N1,N2-dimethyl-N1-{2-[methyl(propan-2-yl)amino]ethyl}-N2-(propan-2-yl)ethane-1,2-diamine

Endpoint summary

Description of key information

The repeated dose toxicity of N-isopropyl-N'-{2-[isopropyl(methyl)amino]ethyl}-N,N'-dimethylethane-1,2-diamine, has been investigated in Sprague Dawley SD rats in a repeated dose, reproductive and developmental screening study conducted according to OECD TG 422. In the study, three respective groups of male and female rats (10 rats per group) were administered the substance daily (5 days/week) at concentrations of 20, 60 or 180 mg/kg bw/day by oral gavage administration for at least five weeks.  Control groups received a reverse osmosis water vehicle. Only not severe effects were observed in female rats treated at 180 mg/kg bw/day during the last part of the lactation period and were expressed by a decrease in body weight gain and food consumption. The No Observed Adverse Effect Level (NOAEL) for the general systemic toxicity of the substance was determined to be 180 mg/kg bw/day.    

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:

repeated dose toxicity: oral, other

Remarks:

OECD 422 - Combined Repeated Dose Toxicity Study and Reproductive/Developmental Toxicity Screening Study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Start: 29 April 2021
Experimental Completion: 28 July 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Justification for type of information:

Annex VIII Data Requirement

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The Sprague Dawley rat was the species and strain of choice because it is accepted by many
regulatory authorities and there are ample experience and background data on this species
and strain.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS srl, San Pietro al Natisone (UD), Italy
- Females (if applicable) nulliparous and non-pregnant: [yes]
- Age at study initiation: 7-8 weeks
- Weight at study initiation: 210-235 g for males and approximately 189 to 199 g for females
- Housing: From arrival to pairing animals were housed up to 5 of one sex to a cage, in clear polycarbonate cages measuring 59.5×38.0×20.0cm with a stainless steel mesh lid and floor
During mating animals were housed one male to one female in clear polycarbonate 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 females were transferred to individual solid bottomed cages measuring 42.5×26.6×18.5 cm (Techniplast Gazzada S.a.r.l.) for the gestation period, birth and lactation. Suitable nesting material was provided and was changed as necessary.
- Diet (e.g. ad libitum): commercially available laboratory rodent diet
- Water (e.g. ad libitum): drinking water
- Acclimation period: 5 weeks

DETAILS OF FOOD AND WATER QUALITY:

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

Route of administration:

oral: gavage

Details on route of administration:

Oral gavage at a constant dose volume of 10 mL/kg

Vehicle:

water

Remarks:

softened water (by reverse osmosis)

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 the analytical method and the formulation procedure (ERBC Study No. A4175). In the same study, the stability of the preparations, at 1 and 200 mg/mL, was assessed after 28 hours at room temperature and after 8 days at 2-8 °C. The results, met the acceptability criteria (recovery 85 to 115% of the theoretical value, with a homogeneity < 10 %), indicating that the suspensions were stable after 28 hours at room temperature and for up to 14 days at 2-8 °C. Samples of the formulations prepared during the current study (the first and the last week of treatment where possible) were analysed to check the concentration and homogeneity. Chemical analysis was carried out by the Analytical Chemistry Department.

Duration of treatment / exposure:

Males: Animals were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing,, through the pairing period and thereafter until the day before necropsy, for a minimum of 32 days.
Females:Animals were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing and thereafter during pairing, post coitum and post partum periods until Day 13
post partum or the day before sacrifice, for minimum of 51 days

Frequency of treatment:

Once daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

20 mg/kg bw/day (nominal)

Dose / conc.:

60 mg/kg bw/day (nominal)

Dose / conc.:

180 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before commencement of treatment and at least once daily during the study

BODY WEIGHT: Yes / No / Not specified
- Time schedule for examinations: Males: weekly
Females: 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 to necropsy

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
The weight of food consumed by each cage of males and females were recorded weekly (whenever possible) during the pre-mating period starting from Day 1 of dosing up to mating. Individual food consumption for females with positive identification of mating were measured on gestation Days 7, 14 and 20 post coitum starting from Day 0 post coitum and on Day 7 and 13 post partum starting from Day 1 post partum.

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 specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not specified

OPHTHALMOSCOPIC EXAMINATION: Not specified

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples for haematology, coagulation and clinical chemistry were collected, at the end of treatment period
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (food deprivation)
- How many animals: 5 males and 5 females (with viable litters) of each group
- Parameters checked in table [No.10] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples for haematology, coagulation and clinical chemistry were collected, at the end of treatment period
- Animals fasted: Yes (food deprivation)
- How many animals: 5 males and 5 females (with viable litters) of each group
- Parameters checked in table [No.12] were examined.

PLASMA/SERUM HORMONES/LIPIDS: Yes
- Time of blood sample collection: at termination, the timing of the blood collection for thyroid hormone determination was as close as possible between animals and at the same time of the day in case of sampling on different days.
- Animals fasted: Not specified


URINALYSIS: Yes (only males)
- Time schedule for collection of urine: during the last week of treatment
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: No
- Parameters checked in table [No.13] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once during the study, toward the end of treatment
- Dose groups that were examined: all groups
- Battery of functions tested: sensory activity / grip strength / motor activity / other:

Sacrifice and pathology:

METHOD OF SACRIFICE

Parental animals killed for humane reasons were sacrificed under carbon dioxide asphyxiation.
Parental animals that had completed the scheduled test period, were killed by exsanguination under isoflurane anaesthesia.
offspring: Intraperitoneal injection of Sodium Thiopenthal

NECROPSY
All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

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.
Intergroup differences between the control and treated groups were assessed by the nonparametric
version of theWilliams test. The mean values, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Animals of both sexes of Group 4 (180 mg/kg/day) showed reaction to treatment such as rales. For males (5/10) the sign was seen before and /or mating periods and again before and after dosing. For females it was recorded on occasion before mating (4/10) and during mating (1/10). No clinical signs were seen during the gestation and lactation periods, apart of one female (no. 67) in which rales were noted for few days of the gestation period. One male receiving 60 mg/kg/day (Group 3) showed rales for some days before mating (starting from Day 8) and during the mating phase. The sign was seen sometimes and recorded before and after treatment (up to 15-30 minutes after dosing).
No clinical signs were noted in males receiving 20 mg/kg/day and females receiving the dose levels up to 60 mg/kg/day.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Two females receiving 180 mg/kg/day were sacrificed for humane reasons, one on Day 22 post coitum and one on Day 6 of the premating phase (after 5 days of dosing).
Female no. 71 (180 mg/kg/day), was sacrificed for humane reasons on Day 22 of gestation, since the impairment during the terminal phase of parturition, confirmed by the prolapse of the uterus, was noted. This female showed 5 dead pups in the cage and 12 live pups which were sacrificed for humane reasons with the dam.
As reported in the Pathology report (see Addendum 6), at post mortem examination, reduced size of the thymus and prolapse of the uterus with red staining in the urogenital region were noted. In the uterus and cervix, this corresponded microscopically to moderate oedema with congestion. The factors contributory to illness of this female could not be determined.
For female no. 79 (180 mg/kg/day), at post mortem examination, moderate distension with gas contents in parts of the gastrointestinal tract (stomach, duodenum and jejunum), without microscopic correlation, were noted. Also in this case the factors contributory to illness of this female could not be determined.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No relevant changes in body weight were observed during the study in the treated males, when compared to controls.
Body weight of females were generally comparable between the treated and control groups, before mating and during gestation and post-partum periods. On Day 4 post partum an isolated statistically significant decrease in body weight was noted in females receiving 60 mg/kg/day, not followed by other changes and, hence, it was not considered to be toxicologically relevant.
Before pairing, on Day 8, females receiving 180mg/kg/day showed a decrease in body weight gain compared to the control group. As for males this isolated change was followed by a regular growth of the animals and, hence, it was not considered to be toxicologically relevant.
On Day 13 post partum, a statistically significant decrease in body weight gain was noted in
females receiving 180mg/kg/day (-55%) compared to the control group

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

No changes of toxicological significance were observed in food consumption during the study in treated males when compared to the control.
Decreases (not greater -13%) in food consumption were observed in females dosed at 180 mg/kg/day on Days 7 and 13 post partum. This change was significant, at statistical analysis,
on Day 13 post partum.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Bile acids were increased in females receiving 180 mg/kg/day (2.3 fold). Due to the absence of other related changes (e.g. liver markers), this finding was considered to be unrelated to treatment.
The other statistically significant differences recorded between control and treated animals (cholesterol and chloride in males, albumin/globulin ration and sodium in females) were not dose-related and/or due to biological variation, therefore they were considered to be incidental.

Endocrine findings:

no effects observed

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

The increase of urinary protein recorded in animals receiving 60 mg/kg/day was not dose related, therefore it was considered to be incidental.The statistically significant decrease of urinary volume recorded in animals dosed at 180mg/lg/day was within the range of expected biological variation, therefore it was considered to be incidental.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

No alterations in motor activity was recorded in any treatment group at the examination performed at the end of treatment.
A slight decrease (up to -16%) in grip strength measurement (second trial and mean values), performed toward the end of treatment was recorded in treated males when compared to control. No toxicological relevance was attributed to these alterations, considering that these values were within the historical control data (HCD).Moreover they were observed in one sex.

Not dose related decrease (up to -21%) in landing foot splay measurement (second trial) performed toward the end of treatment was recorded in treated males when compared to control. Mean decrease in Group 2 males was significant, at statistical analysis. In all cases the changes were within the historical control data (HCD), observed in only one sex and then without toxicological relevance.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

>= 180 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

mortality

organ weights and organ / body weight ratios

Critical effects observed:

no

Conclusions:

Administration of N-isopropyl-N'-{2-[isopropyl(methyl)amino]ethyl}-N,N'-dimethylethane-1,2-diamine at 180 mg/kg/day resulted in the premature death of two females, but the factors contributory to illness of these two females could not be determined.
Treatment-related effects were observed during the in-life phase (rales as clinical signs in males and females and reduction in growth factors in females, such as body weight gain and food consumption) at the dose level of 180 mg/kg/day. These not severe effects, were seen in females during the last part of lactation period and were expressed by a statistically significant decrease in body weight gain and food consumption on Day 13 post partum.

Based on the results of this study it was concluded that the No Observed Adverse Effect Level (NOAEL) for general, reproductive and developmental toxicity was considered 180mg/kg/day for males and females.

Executive summary:

INTRODUCTION

 

The toxicity, as well as any possible effects of PU-2019-872, when administered by oral route at dose levels of 20, 60 and 180mg/kg/day, on male and female Sprague Dawley rats on the reproductive performance (such as gonadal function, mating behaviour, conception, development of conceptuses, parturition and early lactation of the offspring) were investigated in this study.

 

MATERIALS AND METHODS

 

Males were treated for 2 consecutive weeks prior to pairing and during pairing with females until the day before necropsy, for a total of 32/33 days. Females were treated for 2 consecutive weeks prior to pairing, during pairing and throughout the gestation and lactation periods until Day 13 post partum, for a period comprised from 51 to 60 days.

 

During the study, clinical condition, detailed physical examination and arena observations, sensory reactivity observations, grip strength, motor activity, body weight, food consumption, hematology (peripheral blood), blood chemistry, thyroid hormone analysis, estrous cycles, pre-coital interval, mating performance, fertility, gestation length, organ weight and macroscopic pathology and histopathology investigations were undertaken.

 

The clinical condition, litter size and survival, sex ratio, body weight, anogenital distance and macropathology for all offspring were also assessed. Nipple counts were performed on male offspring on Day 13 of age. Blood samples were collected from selected offspring on Day 4 (not analyzed) and Day 13 of age for thyroid hormone analysis.

 

 

RESULTS

 

Two cases of unscheduled deaths occurred during the premating or gestation phases at the dose of 180 mg/kg/day. The Study Pathologist concluded that the factors contributory to illness of these two females could not be determined on the basis of the macro- and microscopic findings.
Rales was the principal treatment-related clinical sign observed in males and females receiving 180 mg/kg/day particularly evident before and during mating. During gestation period one female receiving 180 mg/kg/day showed rales for some days.
No signs of neurotoxicity (weekly observations at removal from the cage and in an open arena, alterations in motor activity, grip strength and sensory reactivity to stimuli) were observed during the study in parental males and females.
No relevant differences in body weight were found between control and treated animals of both sexes throughout the study and for body weight gain in treated males.
Females receiving 180 mg/kg/day showed a statistically significant decrease in body weight gain on Day 13 post partum.
No effects on food consumption were observed in treated males during the treatment period.
Females receiving 180 mg/kg/day showed a significant decrease in food consumption on Days 13 post partum.
No treatment-related changes were observed in haematological (including coagulation), clinical chemistry parameters or urinalysis (only for males).
Thyroid hormone evaluation in parental animals of both sexes, as well as, in pups at Day 14 post partum did not show treatment-related changes.
The number of females with live pups on Day 14 post partum was 7 in the control group, 10 in the group dosed at 20 and 60mg/kg/day and 7 at 180mg/kg/day. Three females, two in control group and one in Group 4 , were found not pregnant and one control female showed unilateral total resorption. One female of Group 4 (180 mg/kg/day) sacrificed on Day 22 of the gestation showed an impairment of the parturition during the terminal phase, confirmed by the prolapse of the uterus.

No treatment-related anomalies were noted in the oestrous cycle of the treated females, when compared to controls. Copulatory and fertility indices did not show any treatment related differences among treated and control groups.
Mean gestation periods were similar in treated groups and controls. All pregnant dams gave birth between Days 22 and 23 post coitum.
Corpora lutea, number of implantations sites, live litter size, pre-implantation and pre-natal losses of treated groups appeared comparable to control values.
Females receiving 180 mg/kg/day showed decreased, but not statistically significant values of total litter size, live litter size and thus an increase in post-natal loss (expressed as percentage), compared to the controls, between the day of birth and Day 4 of lactation.
Changes ranged within the upper and lower historical control values.
OnDay 13 post partum, a trend toward a decrease in litterweight was seen at 180mg/kg/day, although the mean pup weight was comparable to the control.
The percentage of males in treated groups were similar to control at birth and on Days 4 and 14 post partum.
No significant differences in the anogenital distance (normalised value) were recorded between control and treated groups both for male and female pups. No nipple were count in male pups. After culling litter data remained quite similar with control and no additional lost of pups occurred.
Necropsy findings and thyroid weight in pups did not reveal any treatment-related effect. No treatment changes were observed in terminal body weight or absolute and relative organ weights of treated animals of both sexes, when compared to the controls.
No treatment-related changes were observed at post mortem macroscopic observations and microscopic evaluation.
No treatment-related changes were observed in reproductive and developmental parameters.

 

 

CONCLUSION

 

Treatment-related effects were observed during the in-life phase (rales as clinical signs in males and females and reduction in growth factors, such as body weight and food consumption, in females) at the dose level of 180 mg/kg/day. These effects, not severe, were seen in females during the last part of lactation period and were expressed by a statistically significant decrease in body weight gain and food consumption on Day 13 post partum.
Although not conclusive a possible correlation with the pups weight could be possible considering that the litter weight, but not the mean pups weight, appeared lower than control. Moreover, a not statistically significant increase in the total number of lost pups (found dead and missing pups) after birth was noted at 60 and 180 mg/kg/day, when compared with control. This was attributed to single females, for each group, respectively.

 

Therefore in light of this and based on the results of the present study, the NOAEL (No Observed Adverse Effect Level) for general, reproductive and developmental toxicity was considered 180 mg/kg/day for males and females.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

180 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Study conducted according to GLP and OECD Test Guidelines

Additional information

The repeated dose toxicity of N-isopropyl-N'-{2-[isopropyl(methyl)amino]ethyl}-N,N'-dimethylethane-1,2-diamine, has been investigated in Sprague Dawley SD rats in a repeated dose, reproductive and developmental screening study conducted according to OECD TG 422. In the study, three respective groups of male and female rats (10 rats per group) were administered the substance daily (5 days/week) at concentrations of 20, 60 or 180 mg/kg bw/day by oral gavage administration for at least five weeks.  Control groups received a reverse osmosis water vehicle. Only not severe effects were observed in female rats treated at 180 mg/kg bw/day during the last part of the lactation period and were expressed by a decrease in body weight gain and food consumption. The No Observed Adverse Effect Level (NOAEL) for the general systemic toxicity of the substance was determined to be 180 mg/kg bw/day.    

Justification for classification or non-classification

Based on the findings of this study, no classification is required.