Propanol, iminobis-, N-(C16-18 and C18-unsatd. alkyl) derivs.

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

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 current, 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.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

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
DUODENUM^a	10	10	9	10	10	10	10	10
Foamy macrophage accumulation
Minimal	-	-	4	3	-	-	-	3
Slight	-	-	-	6	-	-	-	3
JEJUNUM^a	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
ILEUM^a	10	10	10	10	10	10	10	10
Macrophage accumulation
Minimal	-	-	-	-	-	-	-	2
MESENTERIC LN^a	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)

 

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2018

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1998

Deviations:

no

Test guideline 5

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

Test guideline 6

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:

In addition, the procedures described in this report essentially conform to the following guidelines:
- EPA OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, 2000.
- OECD 421, Reproduction/Developmental Toxicity Screening Test, 2016.
- EPA OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, 2000.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-[(2-hydroxypropyl)(C16-18 sat. C18 unsat. alkyl)amino]propan-1-ol

EC Number:

695-977-9

Cas Number:

1309955-79-0

Molecular formula:

Not applicable UVCB

IUPAC Name:

2-[(2-hydroxypropyl)(C16-18 sat. C18 unsat. alkyl)amino]propan-1-ol

Test material form:

liquid

Details on test material:

Chemical name: 2-[(2-hydroxypropyl)(C16-18 sat. C18 unsat. alkyl)amino]propan-1-ol
EC no.: 695-977-9

To the best of knowledge, the sample used is representative to the boundary composition shared and agreed by each registrant

Test animals

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.
- 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. During locomotor activity monitoring, 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

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

peanut oil

Details on oral 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

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.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

Low dose group

Doses / concentrations 2

Dose / conc.:

150 mg/kg bw/day (nominal)

Remarks:

Mid dose group

Doses / concentrations 3

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

Examinations

Observations and examinations performed and frequency:

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.

Sacrifice and pathology:

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.

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.

Results and discussion

Results of examinations

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):

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).
See also table 6 under ''Any other information on materials and methods 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 7 under ''Any other information on materials and methods 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

Description (incidence and severity):

There were no direct test material-related alterations in organ weights.
Many organ weight differences (absolute and/or relative) in males were statistically significant when compared to the control group but were considered to be the result of a test
material-related effect on final body weight (-14% at 150 mg/kg bw/day, -28% at 300 mg/kg bw/day). At 150 and/or 300 mg/kg bw/day a statistically significant lower absolute weight and/or higher weight when expressed relative to body weight was recorded for brain, pituitary gland, heart, liver, thymus, kidneys, adrenal gland, spleen, testes, epididymides and prostate gland.
For females, the body weight at 300 mg/kg bw/day was lower compared to the control group (-9%) but did not reach statistical significance. Statistically significant lower absolute values were recorded in kidney and spleen and a higher relative weight was recorded for the liver at 300 mg/kg bw/day. These organ weight differences were regarded to be related to the lower final body weight and not directly related to the treatment with the test material.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

There were no direct test material-related alterations in organ weights.
Many organ weight differences (absolute and/or relative) in males were statistically significant when compared to the control group but were considered to be the result of a test
material-related effect on final body weight (-14% at 150 mg/kg bw/day, -28% at 300 mg/kg bw/day). At 150 and/or 300 mg/kg bw/day a statistically significant lower absolute weight and/or higher weight when expressed relative to body weight was recorded for brain, pituitary gland, heart, liver, thymus, kidneys, adrenal gland, spleen, testes, epididymides and prostate gland.
For females, the body weight at 300 mg/kg bw/day was lower compared to the control group (-9%) but did not reach statistical significance. Statistically significant lower absolute values were recorded in kidney and spleen and a higher relative weight was recorded for the liver at 300 mg/kg bw/day. These organ weight differences were regarded to be related to the lower final body weight and not directly related to the treatment with the test material.

Neuropathological findings:

not examined

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.

Histopathological findings: neoplastic:

not examined

Details on results:

As only 4 females at 300 mg/kg bw/day survived to scheduled sacrifice, clinical chemistry haematology and coagluation 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.

Effect levels

Key result

Dose descriptor:

NOAEL

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.

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

150 mg/kg bw/day (nominal)

System:

gastrointestinal tract

Organ:

duodenum

jejunum

mesenteric lymph node

stomach

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Any other information on results incl. tables

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
DUODENUM^a	10	10	9	10	10	10	10	10
Foamy macrophage accumulation
Minimal	-	-	4	3	-	-	-	3
Slight	-	-	-	6	-	-	-	3
JEJUNUM^a	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
ILEUM^a	10	10	10	10	10	10	10	10
Macrophage accumulation
Minimal	-	-	-	-	-	-	-	2
MESENTERIC LN^a	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 – 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 7 – 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)

Applicant's summary and conclusion

Conclusions:

In conclusion, based on the results of this combined 90-day repeated dose toxicity study, the following No Observed Adverse Effect Level (NOAEL) after repeated exposure to Tallow amine propoxylate was established:
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)

Executive summary:

The objective 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.
In addition the No Observed Adverse Effect Levels (NOAELs) after repeated exposure was 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.

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).

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)