Registration Dossier

Diss Factsheets

Administrative data

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
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:
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
Justification for study design:
not applicable
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
Vehicle:
CMC (carboxymethyl cellulose)
Details on 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
Details on mating procedure:
- M/F ratio per cage: 1:1 monogamous
- Length of cohabitation: 14 days
- Proof of pregnancy: spermidentification, vaginal plug in situ or copulation plug found in the cage tray
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): individual solid bottomed cages (measuring 42.5×26.6×18.5 cm) for the gestation period, birth and lactation.
Nesting material was provided inside suitable bedding bags.
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
Details on study schedule:
- Age at mating of the mated animals in the study: 12-13 weeks
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.


Positive control:
not applicable
Parental animals: Observations and examinations:
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

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







Oestrous cyclicity (parental animals):
Oestrous cycle was monitored by vaginal smears.
Vaginal smears were taken daily in the morning starting two weeks before pairing throughout the mating period, until a positive identification of copulation was made.

The vaginal smear data were examined to determine the following:
1. anomalies of the oestrous cycle
2. the pre-coital interval (i.e., the number of nights paired prior to the detection of mating)

Vaginal smears were also taken from all females, before despatch to necropsy.
Sperm parameters (parental animals):
The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed in all parental males.
Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
On Day 4 post partum, all pups were weighed and litters in excess of 8 offspring were culled to 8 (4 males and 4 females, where possible) by a random selection.
At least two pups (males or females, selected for culling) were sacrificed for blood collection.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
As soon as possible after parturition was considered complete (Day 0 post partum), all pups (live and dead) were counted, sexed and only live pups were identified.
Live pups were individually weighed on Days 1, 4, 7 and 13 post partum.

The AGD of each pup was measured on Day 1 post partum. The AGD was normalized to the cube root of body weight collected on Day 1 post partum.
On Day 13 post partum, the presence of nipples/areolae in each male pup was checked and recorded.

On Day 4 post partum, blood samples (approximately 0.2 mL) were collected from two of the non selected pups (1 sample/sex where possible) of the main groups. Blood samples were withdrawn under light ether anaesthesia from the heart (by intracardiac puncture).
On Day 14 post partum, blood samples (approximately 0.5 mL) were withdrawn under light ether anaesthesia from the heart (by intracardiac puncture) from at least two pups (1 sample/sex where possible) per litter.

All pups found dead in the cage were examined for external and internal abnormalities.
All culled pups sacrificed on Days 4 and 14 post partum were subjected to an external examination.
Sex was determined by internal gonads inspection.

The ventral region of pups was checked for presence of nipples/areolae. No nipples were found in pups to be retained on Day 14 post partum.
Data were not tabulated, but will be archived with all raw data.

Postmortem examinations (parental animals):
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 1)(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 1 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).
Postmortem examinations (offspring):
SACRIFICE
Pups that had completed the scheduled test period (Day 4 or 14 post partum) were euthanised by intraperitoneal injection of Sodium Thiopenthal. Pups selected for blood collection for hormone determination were killed on the day of blood sampling

GROSS NECROPSY
All pups found dead in the cage were examined for external and internal abnormalities.
All culled pups sacrificed on Days 4 and 14 post partum were subjected to an external examination.
Sex was determined by internal gonads inspection.

Thyroid was weighed from one male and one female pups selected for blood collection of hormone determination and preserved in 10% neutral buffered formalin. The thyroid weights were determined after fixation.

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 theWilliams test. The mean values, standard deviations and statistical analysiswere calculated fromactual values in the computer without rounding off.
Reproductive indices:
Copulation Index males (%) = (No. of males mated / No. of males paired) X 100
Fertility Index males (%) = (No. of males which induced pregnancy / No. of males paired) X 100
Copulation Index females (%) = (No. of females mated / No. of females paired) X 100
Fertility Index females (%) = (No. of pregnant females / No. of females paired) X 100
Offspring viability indices:
Pre-implantation loss % = (no. of corpora lutea − no. of visible implantations/no. of corpora lutea) x 100
Pre-natal loss on Day 0 post-partum %: = (no. of visible implantations – total litter size ate birth/ no. of visible implantations) x 100
Pup loss at Day 0 post-partum %: = (total litter size at birth – live litter size at birth/ total litter size at birth) x 100
Post-natal loss on Day 4 post-partum (before culling) %: = (total litter size at birth – live litter size at day 4, before culling/ total litter size at birth) x 100
Post-natal loss on Day 13 post-partum (after culling) %: = (live litter size on day 4(after culling) – live litter size on day 14/ live litter size on day 4 (after culling) x 100
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.
Dermal irritation (if dermal study):
not examined
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.
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. The 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
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.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
The oestrous cycle cyclicity of the treated females monitored during the pre-mating period, for a total of 14 days, was not affected by treatment.
The mean number of oestrous cycles observed in treated animals and control group were comparable.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages. Regular layering in the germinal epithelium was noted.
Description (incidence and severity):
An increased incidence in mean number of days for the pre-coital interval was observed in the treated groups compared to controls. The mean pre-coital interval was 3.2 days in the control group, 4.5 days in the low dose group, 7 days in the mid-dose group and 4.8 days in the high dose group. In particular, in the mid- and high dose groups the numbers of females conceiving after 5 days were more than 50% of animals. A reduction in the number of copulation plugs was also observed. The fertility index in treated animals was reduced compared to controls without dose relation. In the absence of dose relationship and without a statistically significance, the relation remained unclear.
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
Clinical signs:
no effects observed
Description (incidence and severity):
Clinical signs of pups were unaffected by treatment.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
Implantation, pre-birth loss data and gestation length of females were unaffected by treatment.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Litter data and sex ratios were unaffected by treatment.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No changes were seen in pups sacrificed on Days 4 and 14 post partum in T4 and TSH (T3 of all treated pups were found below the limit of quantification).
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Description (incidence and severity):
Anogenital distance in treated pups was comparable to control pups.
No nipples were observed in male pups on Day 14 post partum.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No significant differences were noted in thyroid weight between control and pups of treated groups.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No findings were described in any pups at necropsy.
Histopathological findings:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Generation:
F1
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
Key result
Reproductive effects observed:
no
Conclusions:
Based on the results of the present study, the NOAEL (No Observed Adverse Effect Level) for general toxicity and reproductive and developmental toxicity was considered to be 1000 mg/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.

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. Details are reported in section 7.5 of IUCLID dataset.

 

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.

 

Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages. Regular layering in the germinal epithelium was noted.

 

The oestrous cycle cyclicity of the treated females monitored during the pre-mating period, for a total of 14 days, was not affected by treatment. The mean number of oestrous cycles observed in treated animals and control group were comparable.

An increased incidence in mean number of days for the pre-coital interval was observed in the treated groups compared to controls. The mean pre-coital interval was 3.2 days in the control group, 4.5 days in the low dose group, 7 days in the mid-dose group and 4.8 days in the high dose group. In particular, in the mid- and high dose groups the numbers of females conceiving after 5 days were more than 50% of animals. A reduction in the number of copulation plugs was also observed. The fertility index in treated animals was reduced compared to controls without dose relation. In the absence of dose relationship and without a statistically significance, the relation remained unclear.

 

Two control, 4 low dose, 2 mid-dose and 3 high dose females which showed evidence of copulation were found not pregnant at necropsy. One mid-dose and one high dose females showed no evidence of copulation and were found not pregnant at necropsy (did not mate).

One low dose dam lost its litter on the day of parturition (Day 0 post-partum), one high dose female showed dystocia and was humanely killed.

The total number of pregnant females/group was: 8 in the control, 4 in the low dose, 7 in the mid-dose and 6 in the high dose groups.

The number of females with live pups on Day 14 post-partum was: 8 in the control, 3 in the low dose, 7 in the mid-dose and 5 in the high dose groups.

 

Implantation, pre-birth loss data and gestation length of females were unaffected by treatment.

Furthermore litter data and sex ratios were unaffected by treatment. Clinical signs of pups were unaffected by treatment. No nipples were found in male pups on Day 13 post-partum.

No effects on anogenital distance were seen between control and treated pups.

No necropsy findings were described in in decedent pups and in any pups sacrificed on Days 4 and 14 post-partum.

 

No changes were seen in pups sacrificed on Days 4 and 14 post-partum in T4 and TSH (T3 of all treated pups were found below the limit of quantification). No significant differences were noted in thyroid weight between control and pups of treated groups.

 

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.

The NOAEL reproductive toxicity of females and pups was 1000 mg/kg/day.

The NOAEL reproductive toxicity of males was 1000 mg/kg/day, even if an increased incidence in the precoital interval was observed in all treated animals.

The cause of this effect was unclear and conclusion cannot be achieved, considering that this test (as indicated in the guideline) is designed to generate limited information and does not provide complete information on all aspects of reproduction.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2019

Materials and methods

Test guideline
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

Test material

Constituent 1
Chemical structure
Reference substance name:
N,N'-[(methylimino)bis(trimethylene)]bis(stearamide)
EC Number:
236-793-7
EC Name:
N,N'-[(methylimino)bis(trimethylene)]bis(stearamide)
Cas Number:
13483-58-4
Molecular formula:
C43H87N3O2
IUPAC Name:
N-{3-[methyl(3-octadecanamidopropyl)amino]propyl}octadecanamide

Test animals

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

Administration / exposure

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
Doses / concentrationsopen allclose all
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

Examinations

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.

Results and discussion

Results of examinations

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.
 

Effect levels

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

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

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.

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