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

Endpoint summary

Description of key information

There is a 90-day subchronic repeat-dose study on di-(2-hydroxypropyl) C16-C18 (evennumbered), C18 unsaturated-alkyl amine CAS 1309955-79-0 in rats available. This 90-day study was a combination study with a OECD TG 422 study which resulted in a 10 week premating period follwed by maternal female rats being dosed several weeks longer through pregnancy and lactation. Dosing of the test substance occured at 50, 150 and 300 mg/kg bw/day. This study adheared to both the OECD 408 and 422 testing guidelines with only minor deviations.

 

There are two addtional 90-day oral toxicity studies for the analouge substance Ethanol, 2,2’-iminobis-, N-tallow alkyl derives CAS No 61791-44-4 registered under 2,2’-(C16-18 (even numbered, C18 unsaturated) alkyl imino) diethanol CAS No 1218787-32-6.  There is a 90 day dietary study in rats and a 90 Day study in dogs where the test substance was added to the diet in a solution in maize oil.  The Dog study was dosed at 13, 40, 120 mg/kg/day with a maize oil vehicle control.  The rats were feed diets containing 170, 500, 1’500 and 4’500ppm of the tests substance.  Both studies provide NOAEL values.  These studies were carried out in 1965, but are sufficiently well documented including information on the test substance to be considered suitable for use for REACH. Due to issues with sporadic vomiting and anorexia in the dog study, the data from the rat study is considered more reliable.

 

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

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.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2018

Deviations:

no

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

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1998

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:

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

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

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.

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

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.

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.

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.

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

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)

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)

 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

50 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The test used for establishing NOAELs for repeat exposure was performed with a valid test sample of Propanol, iminobis-, N-C16-C18 (evennumbered), C18 unsatd. alkyl) derivs. that was within specifications and under appropriate OECD guidlines.

System:

gastrointestinal tract

Organ:

ileum

jejunum

stomach

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

A study 90-day repeat dose combination with a OECD 422 study design on Propanol, iminobis-, N-C16-C18 (evennumbered), C18 unsatd. alkyl) derivs. resulted in a NOAEL of 50 mg/kg/day (based on clinical signs, lower body weights and food consumption, macroscopic and microscopic findings in the stomach, small intestine and mesenteric lymph nodes). Dosing groups consisted of control, 50, 150 and 300 mg/kg bw/day with an Arachis oil vehicle at a volume of 4mL/kg. This studies design allowed followed both the OECD 422 and OECD 408 guidlines with only minor variations and is suitable for a Klimisch score of 1. Minor variations consited of initiation of dosing at 6 weeks of age (422 variation), lack of pre-dose oesterous cycle determination (422 variation), change in blood chemistry timeline (post-partium day 13), maternal condition of females, lack of fasting in females (408 variation). This test was considered suitable to determine DNEL and hazard classification.

 

There are two 90 day oral toxicity studies for the read across substance Ethanol, 2,2’-iminobis-, N-tallow alkyl derives CAS No 61791-44-4 registered under2,2’-(C16-18 (even numbered, C18 unsaturated) alkyl imino) diethanol CAS No 1218787-32-6. There is a 90 day dietary study in rats and a 90 day study in dogs where the test substance was added to the diet in a solution in maize oil. The Dog study was dosed at 13, 40, 120 mg/kg/day with a maize oil vehicle control. The rats were feed diets containing 170, 500, 1’500 and 4’500ppm of the tests substance. Both studies provide NOAEL values. These studies were carried out in 1965, but are sufficiently well documented including information on the test substance to be considered suitable for use for REACH as Klimisch 2.

 

The 90 Day dog study, where the dogs were dosed by mixing a solution of the test substance in maize oil into the dry dog diet, suffered from serious problems with acceptance of the diet. This was due to corrosive/irritant nature of the test substance. The highest dose level of 120mg/kg/day vomited 2 to 3 hours after feeding and then began to refuse their food. Despite attempts to acclimatise them to the test substance this group had to be terminated after 5-6 weeks due to their poor condition, with 20% bodyweight loss. There was also sporadic vomiting and reluctance to eat all their food in the 40mg/kg/day group. This left the 13 mg/kg/day group as the NOAEL. In the 90 day rat study some decrease in food consumption was evident at the higher dose levels the 500ppm in the diet level was a clear NOAEL. This was calculated to be ca. 35 mg/kg/day. Due to these problems with vomiting and reduced food consumption etc. in the dog study, the NOAEL from the rat study has been selected as being more reliable. This is supported by the findings of no clear systemic toxicity in either study, the effects seen in both studies be associated with local irritant effects in the gastrointestinal tract. The 35mg/kg/day NOAEL value will therefore be used for key value for the chemical safety report for read across to Propanol, iminobis-, N-C16-C18 (evennumbered), C18 unsatd. alkyl) derivs.

 

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:

The rat study on the substance was selected as the dose level.

 

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:

Inhalation is not an expected route of exposure due to the low vapour pressure of the substnace and its corrocive properties to skin would make it vety technically difficult to perform.

 

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:

Inhalation is not an expected route of exposure due to the low vapour pressure of the substnace and its corrocive properties to skin would make it vety technically difficult to perform.

 

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:

Due the test substance being considered to be corrosive to skin based on the read across substance, it is not possible to conduct repeat dose dermal toxicity studies due to animal welfare considerations.  Data from the repeat dose oral studies can be used in the setting of DNELs.

 

Justification for selection of repeated dose toxicity dermal - local effects endpoint:

Due the test substance being considered to be corrosive to skin based on the read across substance, it is not possible to conduct repeat dose dermal toxicity studies due to animal welfare considerations.  Data from the repeat dose oral studies can be used in the setting of DNELs.

 

Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: ileum; digestive: jejunum

Justification for classification or non-classification

The toxic effects seen can be attributed to dietary administration with a corrosive test substance which caused damage/irritation due to the direct contact with the gastrointestinal tract. The no effect level in the rats was 50 mg/kg/day, from the 90 day dietary study in rats was outside the range of 10-30 mg/kg/day where a classification as Category 2 for specific target organ toxicity after repeated exposure is required. Also the effects seen were not systemic such as serious organ damage but local effects in the gastrointestinal tract. As the only effects seen were none specific direct local effects of irritation and the NOAEL was above 30mg/kg/day, 2,2’-(C16-18 (even numbered, C18 unsaturated) alkyl imino) diethanol CAS No 1218787-32-6 does not require classification for specific target organ toxicity.