed this is likely to compromise function and indicates an adverse effect for the males and females administered 70 or 150 mg/kg/day.
  • In the F1 adults the intestine showed diffuse accumulation of the foamy cells within the lamina propria and lymphangiectasis (duodenum, jejunum and ileum) accompanied by villous hypertrophy (duodenum and ileum), with the jejunum being  most severely affected. At the severity noted is likely to compromise function and indicates an adverse effect for the males and females administered 70 or 150 mg/kg/day.  The mesenteric lymph nodes showed accumulation of the foamy cells and dilation of the sinuses: this change is secondary to the intestinal findings, reflecting the drainage function of these lymph nodes.

For both the F0 and F1 adult animals the reductions observed inlife on both body weight gain and food consumption are therefore considered to be secondary to these histopathological findings.  As a consequence of these effects on the F0 adult animals the body weight and body weight gain of F1 offspring was low at 150 mg/kg/day and the F1 adults showed atrophy/involution of the thymus.

Body weight relative kidney weights for both F0 and F1 adult animals were marginally high when compared with Controls but absolute kidney weights were generally unaffected.  F0 males at 70 and 150 mg/kg/day and F1 males at 150 mg/kg/day had marginally high plasma urea levels.  There was no histopathological correlate  for these findings and no adverse or conclusive effect on renal function was inferred.

There were a number of minor changes in haematology, blood chemistry and urinalysis and organ weight parameters, but none were supported by any test item related microscopic changes in the full range of tissues examined and are of uncertain significance and limited biological relevance and may be incidental or minor consequences to the primary pathology observed and likely not direct effects of the test substance.

The stomach changes are considered to represent a local effect of the irritancy of the test item rather than any systemic effect of toxicity however in view of the mortality at 150 mg/kg/day and the extent of the histopathological changes in the stomach at all doses and small intestine at 70 and 150 mg/kg/day these findings are considered as adverse.  Therefore the No Observed Adverse Effect Level (NOAEL) for the local irritant effect was not established and the NOAEL for both systemic and reproductive toxicity is considered to be 150 mg/kg/day.

 

Conclusion

Based on the results of this study, it is concluded that the No Observed Adverse Effect Level (NOAEL) for both systemic and reproductive toxicity in the Han Wistar rat is 150 mg/kg/day and the NOAEL for the local gastrointestinal irritant effect was not established and lies below 30 mg/kg/day.

 

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
High quality OECD 422 study on the target substance and a read across for the conducted EOGRT on 2, 2’-(Octadec-9-enylimino) bisethanol CAS No 25307-17-9 which is the most appropriate read across to for di‐(2‐hydroxypropyl) C16‐C18 (evennumbered), C18 unsaturated‐alkyl amine allows for the comprehensive assessment of reproductive/developmental endpoints. The OECD 422 being conducted on di‐(2‐hydroxypropyl) C16‐C18 (evennumbered), C18 unsaturated‐alkyl amine CAS 1309955‐79‐0 helps to re-enforce conclusions form the current read-across to the EOGRTS.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

There is specific data on toxicity to reproduction for di‐(2‐hydroxypropyl) C16‐C18 (evennumbered), C18 unsaturated‐alkyl amine CAS 1309955‐79‐0 from a OECD 422 with an extended premating period of 10 weeks. In this study there were indications of gastointestinal irritation resulting in a local NOAEL of 50mg/kg/day. However, reproductive and developmental NOAELs were determined to be 150 mg/kg/day (based on the lower number of corpora lutea and implantation sites) and 150 mg/kg/day (based on the small live litter sizes, high pup mortality and low pup body weights) respectively.


There is also addtional information on 2, 2’-(Octadec-9-enylimino) bisethanol CAS No 25307-17-9 from a OECD 443 study which is considered to be the most appropriate read across substance.  2, 2’-(Octadec-9-enylimino) bisethanol CAS No 25307-17-9 is predominantly C18 unsaturated and C16 and di‐(2‐hydroxypropyl) C16‐C18 (evennumbered), C18 unsaturated‐alkyl amine which is predominately C18, C18 unsaturated and C16. In the OECD 443 there were indications of gastrointestinal irritation in F0 and F1 animals leading to observable reductions in bodyweight gain. However in this study there were no indications of any effects on reproductive performance, fertility, or development in other key parameters. There for the NOAEL for developmental/ reproductive toxicity was determined to be 150 mg/kg/day with the effects on the gastrointestinal tract leading to a systemic LOEAL of 30 mg/kg/day. 


 As 2, 2’-(Octadec-9-enylimino) bisethanol CAS No 25307-17-9 is a high volume substance >1000 tonnes with a high volume of reliable and the substances relevance for read-across on the basis of structure adequate conclusions can be drawn for di‐(2‐hydroxypropyl) C16‐C18 (evennumbered), C18 unsaturated‐alkyl amine CAS 1309955‐79‐0 ability to effect developmental/reproductive endpoints.


 


Short description of key information:


Combine 90-day repeat-dose toxicity study with reproductive toxicity screening (OECD 422/OECD 408)


An extended OECD 422 was conducted on di‐(2‐hydroxypropyl) C16‐C18 (evennumbered), C18 unsaturated‐alkyl amine CAS 1309955‐79‐0. This modified study design from the normal OECD 422 added an addtional 10 weeks premating instead of the standard 2 weeks. Dosing groups consisted of control, 50, 150 and 300 mg/kg bw/day with an Arachis oil vehicle at a volume of 4mL/kg. This studies design allowed followed both the OECD 422 and OECD 408 guidlines with only minor variations and is suitable for a Klimisch score of 1. Minor variations consited of initiation of dosing at 6 weeks of age (422 variation), lack of pre-dose oesterous cycle determination (422 variation), change in blood chemistry timeline (post-partium day 13)(408 variation), maternal condition of females, lack of fasting in females before clinical chemistry sampling (408 variation). These minor variations were determined to not effect the reliability of the results and provided addtional benifits to the study that would not be seen in a tradtional OECD 422 design such as, reduced animal usage, prolonged male exposure resulting in a full spermatogensis cycle, prolonged premating female exposure closely mimicing that utilized in EOGRTS, the addtional benifit of examining sub-chronic exposure on fertility and possible bioaccumulation effects on development.


The dose levels in this study were selected to be 0, 50, 150, 300 mg/kg/day, based on the results of the Dose Range Finder and a Prenatal Developmental Toxicity Study. The DRF study proceeded with dose levels of 150, 200, 250, 300, 450, and 600 mg/kg bw/day with three males assigned to each group. the DRF did not showed reduced bodyweight gain in all dose groups except 150mg/kg bw/day, with bodyweight loss at 600 mg/kg bw/day being so severe that all three animals in the dose group were sacrificed for wellfare reasons. Compared to 150 and 300 mg/kg/day, absolute mean body weight was reduced 22% and 15% at 450 mg/kg/day, respectively on Day 14 of treatment. These results and the results of the OECD 414 which resulted in high maternal mortality at 375 mg/kg bw/day concluded that 300 mg/kg bw/day was likely the highest tolerable dose for the OECD 422.


During the OECD 422 two premature sacrifices occurred in maternal females at 300 mg/kg/day at 18 and 20 days post-coitum. At 300 mg/kg/day, non-adverse effects were seen on estrous cycle regularity in 9/10 females
which displayed an irregular estrous cycle or in which the estrous cycle could not be determined as only one estrous cycle was completed during the observation period. All females at this dose level, except the one female with a regular cycle, became pregnant. No test material-related changes were noted in any of the remaining reproductive parameters investigated in this study (i.e., mating and fertility indices, precoital time, spermatogenic profiling, and histopathological examination of reproductive organs). The reproductive NOAEL was determined to be 150 mg/kg/day (based on the lower number of corpora lutea and implantation sites).


At 150 mg/kg/day, the lower pup body weights on PND 4 and 7 were considered non-adverse as the pup body weights recovered on PND 13. At 300 mg/kg/day, the lower live litter sizes and low pup body weights were considered adverse and could be a secondary effect of maternal toxicity although a direct effect of the test material cannot be excluded. In addition, adverse lower viability and lactation indices were noted that could be attributed to the lack of maternal care of three females resulting from a poor condition of these females leading up to and following parturition.No test material-related changes were noted in any of the other developmental parameters investigated in this study (i.e., gestation index, duration of gestation, parturition, post-implantation survival index, live birth index, sex ratio, and some early postnatal pup development parameters consisting of anogenital distance, areola/nipple retention, T4 thyroid hormone levels and macroscopic examination). The developmental NOAEL was determined to be 150 mg/kg/day (based on the small live litter sizes, high pup mortality and low pup body weights).


This study resulted in a local NOAEL of 50 mg/kg/day (based on clinical signs, lower body weights and food consumption, macroscopic and microscopic findings in the stomach, small intestine and mesenteric lymph nodes). This NOAEL is used for the basis of the repeat-dose conclusions and hazards classifications (Section 7.5). This test was considered suitable to determine DNEL and hazard classification for reproductive toxicity endpoints. Addtional evidence for conclusions is given with the OECD 443 on 2, 2’-(Octadec-9-enylimino) bisethanol CAS No 25307-17-9.


 


EOGRT (OECD 443):


2, 2’-(Octadec-9-enylimino) bisethanol CAS No 25307-17-9 is the most appropriate read across substance for di‐(2‐hydroxypropyl) C16‐C18 (evennumbered), C18 unsaturated‐alkyl amine CAS 1309955‐79‐0.


Dose levels were selected after evaluation in a RF in which four groups of eight male and eight female rats received2,2’‑(Octadec-9-enylimino)bisethanolat doses of 0, 30, 70 or 150 mg/kg/day by oral gavage. Males were treated daily for 15 days before pairing and until termination. Females were treated daily for 15 days before pairing, throughout pairing, gestation and lactation. The F1 generation comprised of 10 male and 10 female progeny from each group, and they continued to receive the relevant dose, as per the F0 generation, throughout the study until termination following sexual maturation at approximately eight weeks of age. The oraladministration at dose levels of 30, 70 or 150 mg/kg/day had no adverse effect on general clinical condition (F0/F1), mating performance and fertility (F0), organ weights (F0) or macropathology (F0/F1). However, at 150 mg/kg/day clear effects were apparent on both F0 and F1 body weight performance and two out of the eight litters at this dose level were compromised resulting in high mortality/litter death. It was not clear whether this is indicative for a too severe perinatal toxicity or mostly accidental as suggested based on results on similar structures. Based on the findings in this preliminary study it was decided to use of 150 mg/kg/day as the high dose in the subsequent OECD 443 study.


In the F0 generation of the EOGRTS, three groups of 25 male and 25 female Han Wistar rats received 2,2'‑(Octadec‑9‑enylimino)bisethanol at dose levels of 30, 70 or 150 mg/kg/day at a volume dose of 4 mL/kg/day. Males were treated for ten weeks before pairing, up to necropsy after litters were weaned. Females were treated for ten weeks before pairing, throughout pairing up to necropsy on Day 28 of lactation. The F1 generation comprised of two cohorts: Cohort 1A and 1B, both consisting of 20 male and 20 female progeny. Cohort 1A continued to receive test substance from weaning until scheduled sacrifice at approximately Week 13 of age, and Cohort 1B from weaning until scheduled sacrifice at approximately Week 14 of age. 


Results: Oral administration at dose levels of 30, 70 and 150 mg/kg/day showed no treatment-related effects on general condition (F0/F1), estrous cycles (F0/F1), thyroid hormones(F0/F1), sperm parameters (F0/F1), mating performance (F0), fertility (F0), gestation length (F0), ano genital distance (F1), spleen cell immunophenotyping (F1) or sexual maturation (F1).


There was a total of four premature deaths at 150 mg/kg/day that were considered to relate to treatment; three F0 females and one F1 male.  Macroscopic and histopathological examination of these animals revealed findings related to the stomach and small intestine.For both the F0 and F1 adult animals the reductions observed inlife on both body weight gain and food consumption were considered to be secondary to these histopathological findings. As a consequence of these effects on the F0 adult animals the body weight and body weight gain of F1 offspring was low at 150 mg/kg/day and the F1 adults showed atrophy/involution of the thymus.


The stomach changes are considered to represent a local effect of the irritancy of the test item rather than any systemic effect of toxicity however in view of the mortality at 150 mg/kg/day and the extent of the histopathological changes in the stomach at all doses and small intestine at 70 and 150 mg/kg/day these findings are considered as adverse. Therefore the No Observed Adverse Effect Level (NOAEL) for the local irritant effect was not established and the NOAEL for both systemic and reproductive toxicity is considered to be 150 mg/kg/day.


 


Justification for selection of Effect on fertility via inhalation route:


The low vapour pressure of the substance means inhalation is not considered to be relevant route of exposure so not testing is required.


 


Justification for selection of Effect on fertility via dermal route:


The corrosive properties of this substance mean the repeated dose dermal studies are not scientifically justified due to concerns for animal welfare.

Effects on developmental toxicity

Description of key information

There is an OECD414 pre-natal developmental toxicity study on the substance di‐(2‐hydroxypropyl) C16‐C18 (evennumbered), C18 unsaturated‐alkyl amine CAS 1309955‐79‐0. This study showed no indication of developmental toxicity in the foetuses at the NOAEL being 100mg/kg (lowest dose tested).  There was however observable effects of decreased food consumption and body weight gain of maternal animals and decreased mean foetal bodyweights at both 300 and 375 mg/kg bw/day.  This study does not include dosing during the implantation period, the only available information concerning this is read across to the OECD443 study on 2, 2’-(Octadec-9-enylimino) bisethanol CAS No 25307-17-9 which is predominantly C18.  This OECD 443 did not show any signs effects of the test material on preimplantation or development of exposed rats.


A current OECD 422 reproductive screening test study is running for di‐(2‐hydroxypropyl) C16‐C18 (evennumbered), C18 unsaturated‐alkyl amine CAS 1309955‐79‐0 which will provide more directly relevant information on the implantation period of test animals exposed to the substance.  

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 Oct 2021 - 17 Dec 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Reliability 1
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
June 2018
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
May 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
August 1998
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
Crl: WI(Han)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 10-14 weeks old
- Weight at study initiation: 187 – 269 g
- Fasting period before study: not applicable
- Housing: Individually in polycarbonate cages (Makrolon type MIII, height 18 cm) containing sterilized wooden fibers as bedding material (Lignocel S 8-15, JRS-J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles.
- Diet: ad libitum, SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany. Results of analysis for nutritional components and environmental contaminants were provided by the Supplier. It is considered that there are no known contaminants in the feed that would interfere with the objectives of the study.
- Water: ad libitum, municipal tap water. Periodic analysis of the water was performed. It is considered that there are no known contaminants in the water that could interfere with the outcome of the study.
- Acclimation period: 5-6 days
- Justification of Test System and Number of Animals: At this time, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models, which do not use live animals, currently do not exist. The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for developmental toxicity testing by regulatory agencies. The test facility has historical data on the background incidence of fetal malformations and developmental variations in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of developmental toxicants. The total number of animals to be used in this study is considered to be the minimum required to properly characterize the effects of the test item. This study has been designed such that it does not require an unnecessary number of animals to accomplish its objectives.


ENVIRONMENTAL CONDITIONS
- Temperature: 21°C (actual daily mean)
- Humidity: 39-70% (actual daily mean)
- Air changes: At least 10 air changes per hour
- Photoperiod (hrs dark / hrs light):12 h light /12 h dark

IN-LIFE DATES: From: 18 Oct 2021 To: 05 Nov 2021
Route of administration:
oral: gavage
Vehicle:
peanut oil
Remarks:
Specific Gravity: 0.91
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared at least weekly. Formulations were divided into aliquots and stored in the refrigerator (4°C) until use. Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. Adjustment were made for specific gravity of the vehicle and test item. No correction was made for the purity/composition of the test item.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Trial preparations were performed to select the suitable vehicle and to establish a suitable formulation procedure.
- Concentration in vehicle: 0, 25, 75, 93.75 mg/mL
- Amount of vehicle: 4 mL/kg
- Lot/batch no.: not reported
- Specific Gravity: 0.91
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
During week 1 of treatment dose formulation samples were collected for analysis.

For concentration analysis, samples from the middle part of the dosing container of all dose groups were taken (2 x approximately 500 mg). For concentration, all mean sample concentration results within or equal to ± 10% (solutions) of theoretical concentration were considered acceptable.

For homogeneity analysis, samples from the top, middle and bottom parts of the dosing container of groups 2 and 4 (low- and high-dose groups) were taken (2 x approximately 500 mg). Samples were stored at a temperature set to maintain 18-22°C. For homogeneity, a relative standard deviation (RSD) of concentrations of ≤10% for each group was considered acceptable.

Stability analyses performed previously in conjunction with the method development and validation study (Test Facility Study No. 20279388) demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.
Details on mating procedure:
Untreated females were mated at the Supplier and were at Day 0 or 1 post-coitum on arrival at the Test Facility (Day 0 post-coitum is the day of successful mating).
Duration of treatment / exposure:
Day 6 to Day 20 post-coitum
Frequency of treatment:
once daily
Duration of test:
from Day 2 to Day 21 (scheduled euthanasia)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Group 2 - Low dose group
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
Group 3 - Mid dose group
Dose / conc.:
375 mg/kg bw/day (actual dose received)
Remarks:
Group 4 - High dose group
No. of animals per sex per dose:
22 females per group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The oral route of exposure was selected because this is a possible route of human exposure during manufacture, handling or use of the test item.
The dose levels were selected based on information provided by the Sponsor and the results of a Dose Range Finding Study with oral exposure of Tallow amine propoxylate in male rats (Test Facility Study No. 20279383), and in an attempt to produce graded responses to the test item. In the Dose Range Finding Study, 3 male rats per group were treated with 150, 200, 250, 300, 450 and 600 mg/kg bw/day. At 600 mg/kg bw/day, all 3 males were sacrificed in extremis on Day 12 of treatment with signs of erected fur, hunched posture, decreased activity, pale tail and salivation. Body weight loss (up to 18%) and reduced food consumption were noted. Macroscopic examination showed decreased adipose tissue in 3/3 males and dark red discoloration of the adrenals in 1/3 males. At 450 mg/kg bw/day, no mortality occurred. Clinical signs that were noted included erected fur, hunched posture and salivation on several days. Reduced body weight gain and food consumption were noted during Day 1-14 of treatment. Absolute mean body weight was reduced by 22% and 15% compared to treatment at 150 and 300 mg/kg bw/day, respectively. Macroscopic examination revealed no abnormalities; relative kidney and liver weights were slightly decreased. At 300 mg/kg bw/day, all animals were noted with erected fur, hunched posture and salivation on selective days only. Reduced body weight gain and food consumption was noted during Day 1-4 of treatment, which remained slightly lower between Days 4-14 of treatment. Macroscopic examination revealed no abnormalities; relative kidney and liver weights were slightly decreased. The high-dose level should produce some toxic effects, but not excessive lethality that would prevent meaningful evaluation. The mid-dose level is expected to produce minimal to moderate toxic effects. The low-dose level should produce no observable indications of toxicity.

- Fasting period before blood sampling for (rat) dam thyroid hormones: Animals were not fasted

- Time of day for (rat) dam blood sampling: Sampled between 07.00 and 09.00 from the jugular vein on necropsy day
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Mortality was checked at least twice daily beginning upon arrival through termination/release. Except on days of receipt and necropsy where frequency was at least once daily. Cageside observations were performed twice daily starting on Day 6 post-coitum up to the day prior to necropsy. Observations were made directly pre-dose and 1 to 2 hours postdose.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: On Days 2, 6, 15 and 21 post-coitum

BODY WEIGHT: Yes
- Time schedule for examinations: On Days 2, 6, 9, 12, 15, 18 and 21 post-coitum.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes
- Time schedule for examinations: Over Days 2-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post-coitum.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Regular basis throughout the study. Water consumption was monitored by visual inspection of the water bottles.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21 post-coitum using carbon dioxide inhalation. All animals (including animals found dead or sacrificed before planned necropsy and females with delivery prior to necropsy) were subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs.

OTHER:
- Tissue Collection: The thyroid gland and macroscopic abnormalities were collected from all animals and preserved in 10% buffered formalin.
- Microscopic evaluation: Thyroid gland of all animals was examined microscopically.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Number and distribution of live and dead fetuses. Distribution of early and late resorptions.
Blood sampling:
- Plasma: No
- Serum: Yes
- Volume collected: 1 mL
- Serum was collected for T3, T4 and TSH analysis
Fetal examinations:
- External examinations: Yes: all per litter (for viable and non-viable fetuses of dam surviving until scheduled necropsy on Day 21 post-coitum, late resorptions and fetuses of dams not surviving until Day 21 post-coitum)

- Soft tissue examinations: Yes: half per litter (for viable and non-viable fetuses of dam surviving until scheduled necropsy on Day 21 post-coitum)

- Skeletal examinations: Yes: half per litter (for viable and non-viable fetuses of dam surviving until scheduled necropsy on Day 21 post-coitum)

- Head examinations: Yes: half per litter(for viable and non-viable fetuses of dam surviving until scheduled necropsy on Day 21 post-coitum)

- Anogenital distance of all live rodent pups: Yes, for viable fetuses of dam surviving until scheduled necropsy on Day 21 post-coitum

- Internal and external sex determination: Yes: all per litter (for viable and non-viable fetuses of dam surviving until scheduled necropsy on Day 21 post-coitum)

- Body weight: Yes: all per litter (for viable and non-viable fetuses of dam surviving until scheduled necropsy on Day 21 post-coitum)
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons have been conducted using two sided tests and will be reported at the 1% and 5% levels, unless otherwise noted.
The pairwise comparisons of interest are listed below:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1

- Parametric/Non-Parametric: Levene’s test was used to assess the homogeneity of group variances. The groups was compared using an overall one-way ANOVA F-test if Levene’s test is not significant or the Kruskal-Wallis test if it is significant. If the overall F-test or Kruskal-Wallis test is found to be significant, then pairwise comparisons was conducted using Dunnett’s or Dunn’s test, respectively.

- Non-Parametric: The groups were compared using an overall Kruskal-Wallis test. If the overall Kruskal-Wallis test is found to be significant, then the above pairwise comparisons were conducted using Dunn’s test.

The data corresponding to a response variable of interest and to a related covariate was submitted to an analysis of covariance (ANCOVA), including only groups with at least three non missing paired values and if found to be significant, then pairwise comparisons was conducted using Dunnett’s test.

- Incidence: A Fisher’s exact test was used to conduct pairwise group comparisons of interest.
Indices:
- Body Weight Gains: Calculated for the following intervals: Days 6 to 9, 9 to 12, 12 to 15, 15 to 18, 18 to 21 and 6 to 21 post-coitum.

- Corrected Body Weight Gain: Body weight on Day 21 post-coitum - body weight on Day 6 post-coitum - gravid uterus weight.

- Overall Food Consumption: Calculated between each scheduled interval (individual data only) and as specified above for body weight gains. Summarization and statistical analysis intervals reflect the same intervals as the body weight gains.

- Pregnancy Rate (%): No. of pregnant females x 100 / No. of mated females

- Organ Weight Relative to Body Weight: Calculated against body weight recorded on Day 21 post-coitum

- Male Fetuses (%): No. of male fetuses x 100 / No. of fetuses

- Female Fetuses (%): No. of female fetuses x 100 / No. of fetuses

- Pre-Implantation Loss (%): No. of corpora lutea – No. of implantations x 100 / No. of corpora lutea

- Post-Implantation Loss (%): No. of implantations – No. of live fetuses x 100 / No. of implantations

- Litter % of Fetuses with Abnormalities: No. of fetuses in litter with a given finding x 100 / No. of fetuses in litter examined
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At 375 mg/kg bw/day, erected fur and/or a hunched posture was noted for all females on most days of the Dosing Period. It should be noted that on most days, these post-dose findings were continuously observed pre-dose on the following day. In addition, 2/9 females had black discoloration of their feces on post-coitum Days 13 and 14.

At 300 mg/kg bw/day, all females presented with erected fur on several occasions during the Dosing Period and a hunched posture was noted for 6/19 females on 1-5 days. These postdose findings were continuously observed pre-dose on the following day. No discoloration of the feces was observed at this dose level.

At 100 mg/kg bw/day, erected fur was noted for 4/22 females on 1-3 days. For comparison, a single female in the control group was noted with erected fur on a single day. Additionally, black feces was noted for 8/22 females of the 100 mg/kg/day dose group on 1-3 days.

Salivation seen after dosing among 2/22, 17/19 and 9/9 females of the 100, 300 and 375 mg/kg bw/day dose groups mostly from the second week of treatment onwards was considered not toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response rather than a sign of systemic toxicity.

Incidental findings that were noted included fur loss, and scabbing, red discoloration of the skin and/or slight swelling of several parts of the body. 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 and did not show any apparent dose-related trend. At the incidence observed, these were considered not to be signs of toxicological relevance.
Mortality:
mortality observed, treatment-related
Description (incidence):
In total, 16 females did not survive until scheduled necropsy, of which 13 females were treated at 375 mg/kg bw/day and 3 females were treated at 300 mg/kg bw/day. Details on these females are given in the text table below. At 100 mg/kg bw/day, no mortality occurred.

Test item-related mortality at 375 mg/kg bw/day:
Eleven females were euthanized in extremis based on body weight loss (between 9-14% compared to their highest body weights). Food consumption was moderately to severely reduced for all females. Relevant clinical signs included hunched posture, erected fur, decreased activity, reduced production and/or black or orange discoloration of the feces, and/or incidentally salivation. Macroscopic findings that were noted at necropsy included yellow, gelatinous content of the stomach and a large part of the intestine (i.e. duodenum, jejunum, ileum; for one female, and cecum for four females, enlargement of the adrenal glands for four females, black discoloration of the right adrenal gland for one female, and a small thymus for two females. Three female were without necropsy findings. All females were pregnant with live fetuses.
Another two females at 375 mg/kg bw/day were euthanized in extremis, based on clinical signs including decreased activity, closed eyes, hunched posture, erected fur, black and/or orange discolored feces and/or incidentally salivation. Food consumption was severely reduced for both females and body weight loss (4% compared to their highest body weights) was noted. Macroscopic findings that were noted at necropsy included enlargement of the adrenal glands and a small thymus for both females. Both females were pregnant with live fetuses.

Test item-related mortality at 300 mg/kg bw/day:
Three females were euthanized in extremis based on severe body weight loss (between 10-12% compared to their highest body weights). Food consumption was moderately to severely reduced for all females. Clinical signs included hunched posture, erected fur, black or orange discolored feces and/or incidentally salivation. The only macroscopic finding at necropsy was enlargement of the adrenal glands for 2/3 females. These two females were pregnant with live fetuses, the third female was non-pregnant.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 375 mg/kg bw/day, body weight gain was lower throughout the Dosing Period, resulting in a 20% lower mean body weight compared to control, at the end of the Dosing Period.

At 300 mg/kg bw/day, a lower mean body weight gain was observed throughout the Dosing Period (statistically significant between Days 9-12 and 18-21 post-coitum), resulting in 13% lower mean body weight compared to control at the end of the Dosing Period (not statistically significant until Day 13 post-coitum).

At 100 mg/kg bw/day, mean body weight gain was in the same range as control during the observation period. The apparent slightly lower mean body weight was caused by the fact that animals allocated to the low dose group coincidentally had a mean body weight that was 3% lower than control (not statistically significant).

At 300 and 375 mg/kg bw/day, gravid uterus weight showed a decreasing trend with increasing dose concentrations (6% and 17% lower than control, respectively; not statistically significant). It should be noted that due to high toxicity at 375 mg/kg bw/day, data from only nine females were available for evaluation at this highest dose tested. Gravid uterus weight at 100 mg/kg bw/day was in the same range as control.
Note: One female at 100 mg/kg bw/day) and one at 300 mg/kg bw/day were noted with low gravid uterus weight due to small litters (3 and 5 live fetuses, respectively).

Body weight loss when corrected for gravid uterus weight was noted at 300 and 375 mg/kg bw/day (-6 grams and -22 grams). At the individual level, 11/18 females at 300 mg/kg bw/day and all 9 females at 375 mg/kg bw/day were noted with body weight loss (up to 32 and 38%, respectively). For comparison, none of the females in the control and 100 mg/kg bw/day groups showed body weight loss after correction for gravid uterus weight. At 100 mg/kg bw/day, body weight corrected for gravid uterus weight was in the same range as control.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 300 and 375 mg/kg bw/day, food consumption was lower from start of dosing onwards, reaching a maximal difference of 39 and 62% below control, respectively, between Days 18-21 post-coitum.

At 100 mg/kg bw/day, food consumption was slightly lower than control (up to 8% between Days 9-12 post-coitum; not statistically significant).
Endocrine findings:
effects observed, treatment-related
Description (incidence and severity):
At 375 mg/kg bw/day, lower mean serum levels of T3, T4 and TSH were noted (0.50, 0.51 and 0.57x of control mean, respectively). The mean T3 and T4 levels were below the lower limit of the available historical control range. The mean TSH value (0.1397 mU/L; not statistically significantly lower than control) was at the lower limit of the available historical control range.

At 300 mg/kg bw/day, lower mean serum levels of T3 and T4 were noted (0.60 and 0.66x of control mean, respectively). The mean T3 and T4 levels (and individual serum levels of 14/19 and 12/19 females, respectively) were below the lower limit of the available historical control range. The mean TSH level remained within the normal range of biological variation.

The individual values show a very wide and overlapping distribution over all dose groups. Considering the magnitude of the differences between the groups averages with values below or at the lower limit of the available historical control range and an apparent dose-related response, this decrease was considered test item-related. However, the biological consequences of thyroid hormone changes are not assessed within this type of study and therefore possible adversity of the effects on thyroid hormones could not be assessed. As such, this finding was not taken into account when determining the maternal NOAEL.

At 100 mg/kg bw/day, the mean T3, T4 and TSH levels remained within the normal range of biological variation.

Note 1: A single female at 100 mg/kg bw/day was not gravid and therefore excluded from the data tables.

Note 2: The 375 mg/kg bw/day group contained only 9 females.

[- Historical control data for pregnant Wistar Han rats (period 2020-2021): T3 (ng/mL) mean: 0.439; P5 - P95: 0.280 – 0.616 (n = 263); T4 (ng/mL) mean: 23.2; P5 - P95: 16.3 – 35.2 (n = 154)]
[- Historical control data for pregnant Wistar Han rats (period 2016-2020): TSH (mU/L) mean: 0.353; P5 - P95: 0.127 – 0.699 (n = 373)].
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
The mean thyroid gland weight (absolute) was lower compared to control in all treatment groups, but without a dose-related response and great overlap between the groups (reaching statistical significance at 300 mg/kg bw/day only). The relative thyroid gland weight was in the same range as control for all treatment groups.

Note 1: A single female at 100 mg/kg bw/day was not gravid and therefore excluded from the data tables.
Note 2: The 375 mg/kg bw/day group contains only 9 females.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
At 375 mg/kg bw/day, a small thymus was noted for 1/9 females surviving until scheduled necropsy. It should be noted that the same finding was observed in 4/13 females in this high dose group that were euthanized preterm (see mortality section). In addition, in the 13 females that were euthanized in extremis at 375 mg/kg bw/day, test-item related enlargement and/or black discoloration of the adrenal glands (6/13 females), reduction in thymus size (4/13 females) and yellow gelatinous content in different parts of the intestine (4/13 females) was observed.

At 300 mg/kg bw/day macroscopic evaluation revealed test-item related enlargement of the adrenal glands for 2/3 females that were euthanized in extremis.

Remaining findings that were noted among control and/or treated animals were considered to be of no toxicological significance, since they remained within the range of biological variation for rats of this age and strain.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
The recorded microscopic findings in the thyroid gland were within the range of background pathology encountered in rats of this age and strain.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The mean numbers of pre- and post-implantation loss in the control, 100 and 300 mg/kg bw/day groups were similar and in the range of normal biological variation.

At 375 mg/kg bw/day the mean numbers of pre- and post-implantation loss were in the range of normal biological variation.
Early or late resorptions:
no effects observed
Description (incidence and severity):
The mean numbers of early and late resorptions in the control, 100 and 300 mg/kg bw/day groups were similar and in the range of normal biological variation.

At 375 mg/kg bw/day the mean numbers of early and late resorptions were in the range of normal biological variation.
Changes in number of pregnant:
effects observed, treatment-related
Description (incidence and severity):
All females, including those that were necropsied preterm (see section mortality), were pregnant, except for a single female at 100 mg/kg bw/day and 300 mg/kg bw/day respectively. Excluding non pregnant females and females that did not survive until scheduled necropsy, the number of females with viable litters for evaluation was 22, 21, 19 and 9 females in the control, 100, 300 and 375 mg/kg bw/day groups, respectively.
Other effects:
no effects observed
Description (incidence and severity):
The mean numbers of corpora lutea in the control, 100 and 300 mg/kg bw/day groups were similar and in the range of normal biological variation.

At 375 mg/kg bw/day the mean numbers of corpora lutea were in the range of normal biological variation.
Details on maternal toxic effects:
As according to the guideline, a minimum of 16 females with implantations surviving until scheduled necropsy is required for an adequate evaluation of developmental data, the 9 litters available at 375 mg/kg bw/day were considered not sufficient for a robust and valid evaluation of developmental data. Therefore, the developmental data from the 375 mg/kg bw/day group were not used for any conclusions and were reported separately.
Key result
Dose descriptor:
NOAEL
Remarks:
Maternal
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
clinical signs
food consumption and compound intake
gross pathology
mortality
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 300 mg/kg bw/day, male, female and combined fetal body weight was 8, 9 and 8% lower than control, respectively, and below the historical control range of the Test Facility.

At 100 mg/kg bw/day, male, female and combined fetal body weight remained within the normal range of biological variation.

At 375 mg/kg bw/day, male, female and combined fetal weight was 15, 16 and 14% lower than control, respectively, and below the historical control range of the Test Facility.

[Historical control data for fetal body weights of Wistar Han rats (period 2016-2020): Males (g): mean: 5.4; P5 - P95: 5.2 – 5.5 (n= 6774); Females (g): mean: 5.1; P5 - P95: 4.9 – 5.3 (n= 7024); Combined (g): mean: 5.3; P5 - P95: 5.0 – 5.4 (n= 13798)]
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The male : female ratio of control, 100 and 300 mg/kg bw/day groups was within the normal range of biological variation, with great overlap between all groups.

At 375 mg/kg bw/day, the male:female ratio was within the normal range of biological variation.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
Mean litter size of the 100 and 300 mg/kg bw/day groups was in the same range as the control group.

At 375 mg/kg bw/day, the mean litter size was in the same range as the control group.
Anogenital distance of all rodent fetuses:
no effects observed
Description (incidence and severity):
Fetal anogenital distance (both sexes; absolute and normalized for fetal weight) of the 100 and 300 mg/kg bw/day groups was in the same range as the control group.

At 375 mg/kg bw/day, fetal anogenital distance (both sexes; absolute) was slightly lower than control (not statistically significant). The mean fetal anogenital distance normalized for fetal weight was in the same range as control.
External malformations:
no effects observed
Description (incidence and severity):
No external malformations and variations were observed in the control, 100 and 300 mg/kg bw/day group.

At 375 mg/kg bw/day, the fetuses of the females surviving until scheduled necropsy did not show any external malformations or variations.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Two fetuses in the 100 mg/kg bw/day group had a skeletal malformation. The first fetus presented bent limb bones (radius, ulna and femur) and the second fetus had an extra lumbar vertebra.

Regarding skeletal variations, at 100 mg/kg bw/day, the fetal incidence of incomplete ossification of both parietals was significantly increased. This variation occurred in five low-dose fetuses from five litters.

Note: At 300 mg/kg bw/day, there were no fetuses observed with misaligned ilia, resulting in a significantly decreased incidence of this finding compared to the control group.
At 375 mg/kg bw/day, the fetuses of the females surviving until scheduled necropsy did not show any skeletal malformations. Skeletal variations were observed in 33(9) fetuses (litters).
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Visceral malformations were observed in a single fetus of the control group with situs inversus and fused lung lobes.

Visceral variations in the control, 100 and/or 300 mg/kg bw/day groups were limited to supernumerary liver lobes, absent innominate artery and convoluted or dilated ureters.
At 375 mg/kg bw/day, the fetuses of the females surviving until scheduled necropsy did not show any visceral malformations. Visceral variations were observed in 8(3) fetuses (litters). Five fetuses from a single female were noted with absent renal papilla and distended urinary bladder of which three also had related dilated and/or convoluted ureters.
Key result
Dose descriptor:
NOAEL
Remarks:
Developmental
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw (total dose)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes

Dose Formulation Analyses



  1. Accuracy
    The concentrations analyzed in the formulation of Groups 2, 3 and 4 were in agreement with target concentrations (i.e. mean sample concentration results were within or equal to 90-110% of target concentration). No test item was detected in the Group 1 formulation.

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


Table 1 - Accuracy and Homogeneity Test (Week1)



































































































































































Group



Sample position



Concentration

[mg/mL]



Recovery

[%]



Coefficient of variation
[%]



Target



Analyzed



Individual



Mean



1



50% height



0



n.d.



n.a.



n.a.



n.a.



 



 



0



n.d.



n.a.



 



 



2



90% height



25.0



26.0



104



101



2.7



 



 



25.0



25.8



103



 



 



 



50% height



25.0



24.8



99



 



 



 



 



25.0



24.7



99



 



 



 



10% height



25.0



26.1



104



 



 



 



 



25.0



24.6



98



 



 



3



50% height



75.0



81.1



108



110



n.a.



 



 



75.0



84.2



112



 



 



4



90% height



93.75



106



113



105



4.8



 



 



93.75



96.3



103



 



 



 



50% height



93.75



96.5



103



 



 



 



 



93.75



95.9



102



 



 



 



10% height



93.75



93.7



100



 



 



 



 



93.75



103



109



 



 



n.d. Not detected.


n.a. Not applicable.


Table 2: Overview of maternal and foetal findings






















































































































Dose level (mg/kg bw/day)



0



100



300



375



Pregnant/total dams



22/22



21/22



21/22



22/22



-early resorptions


-late resorptions


(% per litter)



11.8


0.7



18.52


0



9.35


0


 



13.94


0



Dams with abortion, early deliveries, stillbirths, resorptions only and/or dead fetuses



0



0



0



0



Pre-implantation loss (number and percent)



19 (6.56 %)



12 (7.22 %)



18 (7.48 %)



10 (7.83 %)



Post-implantation loss (number and percent)



16 (6.26 %)



13 (6.17 %)



10 (3.94 %)



7 (6.20 %)



Body weight on day 21



341.2



329.4



298.1**



272.2**



Body weight gain day 6-21 (%)



31.74



31.87



23.45



16.42



Gravid uterine weight (g)



79.51



79.47



74.63



66.17



Mean live offspring (number)



11.4



11.2



11.5



11.0



Live offspring (percent)



100 %



100 %



100 %



100 %



Mean fetal/pup body weight males (g)



5.391



5.276



4.965**



4.594**



Mean fetal body weight females (g)



5.112



5.049



4.669**



4.277**



Mean fetal body weight (sexes combined) (g)



5.233



5.192



4.814**



4.475**



Malformations (including runts) number and percent of fetuses per litter



 


 


1 (1.14 %)



 


 


2 (2.38 %)



 


 


0 (0 %)



 


 


0 (0 %)



Variations (% per litter)


-external


-soft tissue


-skeletal



 


0


6.55


72.08



 


0


10.48


76.89



 


0


6.89


51.60



 


0


15.56


70.21



 


 


Table 3: Summary of Body Weight Gains (g): Gestation










































































































































Sex: Female



Day(s) Relative to Mating (Litter: A)



6 → 9 [G]



9 → 12 [G]



12 → 15 [G]



15 → 18 [G]



18 → 21 [G]



6 → 21 [G1]



0



Mean



6.6



16.1



16.0



30.7



39.0



108.3



mg/kg/day



SD



4.7



4.6



5.4



5.0



7.8



15.9



Group 1



N



22



22



22



22



22



22



100



Mean



6.9



15.4



15.1



30.6



37.0



105.0



mg/kg/day



SD



2.9



3.0



4.8



7.4



8.1



17.7



Group 2



N



21



21



21



21



21



21



300



Mean



3.0



12.3*



8.8



23.6



16.1**



69.9**



mg/kg/day



SD



4.5



5.4



10.7



13.8



12.6



20.5



Group 3



N



21



21



21



21



19



19



375



Mean



-0.4**



5.5**



1.9**



8.6**



1.0**



44.7**



mg/kg/day



SD



6.6



11.7



8.5



17.0



11.9



15.3



Group 4



N



22



22



16



14



9



9



 


Table 4: Summary of Food Consumption: Gestation. Food Mean Daily Consumption (g/animal/day)








































































































































































Sex: Female



Day(s) Relative to Mating (Litter: A)



6 → 9 [G]



9 → 12 [G1]



12 → 15 [G1]



15 → 18 [G1]



18 → 21 [G1]



6 → 21 [G]



0



Mean



19.26



20.91



20.94



22.79



22.06



21.19



mg/kg/day



SD



2.30



2.48



2.05



1.72



1.40



1.49



Group 1



N



22



22



22



22



22



22



100



Mean



18.79



19.22



19.92



21.37



20.90



20.04



mg/kg/day



SD



2.04



2.22



1.76



1.91



2.50



1.52



Group 2



N



21



21



21



21



21



21



 



%Diff



-2.41



-8.07



-4.87



-6.24



-5.24



-5.43



300



Mean



14.79 **



15.84 **



15.56 **



15.65 **



13.35 **



15.59 **



mg/kg/day



SD



2.83



3.42



4.23



5.44



3.47



2.09



Group 3



N



21



21



21



21



19



19



 



%Diff



-23.18



-24.24



-25.71



-31.32



-39.48



-26.42



375



Mean



12.97 **



13.05 **



12.79 **



10.52 **



8.33 **



14.17 **



mg/kg/day



SD



2.89



5.59



5.40



6.71



4.21



2.02



Group 4



N



22



22



16



14



9



9



 



%Diff



-32.65



-37.61



-38.91



-53.82



-62.23



-33.13



 


 


Table 5: Maternal performance and Mortality





































































































































Sex: Female


 


Day(s) Relative to Mating (Litter: A)



0


mg/kg/day Group 1



100


mg/kg/day Group 2



300


mg/kg/day Group 3



375


mg/kg/day Group 4



Group Size - Females



 



22



22



22



22



Number of Females Pregnant [f]



N+ve



22



21



21



22



 



%



100.0



95.5



95.5



100.0



Female with Live Fetuses [f]



N+ve



22



21



21



22



 



%



100.0



100.0



100.0



100.0



Total Resorptions [f]



N+ve



0



0



0



0



 



%



0.0



0.0



0.0



0.0



Female with all Nonviable [f]



N+ve



0



0



0



0



 



%



0.0



0.0



0.0



0.0



Terminal Euthanasia [f]



N+ve



22



22



19



9**



 



%



100.0



100.0



86.4



40.9**



Unscheduled Death/Euthanasia [f]



N+ve



0



0



3



13 **



 



%



0.0



0.0



13.6



59.1**



Unscheduled Euthanasia [f]



N+ve



0



0



3



13 **



 



%



0.0



0.0



13.6



59.1**



 


Table 6: Summary of Ovarian and Uterine Examinations and Litter Observations







































































































































































































































































































































































































































































































































































































Sex: Female


 


Day(s) Relative to Mating (Litter: A)



0


mg/kg/day Group 1



100


mg/kg/day Group 2



300


mg/kg/day Group 3



375


mg/kg/day Group 4



Female with Live Fetuses



N+ve



22



21



19



9



%



100.0



100.0



100.0



100.0



 



Number of Corpora Lutea [k]



Mean


SD



13.0


2.1



12.7


1.3



13.0


2.3



12.9


1.5



 



N



22



21



19



9



 



%Diff



-



-1.9



0.4



-0.5



 



Number of Implantations [k]



Mean


SD



12.1


2.5



11.8


2.2



12.1


2.5



11.8


1.7



 



N



22



21



19



9



 



%Diff



-



-2.3



-0.3



-2.6



 



Pre-implantation Loss (%) [k]



Mean


SD



6.56


12.96



7.22


13.94



7.48


11.36



7.83


13.99



 



N



22



21



19



9



 



%Diff



-



10.16



14.16



19.44



 



Total Number of Fetuses [k]



Mean


SD



11.4


2.7



11.2


2.7



11.5


2.3



11.0


1.6



 



N



22



21



19



9



 



%Diff



-



-1.1



1.4



-3.2



Number of Live Fetuses [k]



Mean


SD



11.4


2.7



11.2


2.6



11.5


2.3



11.0


1.6



 



N



22



21



19



9



 



%Diff



-



-1.5



1.4



-3.2



 



Number of Dead Fetuses [k]



Mean


SD



0.0


0.0



0.0


0.0



0.0


0.0



0.0


0.0



 



N



22



21



19



9



 



%Diff



-



-



-



-



 



Number of Early Resorptions [k]



Mean


SD



0.7


0.8



0.6


1.2



0.5


0.7



0.8


1.0



 



N



22



21



19



9



 



%Diff



-



-9.2



-22.8



14.1



 



Number of Late Resorptions [k]



Mean


SD



0.0


0.2



0.0


0.0



0.0


0.0



0.0


0.0



 



N



22



21



19



9



 



%Diff



-



-100.0



-100.0



-100.0



 



Total Number of Resorptions [k]



Mean


SD



0.7


0.9



0.6


1.2



0.5


0.7



0.8


1.0



 



N



22



21



19



9



 



%Diff



-



-14.9



-27.6



6.9



Post-implantation Loss (%) [k]



Mean


SD



6.26


7.94



6.17


12.81



3.94


5.01



6.20


7.71



 



N



22



21



19



9



 



%Diff



-



-1.36



-37.04



-0.99



 



Number of Live Male Fetuses [k]



Mean


SD



4.9


2.0



5.9


2.2



5.6


1.3



6.3


2.2



 



N



22



21



19



9



 



%Diff



-



21.4



14.7



30.2



 



Number of Live Female Fetuses [k]



Mean


SD



6.5


2.0



5.3


2.4



5.9


2.1



4.7


1.7



 



N



22



21



19



9



 



%Diff



-



-18.7



-8.5



-28.2



 



Live Male Fetus/Litter (%) [k]



Mean


SD



42.24


12.19



54.79


20.24



49.63


11.93



56.98


16.51



 



N



22



21



19



9



 



%Diff



-



29.71



17.49



34.89



 



Live Female Fetuses/Litter (%) [k]



Mean


SD



57.76


12.19



45.21


20.24



50.37


11.93



43.02


16.51



 



N



22



21



19



9



 



%Diff



-



-21.73



-12.79



-25.52



Mean Fetal Weight males (g) [G]



Mean


SD



5.391


0.279



5.276


0.338



4.965 **


0.330



4.594 **


0.379



 



N



22



21



19



9



 



%Diff



-



-2.129



-7.901



-14.780



 



Mean Fetal Weight females (g) [G]



Mean


SD



5.112


0.267



5.049


0.303



4.669 **


0.314



4.277 **


0.460



 



N



22



20



19



9



 



%Diff



-



-1.223



-8.653



-16.320



 



Mean Fetal Weight all (g) [G]



Mean


SD



5.233


0.254



5.192


0.314



4.814 **


0.313



4.475 **


0.379



 



N



22



21



19



9



 



%Diff



-



-0.780



-8.005



-14.478



 



Mean Fetal AGD males (mm) [G1]



Mean


SD



3.00


0.24



2.98


0.26



2.92


0.27



2.79


0.25



 



N



22



21



19



9



 



%Diff



-



-0.68



-2.56



-6.94



 



Mean Fetal AGD females (mm) [G1]



Mean


SD



1.27


0.16



1.33


0.15



1.27


0.13



1.24


0.18



 



N



22



20



19



9



 



%Diff



-



4.74



0.31



-2.21



Mean Normalized Fetal AGD m [G]



Mean


SD



1.713


0.137



1.713


0.137



1.717


0.166



1.683


0.164



 



N



22



21



19



9



 



%Diff



-



0.029



0.230



-1.730



 



Mean Normalized Fetal AGD f [G]



Mean


SD



0.735


0.092



0.773


0.085



0.761


0.082



0.765


0.117



 



N



22



20



19



9



 



%Diff



-



5.163



3.466



4.099



 


Table 7: Fetal Abnormalities by classification














































Exam Type: Visceral Body


 


 


Number of Fetuses Examined:


Number of Fetuses Evaluated:


Number of Litters Examined:


Number of Litters Evaluated:



0


mg/kg/day Group 1



100


mg/kg/day Group 2



300


mg/kg/day Group 3



375


mg/kg/day Group 4



124


251


22


22



116


235


21


21



110


219


19


19



51


99


9


9



Variation


Number of Fetuses


Litter % of Fetuses [k]


Number of Litters


Malformation


Number of Fetuses


Litter % of Fetuses [k]


Number of Litters


All classifications


Number of Fetuses


Litter % of Fetuses [k]


Number of Litters



 


8


6.55


6


 


1


1.14


1


 


9


7.69


7



 


13


10.48


7


 


0


0.00


0


 


13


10.48


7



 


8


6.89


7


 


0


0.00


0


 


8


6.89


7



 


8


15.56


3


 


0


0.00


0


 


8


15.56


3



Exam Type: Skeletal


 


 


Number of Fetuses Examined:


Number of Fetuses Evaluated:


Number of Litters Examined:


Number of Litters Evaluated:



0


mg/kg/day Group 1



100


mg/kg/day Group 2



300


mg/kg/day Group 3



375


mg/kg/day Group 4



126


251


22


22



119


235


21


21



109


219


19


19



48


99


9


9



Variation


Number of Fetuses


Litter % of Fetuses [k]


Number of Litters


Malformation


Number of Fetuses


Litter % of Fetuses [k]


Number of Litters


All classifications


Number of Fetuses


Litter % of Fetuses [k]


Number of Litters



 


91


72.08


22


 


0


0.00


0


 


91


72.08


22



 


92


76.89


21


 


2


2.38


2


 


92


76.89


21



 


55


51.60


17


 


0


0.00


0


 


55


51.60


17



 


33


70.21


9


 


0


0.00


0


 


33


70.21


9


Conclusions:
Based on mortality (13/22 at 375 mg; 3/22 at 300 mg), clinical signs, reduced food consumption and body weight gain, body weight loss after correcting for gravid uterus weight, on macroscopic findings of the adrenal glands and intestine (at 300 and 375 mg/kg bw/day) and the thymus (at 375 mg/kg bw/day), the maternal No Observed Adverse Effect Level (NOAEL) for Tallow amine propoxylate was established as being 100 mg/kg bw/day.

The developmental NOAEL for Tallow amine propoxylate was established as being 100 mg/kg bw/day, based on decreased fetal body weights at 300 and 375 mg/kg bw/day as only effects.

The developmental data from the 375 mg/kg bw/day group was not used for any conclusions as with 9 evaluable litters the minimum required number of 16 litters for a robust and valid evaluation of the developmental data was not reached.
Executive summary:

The objectives of this study were to determine the potential of Tallow amine propoxylate to induce developmental toxicity after maternal exposure during the critical period of organogenesis and to characterize maternal toxicity at the exposure levels tested when given orally by gavage to time-mated female Wistar Han rats from Days 6 to 20 post-coitum, inclusive. In addition, the No Observed Adverse Effect Levels (NOAELs) for maternal toxicity and developmental toxicity were evaluated. The study was performed according to GLP principles and in agreement with the OECD TG 414.


The dose levels in this study were selected to be 0, 100, 300, 375 mg/kg bw/day, based on information provided by the Sponsor and the results of the Dose Range Finding study.


Chemical analyses of formulations were conducted once during the study to assess accuracy and homogeneity. Formulation analyses confirmed that formulations of test item in peanut oil were prepared accurately and homogenously.


 


Maternal Findings
At 100 mg/kg bw/day, no mortality occurred, and no test item-related findings were noted.


At 300 mg/kg bw/day, three females were euthanized in extremis between post-coitum Days 14 and 19. These females were noted with body weight loss, strongly reduced food consumption and/or clinical signs indicative of poor general condition prior to death. Macroscopic evaluation revealed enlargement of the adrenal glands for 2/3 females. All these findings were considered test item-related.
At 300 mg/kg bw/day, for females that survived until scheduled necropsy, the following, considered test item-related findings were present:
Erected fur and hunched posture were noted in combination with strongly reduced food consumption, reduced body weight gain and body weight loss after correction for gravid uterus weight. Together, based on their severity, these findings were considered adverse. Non-adverse lower gravid uterus weight was noted. This decrease was considered to be related to the observed lower fetal weights (see below).


At 300 mg/kg bw/day, mean serum levels of T3 and T4 were lower compared to control. The individual values show a very wide and overlapping distribution over all dose groups, and microscopical evaluation of the thyroid glands showed no remarkable findings. However, considering that group average values were below the lower limit of the available historical control range and an apparent dose-related response, this decrease was considered test itemrelated. However, the biological consequences of thyroid hormone changes are not assessed within this type of study and therefore possible adversity of the effects on thyroid hormones could not be assessed. As such, this finding was not considered when determining the maternal NOAEL.


At 375 mg/kg bw/day, 13 females were euthanized in extremis between post-coitum Days 12 and 19. For these females, body weight loss, severely reduced food consumption and/or clinical signs indicative of poor general condition were observed prior to death. Macroscopic evaluation revealed enlargement and/or black discoloration of the adrenal glands, reduction in thymus size and yellow gelatinous content in different parts of the intestine. All these findings were considered test item-related.
At 375 mg/kg bw/day, for females that survived until scheduled necropsy, the following, considered test item-related findings were present:
Erected fur, hunched posture, and black and/or orange discolored feces were noted in combination with strongly reduced food consumption, reduced body weight gain and body weight loss after correction for gravid uterus weight. For a single female, a small thymus was noted. As these findings were in line with the findings for females that were euthanized in extremis, and based on their severity, they were considered adverse. Non-adverse lower gravid uterus weight was noted. This decrease was considered to be related to the observed lower fetal weights (see below).
At 375 mg/kg bw/day, mean serum levels of T3, T4 and TSH were lower compared to control. The individual values show a very wide and overlapping distribution over all dose groups, and microscopical evaluation of the thyroid glands showed no remarkable findings. However, considering the magnitude of the differences between the groups averages with values below or at the lower limit of the available historical control range and an apparent dose-related response, this decrease was considered test item-related. However, the biological consequences of thyroid hormone changes are not assessed within this type of study and therefore possible adversity of the effects on thyroid hormones could not be assessed. As such, this finding was not taken into account when determining the maternal NOAEL.
No test item-related changes were noted in any of the remaining maternal parameters investigated in this study (i.e. thyroid gland weights, microscopic evaluation of the thyroid gland, uterine contents, corpora lutea, implantation sites and pre- and post-implantation loss).



Fetal Findings
Note: According to the guidelines, a minimum of 16 females with implantations at scheduled necropsy is required in each group for an adequate evaluation. As in the high dose group (375 mg/kg/day) only 9 females were available for evaluation, the available data was considered not sufficient for a robust and valid evaluation of the developmental data.
At 100 mg/kg bw/day, no test item-related findings were noted.
At 300 mg/kg bw/day, the following test item-related findings were present: Mean fetal body weights (male, female and total) were decreased compared to control, with mean values below the historical control range. Given the magnitude of the effect, this finding was considered adverse.
At 375 mg/kg bw/day, the following test item-related findings were present: Mean fetal body weights (male, female and total) were decreased compared to control and were below the available historical control data.


No test item-related changes were noted in any of the remaining developmental parameters investigated in this study (i.e. litter size, sex ratio, anogenital distance, external, visceral and skeletal malformations and developmental variations).


In conclusion, based on the results of this prenatal developmental toxicity study in time-mated female Wistar Han rats the maternal No Observed Adverse Effect Level (NOAEL) for Tallow amine propoxylate was established as being 100 mg/kg bw/day. This was based on mortality,
clinical signs, reduced food consumption and body weight gain, body weight loss after correcting for gravid uterus weight, on macroscopic findings of the adrenal glands and intestine (at 300 and 375 mg/kg/day) and the thymus (at 375 mg/kg/day). The developmental NOAEL for Tallow amine propoxylate was established as being 100 mg/kg/day, based on decreased fetal body weights at 300 mg/kg/day.


 


Note: The developmental data from the 375 mg/kg bw/day group was not used for any conclusions as with 9 evaluable litters the minimum required number of 16 litters for a robust and valid evaluation of the developmental data was not reached.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
This study is high quality for developmental toxicity but it does not cover the period immediately after implantation so it does not supersede the read across to the OECD443 study on 2, 2’-(Octadec-9-enylimino) bisethanol CAS No 25307-17-9 which is available for read across.
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

 


Short Description of Key Information


Maternal findings


At 100 mg/kg bw/day, no mortality occurred, and no test item-related findings were noted.


At 300 mg/kg bw/day, three females were euthanized in extremis and at 375 mg/kg bw/day thirteen females were euthanized in extremis. These females were noted with body weight loss, strongly reduced food consumption and/or clinical signs indicative of poor general condition prior to death. Macroscopic evaluation revealed enlargement of the adrenal glands for 2/3 females at 300 mg/kg bw/day and enlargement and/or black discoloration of the adrenal glands, reduction in thymus size and yellow gelatinous content in different parts of the intestine at 375 mg/kg bw/day. All these findings were considered test item-related.


At 300 and 375 mg/kg bw/day, for females that survived until scheduled necropsy, the following, considered test item-related findings were present: Erected fur and hunched posture were noted in combination with strongly reduced food consumption, reduced body weight gain and body weight loss after correction for gravid uterus weight. Together, based on their severity, these findings were considered adverse.


At 300 and 375 mg/kg bw/day, mean serum levels of T3 and T4 were lower compared to control. Individual serum values show a very wide and overlapping distribution over all dose groups, and microscopical evaluation of the thyroid glands showed no remarkable findings. However, considering that group average values were below the lower limit of the available historical control range and an apparent dose-related response, this decrease was considered test item related. However, the biological consequences of thyroid hormone changes are not assessed within this type of study and therefore possible adversity of the effects on thyroid hormones could not be assessed. As such, this finding was not considered when determining the maternal NOAEL but are considered to be test item related. This is investigated further in the current OECD 422 on the same substance for which possible adversity can be derived on development and reproduction.


 


Foetal Findings


At 100 mg/kg bw/day, no test item-related findings were noted.


At 300 mg/kg bw/day, the following test item-related findings were present: Mean fetal body weights(male, female and total) were decreased compared to control, with mean values below the historical control range. Given the magnitude of the effect, this finding was considered adverse.


At 375 mg/kg bw/day, the following test item-related findings were present: Mean fetal body weights (male, female and total) were decreased compared to control and were below the available historical control data.


No test item-related changes were noted in any of the remaining developmental parameters investigated in this study (i.e. litter size, sex ratio, anogenital distance, external, visceral and skeletal malformations and developmental variations).

Justification for classification or non-classification

There is specific test data on potential reproductive toxic effect of for di‐(2‐hydroxypropyl) C16‐C18 (evennumbered), C18 unsaturated‐alkyl amine CAS 1309955‐79‐0 in a conducted OECD 422. Indications of developmental and reproductive toxicity seen at 300 mg/kg/day could be secondary to the maternal toxicity and severity of local effects on the gastorintestinal tract and bodyweight loss or direct results of the test substance itself. As such a conservative NOAEL is placed of 150 mg/kg bw/day.


 


Addtionally, there is an OECD 443 available for the appropriate read across substance 2’-(Octadec-9-enylimino) bisethanol CAS No 25307-17-9.  2, 2’-(Octadec-9-enylimino) bisethanol CAS No 25307-17-9 is predominantly C18 unsaturated and di‐(2‐hydroxypropyl) C16‐C18 (evennumbered), C18 unsaturated‐alkyl amine which is predominantly C18, C18 unsaturated and C16. The available study is an OECD 443 extended one-generation reproductive toxicity test. There were no indications of effects on reproductive or developmental toxicity considered to be sufficient for classification though effects were observed on decreased maternal body weight gain but considered to be secondary to gastrointestinal irritation observed. There were no indications of any adverse effects on the reproductive parameters such as fertility etc. in the adult rats of either sex or developmental defects.


 


The OECD 414 pre-natal development study in rats, showed no evidence of developmental toxicity even at 100 mg/kg bodyweight/day, therefore while this study does not include dosing during the pre-mating period until day 4, it does not indicate any potential for developmental toxicity (adverse effects on the morphological development of the foetus) at this dose. At 300 and 375 mg/kg bw/day significant decreases in body weight gain was observed in both maternal animals and foetuses. Which were determined to be adverse in addition to macropathology findings at doses higher than 100 mg/kg bw/day. The available OECD 414 was not sufficient to draw conclusions on observed decreases in thyroid weight and decreased T3, T4, THS serum levels as this study design does not allow for the active assessment of the biological relevance of these decreases. The OECD 422 on this substance allows for a better evaluation of these biological relevant consequences.


 


According to regulation EC 1272/2008 (Section 3.7.2.4.4) maternal mortality over 10% is considered to be excessive and the data for dose levels exceeding this threshold shall not normally be considered for further evaluation. This is reiterated in EC 1272/2008 (Section 3.7.2.5.8) as,”In principle, adverse effects on reproduction seen only at very high dose levels in animal studies (for example doses that induce prostration, severe inappetence, excessive mortality) would not normally lead to classification…


 


Therefore, it is clear that it is not currently necessary to classify this substance for reproductive or developmental toxicity based on current data from both di‐(2‐hydroxypropyl) C16‐C18 (evennumbered), C18 unsaturated‐alkyl amine CAS 1309955‐79‐0 and the read across to 2, 2’-(Octadec-9-enylimino) bisethanol CAS No 25307-17-9.

Additional information