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Description of key information

A NOAEL for systemic toxicity of 1000 mg/kg bw/day was established for N,N’[(methylimino)bis(trimethylene)]bis(stearamide) based on information from an OECD 422 guideline study.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-07-26 to 2017-10-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29 July 2016
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Envigo Netherlands, Kreuzelweg 53, 5961 NM Horst, Netherlands
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 7 to 8 weeks old
- Weight at study initiation: 184 - 200 g for males and 208 - 221 g for females
- Fasting period before study: no
- Housing: From arrival to mating, animals were housed up to 5 of one sex to a cage, in polysulfone solid bottomed cages measuring 59.5×38×20 cm.
During mating, animals were housed one male to one female in clear polysulfone cages measuring 42.5×26.6×18.5 cm with a stainless steel mesh lid and floor.
After mating, the males were re-caged as they were before mating.
The femaleswere transferred to individual solid bottomed cages (measuring 42.5×26.6×18.5 cm) for the gestation period, birth and lactation.
- Diet (e.g. ad libitum): ad libitum, A commercially available laboratory rodent diet (4 RF 21,Mucedola S.r.l., Via G. Galilei, 4, 20019, SettimoMilanese (MI), Italy)
- Water (e.g. ad libitum): ad libitum to each cage via water bottles
- Acclimation period: 21 days, (main groups) and 33 days (recovery groups)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 2°C
- Humidity (%): 55% ± 15%,
- Air changes (per hr): 15 to 20 air changes per hour
- Photoperiod (hrs dark / hrs light): artificial light for 12 hours

Route of administration:
oral: gavage
Details on route of administration:
The test item was administered orally by gavage at a dose volume of 10mL/kg body weight.
Vehicle:
CMC (carboxymethyl cellulose)
Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:
The required amount of test material was suspended in the vehicle.
The formulations were prepared daily (concentrations of 10, 30 and 100 mg/mL).
Concentrations were calculated and expressed in terms of test item as supplied.

- VEHICLE
- Justification for use and choice of vehicle (if other than water): aqueous solution of 0.5 % carboxymethylcellulose
- Concentration in vehicle: The formulations were prepared daily (concentrations of 10, 30 and 100 mg/mL).
- Amount of vehicle (if gavage): 10 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis was performed in a separate study in order to validate both the analytical method and the formulation procedure and to verify the stability of the formulations.
Samples of the formulations prepared on Week 1 and last week were analysed to check the homogeneity and concentration.
Results of the analyses were within the laboratory acceptability limits for suspensions (85-115% for concentration and CV < 10% for homogeneity).
Duration of treatment / exposure:
males: 34/35 days
females: for at least 51 days
Frequency of treatment:
once a day, 7 days a week
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Each main group comprised 10 male and 10 female rats (Groups 1 to 4).
Control and high dose level included 5 additional animals per sex to be sacrificed after 4 weeks of recovery (Groups 5 and 6).
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Dose levels have been selectedbased on information from a preliminary study.

- Rationale for animal assignment:
The rats were allocated to the groups by computerised stratified randomisation to give approximately equal initial group mean body weights.
Positive control:
not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were checked twice daily for mortality. Once before commencement of treatment and at least once daily during the study, each animal was observed and any clinical signs recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before commencement of treatment and at least once a week thereafter, each animal was given a detailed clinical examination.
Data are reported until week 5 of study (males) and until week 6 (females)

BODY WEIGHT: Yes
- Time schedule for examinations: Males were weighed weekly from allocation to termination.
Females were weighed weekly from allocation to positive identification of mating and on Days 0, 7, 14 and 20 post coitum.
Dams were also weighed on Days 1, 4, 7, 13 post partum and just before necropsy.
Recovery groups: Each animal was weighed on the day of allocation to treatment groups, on the day that treatment commenced, weekly thereafter and just prior to necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: no feeding study
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: no feeding study

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: not applicable

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): not applicable

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at sacrificial procedure
- Anaesthetic used for blood collection: Yes; under isofluorane anaesthesia
- Animals fasted: Yes; under condition of food deprivation
- How many animals: 5 males and 5 females randomly selected from each main group
- Parameters checked in table [No.1] were examined.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at sacrificial procedure
- Anaesthetic used for blood collection: Yes; under isofluorane anaesthesia
- Animals fasted: Yes; under condition of food deprivation
- How many animals: 5 males and 5 females randomly selected from each main group
- Parameters checked in table [No.1] were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
-- Time schedule: Once before commencement of treatment and at least once a week thereafter, each animal was given a detailed clinical examination.
Data are reported until week 5 of study (males) and until week 6 (females)
- Dose groups that were examined: 5/10 animals of the main groups selected randomly and in all animals of the recovery groups
- Battery of functions tested: sensory activity / grip strength / motor activity

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
The clinical history of the adult animals was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices).
Changes were noted, the requisite organs weighed (Table 2)(excluding animals sacrificed for humane reasons or found dead) and the required tissue samples preserved in fixative and processed for histopathological examination.

HISTOPATHOLOGY: Yes
Samples of all the tissues listed table 2 were fixed and preserved in 10% neutral buffered formalin (except eyes, optic nerves and Harderian glands, testes and epididymides which were fixed in modified Davidson’s fluid and preserved in 70% ethyl alcohol).
Statistics:
Standard deviations were calculated as appropriate. For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t-test, depending on the homogeneity of data.
The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters and for the evaluation of the hormone thyroid data in pups. Intergroup differences between the control and treated groups were assessed by the non-parametric version of the Williams test. The mean values, standard deviations and statistical analysiswere calculated fromactual values in the computer without rounding off.
Clinical signs:
no effects observed
Description (incidence and severity):
No significant clinical signs were observed throughout the study in all treated animals of both sexes.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
A total of 3 females died or were sacrificed for humane reasons during the treatment period (one animal in each dose group).
Based on the post mortem and histopathological finding, the cause of death of these animals was attributed to a misdosing.
In addition, one female receiving 1000 mg/kg/day was sacrificed for humane reasons on Day 22 post coitum for difficulty in parturition, this isolated case was considered incidental.
No mortality occurred in males.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No differences in body weights and body weights gain were recorded in animals of both sexes compared to the control group, throughout the study, both for main and recovery groups.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No effects on food consumption were observed in either males or females throughout the study, both for main and recovery groups.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
At haematological investigation performed in the animals of the main groups at the end of treatment, leucocytosis was recorded in high dose males.
This finding was considered to be not adverse.
The other statistically significant differences recorded between control and treated animals were not dose-related, therefore they were considered unrelated to treatment.

The statistically significant difference of activated partial thromboplastin time observed between controls and mid-dose males was not dose-related, therefore it was considered to be incidental.

Recovery group: Lymphocytes data were comparable with controls.
Other statistically significant changes recorded were not observed during the treatment period, therefore they were considered incidental.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Main group: The differences recorded in some parameters were not dose-related and/or of minimal severity, therefore they were considered not to be adverse or of no toxicological relevance.
Recovery group: No differences between control and treated animals were recorded. Other statistically significant differences recorded only in females were not observed during the treatment period, therefore they were considered incidental.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No significant alterations in motor activity, grip strength or sensory reaction to stimuli were observed in any treatment group at the examinations performed during the study.
The changes noted in landing foot splay during the recovery period were not considered related to treatment since no changes were observed during the treatment period.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Main group: No changes were observed in terminal body weight or organ weights (absolute and relative) of treated animals, when compared to the controls.

Recovery group: No changes were observed in terminal body weight or absolute organ weights of recovery animals, when compared to the controls.
Changes in relative kidneys or adrenals observed in treated males and females respectively were considered not treatment-related, since the effects were not observed in main groups animals.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No differences were noted at post mortem examination in treated animals sacrificed at the end of treatment and recovery periods, that could be considered treatment-related.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Animals that completed the treatment or recovery period and killed at termination did not show relevant macroscopic changes that could be considered treatment-related.
The sporadic changes such as small prostate observed respectively in two high dose males and one low dose male or single dark depressed area in one high dose male were considered spontaneous and incidental, having a comparable incidence in control and treated groups and/or are characteristically seen in untreated Sprague Dawley SD rats of the same age.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
No treatment-related changes were noted in animals sacrificed at the end of the treatment period nor in the abnormalities observed in animals sacrificed at the end of the recovery period.
Description (incidence and severity):
Males dosed at 300 and 1000 mg/kg bw/day showed statistically significant decreases of tyroxine (T4) and thyroid stimulating hormone (TSH) and no relevant changes of the triiodothyronine (T3) levels were recorded.
Compared with controls, changes were 44% to 55% with some dose-relation.
The concurrent decrease of T4 and TSH observed in mid- and high dose males of main group is usually associated with secondary hypothyroidism (due to pituitary impairment).
However, since no changes of pituitary were recorded at histopathological examination in high dose males, no clear conclusion could be drawn for these findings.

In the recovery group Thyroid hormones determination revealed no significant differences between control and high dose animals that could be considered related to treatment.
 
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: no effects
Key result
Critical effects observed:
no
Conclusions:
Based on the results of the present study, the NOAEL for general toxicity was considered to be 1000mg/kg/day for both males and females.
Executive summary:

In a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening study according to OECD guideline 422, N-N’-[(methylimino)bis-(trimethylene)]bis-(stearamide) was administered to 10 Sprague-Dawley rats /sex/ at dose levels of 0, 100, 300 and 1000 mg/kg bw/day by gavage.

Males were treated for 2 weeks prior to pairing and during pairing with females until the day before necropsy, for a total of 34/35 days. Pregnant females were treated for 2 weeks prior to pairing, during pairing, post-coitum and post-partum periods until Day 13 post-partum (for at least 51 days). The non-pregnant females were dosed up to the day before necropsy (post-coitum from Days 26/28). Females which did not mate were sacrificed after approximately 8 weeks of treatment.

Additionally a recovery group was included (5 rats/sex) for control and high dose with a 4 week treatment-free period in order to assess any delayed toxicity or recovery from any adverse effects observed during the dosing phase. Recovery males were treated for up to 4 consecutive weeks and killed after 4 weeks of recovery period. Recovery females were treated for up to 6 weeks, when most of the main group females were killed. The females were sacrificed after 4 weeks of recovery period.

A total of 3 females (two of the main groups and one of the recovery groups) died or were sacrificed for humane reasons during the treatment period. Based on the post mortem and histopathological findings (clear and/or white fluid or creamy content in the thoracic cavity, adherence to heart, aorta and lungs with or without evident thymus), the cause of death of these animals was attributed to a mis-dosing.

In addition, one female receiving 1000 mg/kg/day was sacrificed for humane reasons on Day 22 post coitum for difficulty in parturition, this isolated case was considered incidental.

No mortality occurred in males.

 

No significant clinical signs were observed throughout the study in all treated animals of both sexes. Neurotoxicity assessment was unaffected by treatment. No differences in body weights and body weights gain were recorded in animals of both sexes compared to the control group, throughout the study, both for main and recovery groups.

 

No effects on food consumption were observed in either males or females throughout the study, both for main and recovery groups.

 

At haematological investigation performed in the animals of the main groups at the end of treatment, leucocytosis was recorded in high dose males. This finding was considered to be not adverse. The other statistically significant differences recorded between control and treated animals were not dose-related, therefore they were considered unrelated to treatment.

The statistically significant difference of activated partial thromboplastin time observed between controls and mid-dose males was not dose-related, therefore it was considered to be incidental. Recovery groups animals Lymphocytes data were comparable with controls.

The other statistically significant changes recorded were not observed during the treatment period, therefore they were considered incidental.

 

The differences recorded in some clinical chemistry parameters in main group animals were not dose-related and/or of minimal severity, therefore they were considered not to be adverse or of no toxicological relevance. No differences between control and treated animals of recovery groups were recorded. The other statistically significant differences recorded only in females were not observed during the treatment period, therefore they were considered incidental.

 

The concurrent decrease of T4 and TSH observed in mid- and high dose males of main group is usually associated with secondary hypothyroidism (due to pituitary impairment). However, since no changes of pituitary were recorded at histopathological examination in high dose males, no clear conclusion could be drawn for these findings.

In the recovery group Thyroid hormones determination revealed no significant differences between control and high dose animals that could be considered related to treatment.

 

No changes were observed in terminal body weight or organ weights (absolute and relative) of treated animals of main group, when compared to the controls. No changes were observed in terminal body weight or absolute organ weights of recovery animals, when compared to the controls. Changes in relative kidneys or adrenals observed in treated males and females respectively were considered not treatment-related, since the effects were not observed in main group animals.

 

No differences were noted at post mortem examination in treated animals sacrificed at the end of treatment and recovery periods, that could be considered treatment-related.

 

Animals that completed the treatment or recovery period and killed at termination did not show relevant macroscopic changes that could be considered treatment-related. The sporadic changes such as small prostate observed respectively in two high dose males and one low dose male or single dark depressed area in one high dose male were considered spontaneous and incidental, having a comparable incidence in control and treated groups and/or are characteristically seen in untreated Sprague Dawley SD rats of the same age.

 

Conclusion:

Based on the results of the present study, the NOAEL for general toxicity was considered to be 1000 mg/kg/day for both males and females.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
OECD 422 Guideline with GLP

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

In a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening study according to OECD guideline 422, N-N’-[(methylimino)bis-(trimethylene)]bis-(stearamide) was administered to 10 Sprague-Dawley rats /sex/ at dose levels of 0, 100, 300 and 1000 mg/kg bw/day by gavage.

Males were treated for 2 weeks prior to pairing and during pairing with females until the day before necropsy, for a total of 34/35 days. Pregnant females were treated for 2 weeks prior to pairing, during pairing, post-coitum and post-partum periods until Day 13 post-partum (for at least 51 days). The non-pregnant females were dosed up to the day before necropsy (post-coitum from Days 26/28). Females which did not mate were sacrificed after approximately 8 weeks of treatment.

Additionally a recovery group was included (5 rats/sex) for control and high dose with a 4 week treatment-free period in order to assess any delayed toxicity or recovery from any adverse effects observed during the dosing phase. Recovery males were treated for up to 4 consecutive weeks and killed after 4 weeks of recovery period. Recovery females were treated for up to 6 weeks, when most of the main group females were killed. The females were sacrificed after 4 weeks of recovery period.

A total of 3 females (two of the main groups and one of the recovery groups) died or were sacrificed for humane reasons during the treatment period. Based on the post mortem and histopathological findings (clear and/or white fluid or creamy content in the thoracic cavity, adherence to heart, aorta and lungs with or without evident thymus), the cause of death of these animals was attributed to a mis-dosing.

In addition, one female receiving 1000 mg/kg/day was sacrificed for humane reasons on

Day 22 post coitum for difficulty in parturition, this isolated case was considered incidental.

No mortality occurred in males.

 

No significant clinical signs were observed throughout the study in all treated animals of both sexes. Neurotoxicity assessment was unaffected by treatment. No differences in body weights and body weights gain were recorded in animals of both sexes compared to the control group, throughout the study, both for main and recovery groups.

 

No effects on food consumption were observed in either males or females throughout the study, both for main and recovery groups.

 

At haematological investigation performed in the animals of the main groups at the end of treatment, leucocytosis was recorded in high dose males. This finding was considered to be not adverse. The other statistically significant differences recorded between control and treated animals were not dose-related, therefore they were considered unrelated to treatment.

The statistically significant difference of activated partial thromboplastin time observed between controls and mid-dose males was not dose-related, therefore it was considered to be incidental. Recovery groups animals Lymphocytes data were comparable with controls.

The other statistically significant changes recorded were not observed during the treatment period, therefore they were considered incidental.

 

The differences recorded in some clinical chemistry parameters in main group animals were not dose-related and/or of minimal severity, therefore they were considered not to be adverse or of no toxicological relevance. No differences between control and treated animals of recovery groups were recorded. The other statistically significant differences recorded only in females were not observed during the treatment period, therefore they were considered incidental.

 

The concurrent decrease of T4 and TSH observed in mid- and high dose males of main group is usually associated with secondary hypothyroidism (due to pituitary impairment). However, since no changes of pituitary were recorded at histopathological examination in high dose males, no clear conclusion could be drawn for these findings.

In the recovery group Thyroid hormones determination revealed no significant differences between control and high dose animals that could be considered related to treatment.

 

No changes were observed in terminal body weight or organ weights (absolute and relative) of treated animals of main group, when compared to the controls. No changes were observed in terminal body weight or absolute organ weights of recovery animals, when compared to the controls. Changes in relative kidneys or adrenals observed in treated males and females respectively were considered not treatment-related, since the effects were not observed in main group animals.

 

No differences were noted at post mortem examination in treated animals sacrificed at the end of treatment and recovery periods, that could be considered treatment-related.

 

Animals that completed the treatment or recovery period and killed at termination did not show relevant macroscopic changes that could be considered treatment-related. The sporadic changes such as small prostate observed respectively in two high dose males and one low dose male or single dark depressed area in one high dose male were considered spontaneous and incidental, having a comparable incidence in control and treated groups and/or are characteristically seen in untreated Sprague Dawley SD rats of the same age.

 

Conclusion:

Based on the results of the present study, the NOAEL for general toxicity was considered to be 1000 mg/kg/day for both males and females.

 

 

Justification for classification or non-classification

Repeated dose toxicity of N,N’[(methylimino)bis(trimethylene)]bis(stearamide) was assessed based on data from a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening study.

The no-observed-adverse-effect-level (NOAEL) for systemic toxicity was considered to be 1000 mg/kg/day, the highest tested dose.

Evaluating information from the sub-acute toxicity study a classification with STOT-RE according to GHS Regulation EC No 1272/2008 for N,N’[(methylimino)bis(trimethylene)]bis(stearamide) is not warrant.

 

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