2,2,4,4-tetramethyl-7-oxa-3,20-diazadispiro[5.1.11.2]-henicosan-21-one

Endpoint summary

Description of key information

Based on the changes observed in the available 13 week oral toxicity study (OECD 408) performed in Sprague Dawley rats the NOAEL was considered to be 225 mg/kg bw/d in males and 75 mg/kg bw/d in females (highest doses tested)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Sept 2008 - Jan 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Batch No.: DEFD097971
Purity: > 99.9%
Expiry date: 26. July 2012

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Hsd: SD

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Italy S.r.l, San Pietro al Natisone, Italy
- Age at study initiation: 27-29 days
- Weight at study initiation: 75-99 g
- Housing: up to 5 of one sex in clear polycarbonate cages with stainless steel mesh lid and floor
- Diet (e.g. ad libitum): laboratory rodent diet (4 RF 21, Mucedola S.r.l, Settimo Milanese, Italy), ad libitum
- Water (e.g. ad libitum): drinking water, ad libitum
- Acclimation period: 18 days

DETAILS OF FOOD AND WATER QUALITY:
Records of analyses of water and diet are kept on file at RTC

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-2°C
- Humidity (%): 55+/-15%
- Air changes (per hr): 15-25
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
From: 23. Sept. 2008 To: 26. Jan. 2009

Route of administration:

oral: gavage

Vehicle:

other: sesame oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): solubility
- Concentration in vehicle:
males: 2.5, 7.5 and 22.5 mg/mL
females: 1.0, 2.5 and 7.5 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Prior to commencement of treatment the proposed formulation procedure for the test item was checked in the range 1 - 22.5 mg/mL (concentration and homogeneity) and stability by chemical analysis.
Samples of the formulations prepared in weeks 1 and 13 of the study were analysed to check the homogeneity and concentration.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

daily

Dose / conc.:

25 mg/kg bw/day (actual dose received)

Remarks:

males

Dose / conc.:

75 mg/kg bw/day (actual dose received)

Remarks:

males

Dose / conc.:

225 mg/kg bw/day (actual dose received)

Remarks:

males

Dose / conc.:

10 mg/kg bw/day (actual dose received)

Remarks:

females

Dose / conc.:

25 mg/kg bw/day (actual dose received)

Remarks:

females

Dose / conc.:

75 mg/kg bw/day (actual dose received)

Remarks:

females

No. of animals per sex per dose:

10 + 5 animals/sex for 4 weeks recovery (control and high dose group)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Based on the results of a 28 day dose range finder
- Post-exposure recovery period in satellite groups:
4 weeks

Positive control:

none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily for clinical signs, twice daily for mortality

DETAILED CLINCAL EXAMINATION:
weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

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: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / Not specified
- Time schedule for examinations:

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations:prior to treatment and suring week 13
- Dose groups that were examined: all groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: during week 13 and week 4 of recovery
- Anaesthetic used for blood collection: Yes (isofluorane)
anminals under food and water deprivation
- How many animals: all animals
- Parameters examined:
Haematocrit, haemoglobin, red blood cell count, reticulocyte count, reticulocyte maturation index, mean red blood cell volume, red cell volume distribution width, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, Haemoglobin concentration width, Heinz bodies, white blood cell count, differential leucocyte count, platelets, prothrombin time, activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: during week 13 and week 4 of recovery
- How many animals: all animals
animals under food and water deprivation
- Parameters examined:
Alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, gamma-glutamyltransferase, lactate dehydrogenase, glutamat dehydrogenase, creatine kinase, urea, creatinine, glucose, triglycerides, phosphorus, total bilirubin, total cholesterol, total protein, albumin, globulin, A/G ratio, sodium, potassium, calcium, chloride

URINALYSIS: Yes
- Time schedule for collection of urine: during week 13 and week 4 of recovery (overnight)
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Yes
- Parameters examined:
Appearance, color, volume, specific gravity, pH, protein, total reducing substances, nitrite, glucose, ketones, bilirubin, urobilinogen, blood
Sediment examination:
epithelial cells, erythrocytes, leucocytes, crystals, spermatozoa and precursors, other abnormal components

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during week 12 and once during week 4 of recovery
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity
additionally (weekly):
handling reactivity, lachrymation, palperal closure, salivation, piloerection, rearing, spasms, myclonia, mobility impairment, arousal, vocalisation, stereotypies, unusual repsiratory pattern, bizarre behavior, urination, defecation, tremors, gait

Sacrifice and pathology:

GROSS PATHOLOGY:
Yes, all animals

ORGAN WEIGHT:
From all animals completing the scheduled test period the following organs were weighed:
adrenal glands, brain, epididymides, heart, kidneys, liver, ovaries, parathyroid glands,spleen, testes, thymus, thyroid, uterus

HISTOPATHOLOGY: Yes
from all main phase animals in the control and high dose groups, from all animals in low and medium dose groups died during the treatment and all abnormalities in all main phase animals.
Histopathologically examined organs/tissues:
adrenal glands, aorta, bone, boe marrow, brain, caecum, colon, duodenum, epididymides, heart, ileum, jejunum, kidneys, lungs, lymph nodes (mandibular and mesenteric), mammary gland, oesophagus, ovaries, oviducts, pancreas, prathyroid gland, pituitatry gland, prostate glandm rectum, salivary glands, sciatic nerve, skin, spinal cord, spleen, stomach, testes, thymus, thyroid, trachea, urinary bladder, uterus/cervix, vagina

Statistics:

For continuous variables the significance of the differences amongst groups was assessed by analysis of variance. Differences between each treated group and the control group were assessed by Dunnett’s test. The homogeneity of the data was verified by Bartlett’s test before Dunnett’s test. If data were found to be inhomogeneous a Modified t test (Cochran and Cox) was applied. The mean values, standard deviations and statistical analysis were calculated from the actual values in the computer without rounding off. Statistical analysis of histopathology findings was carried out by means of the non-parametric Kolmogorov-Smirnov
test.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Hairloss was observed in some animals of both sexes of the high dose group. Other minor clinical signs, such as damaged eye, corneal opacity, broken/missing teeth, swollen head and skin/fur staining were observed in individual animals during the treatment period. These signs were not considered to be treatment-related.
No significant clinical signs were noted in recovery animals during the treatment-free period.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Very slight but statistically significant body weight reductions were observed in the high dose males from Day 15 (-4%) to Day 92 (-10%) of the treatment period. Very slight reductions were also observed in the high dose females, treated at 75 mg/kg/day, from Day 85 (-6% to -4%). Terminal body weight showed a statistically significant reduction (- 9%) in the high dose males, treated at 225 mg/kg/day.

Although all the above mentioned body weight reductions were treatment-related, they were considered to be of no toxicological importance as they were very slight (< 10%) and comparable to the historical control data.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Very slight decreases in food consumption (from -9% to -17%) were observed in the high dose males (225 mg/kg/day) from Day 8 to Day 43. Differences in food consumption disappeared by Day 50 and were no longer detected up to the end of the recovery period.
These changes were not considered of toxicological importance as they were slight and comparable to the historical control data.
No significant food consumption differences were observed in female animals at any stage of the study.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No changes of toxicological significance were observed.
Slight leucopenia was observed in all treated females and comprised almost all leukocyte cell types (approximately 30%-35% when compared with controls), whereas the decrement of white blood cells was not dose-related and the results obtained were within the range of historical data.
In addition, a slight increase in Heinz Bodies was observed in treated groups when compared to controls. However, due the lack of relation with the dose and of further related changes (anaemia, reticulocytosis) this change was considered to be of no toxicological significance.
The other statistically significant differences between control and treated animals (increase in haemoglobin in males treated with 25 mg/kg/day, haematocrit and mean corpuscular haemoglobin concentration in males receiving 25 and 225 mg/kg/day, 37% decrease in monocytes in males treated with 25 mg/kg/day and in mean corpuscular haemoglobin concentration) were of low severity (1-5%) and/or with no dose-relation, therefore they were considered to be incidental.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

In general, minor fluctuations of some biochemical parameters were recorded in treated animals as showed below.
Males treated with 225 mg/kg/day showed a slight increase in alkaline phosphatase (+30%), alanine aminotransferase (+45%), urea (+23%), globulin (+10%) and chloride (+2%) and a decrease in bilirubin (-26%), cholesterol (-17%) and sodium (-2%). Cholesterol was also decreased in the other treated male groups and changes in globulin and sodium were observed even in those receiving 75 mg/kg/day. Females treated with 75 mg/kg/day showed an increase in alkaline phosphatase (+29%), cholesterol (+16%), chloride (+2%) and sodium (+2%) and a decrease in lactate dehydrogenase (-48%) and bilirubin (-23%).

Changes were considered to be of no toxicological importance, since they were of low magnitude and the direction of some of the alterations has no pathological importance. In addition, there were no histopathological findings which could be related with the liver and kidney marker changes.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

A slight but statistically significant decrease in absolute and relative weight of the spleen (-23% and -16% respectively) was observed in the males dosed at 225 mg/kg/day. In addition, an increase in testes relative weight (+10%) was noted in the high dose males.
No other changes of toxicological significance were observed.
Partial or total recovery was observed at the end of the 4-week treatment-free period. As no correlated lesion was noted at histology, this change was not considered to be of toxicological importance.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

The histopathological examination of the testes and epididymides did not reveal any evident differences between the findings observed in treated and control animals that could be considered treatment-related.
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 and no abnormalities were noted.

Key result

Dose descriptor:

NOAEL

Effect level:

225 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: no effects of toxicological relevance observed

Key result

Dose descriptor:

NOAEL

Effect level:

75 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no effects of toxicological relevance observed

Key result

Critical effects observed:

no

For clarification on the substance`s mode of action please refer to attached document "Assessment of toxicological mode of action_Hostavin N 20"

Conclusions:

Based on the changes observed in the present 13 week oral toxicity study performed in Sprague Dawley rats the NOAEL was considered to be 225 mg/kg bw/d in males and 75 mg/kg bw/d in females (highest doses tested).

Executive summary:

The oral toxicity of the test substancewhen given by daily administration to rats, has been investigated over a period of 13 consecutive weeks and recovery from any potential treatment-related effects over a period of 4 consecutive weeks.

No mortality occurred and no significant clinical signs were observed during the in vivo phase of the study. The slight reductions in body weight and food consumption observed in the high dose animals were not considered to be of toxicological importance due to the low magnitude (<20%).

The statistically significant changes observed in haematological parameters during dosing and/or recovery phase were considered to be of no toxicological significance, as they were of low severity and not dose-related. Minor fluctuations of some biochemical parameters were recorded in treated animals during the dosing and/or recovery phase. However, changes were of low magnitude and not in a direction to have pathological importance. In addition, there were no histopathological findings which could be related with the liver and kidney markers changes. Therefore they were considered to be of no toxicological importance. No changes of toxicological significance were observed in urine parameters. A slight but statistically significant decrease of the absolute and relative weight of the spleen was observed in the males dosed at 225 mg/kg/day. The toxicologically significance of this change was not supported by the microscopic examination of this organ. No changes of toxicological significance were found at post mortem macroscopic observation and microscopic examination performed at the end treatment and recovery periods. No abnormalities were noted at the evaluation in the seminiferous tubules of the stage in the spermatogenic cycle and in the integrity of the various cell types within the different stages.

On the basis of these results, some slight changes were seen at the high doses of 225 mg/kg/day for males and 75 mg/kg/day for females. However, these changes were of low magnitude and/or inconsistent between sexes. In addition, no lesions were observed at histopathological examination of tissues/organs.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

75 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

reliable without restriction

System:

cardiovascular

Organ:

vascular system

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Data waiving:

other justification

Justification for data waiving:

a short-term toxicity study does not need to be conducted because a reliable sub-chronic (90 days) or chronic toxicity study is available, conducted with an appropriate species, dosage, solvent and route of administration

Endpoint conclusion

Endpoint conclusion:

no study available

Quality of whole database:

n.a.

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Data waiving:

other justification

Justification for data waiving:

a short-term toxicity study does not need to be conducted because a reliable sub-chronic (90 days) or chronic toxicity study is available, conducted with an appropriate species, dosage, solvent and route of administration

Endpoint conclusion

Endpoint conclusion:

no study available

Quality of whole database:

n.a.

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Data waiving:

other justification

Justification for data waiving:

a short-term toxicity study does not need to be conducted because a reliable sub-chronic (90 days) or chronic toxicity study is available, conducted with an appropriate species, dosage, solvent and route of administration

Endpoint conclusion

Endpoint conclusion:

no study available

Quality of whole database:

n.a.

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Data waiving:

other justification

Justification for data waiving:

a short-term toxicity study does not need to be conducted because a reliable sub-chronic (90 days) or chronic toxicity study is available, conducted with an appropriate species, dosage, solvent and route of administration

Endpoint conclusion

Quality of whole database:

n.a.

Mode of Action Analysis / Human Relevance Framework

Likely mode of action based on the read-across approach:

Hostavin N20 belongs to the so called “hindered amine light stabilizer (HALS)” compounds. The most characteristic structural feature of HALS compounds is the 2,2,6,6- tetramethylpiperidine moiety which spontaneously undergoes piperidine nitroxide formation. It should be noted that the piperidine nitroxide moiety is also the active site of heterocyclic nitroxide drugs, which may explain that some of the effects found in toxicity studies with Hostavin N20 are comparable to the pharmacological actions of these nitroxide drugs. A readacross approach therefore may be considered when discussing the mode of action of Hostavin N20 in absence of any other relevant data such as kinectis/metabolism studies.

4-Hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (tempol) is the most extensively studied nitroxide, primarily used for antihypertensive (blood pressure lowering) effect. The underlying mechanism is related to the superoxide dismutase mimic action on the sympathetic nervous system, further related to vasodilatative effect. Among the various effects attibuted to tempol, body weight decreasing effects as well as sex-specific and/or hormone statues dependent antihypertensive effects are known and extensively investigated.

The likelihood emerges that Hostavin N20 exhibited a comparable superoxide dismutase mimic activity, resulting in the impairment of cardiovascular system after repeated exposure. The observed higher susceptibility of females after exposure to Hostavin N20 would be then comparable to the observed gender different response as well as hormone statues dependent responses to tempol application in animal studies. Even more enhanced susceptibility of pregnant animals could be attributed to the changed physiological conditions of pregnancy such as increased hypervolemia, increased cardiac output, and a decreased total peripheral resistance. The evident clinical findings of Hostavin N20 were emaciation, which seems to be comparable to the body weight decreasing effect of tempol. The decreased value of LDH then can be interpreted as a result of superoxide dismutase mimicking activity of Hostavin N20.

Additional information

Oral:

Relevant NOAEL (OECD 408, gavage, rat): 225 mg/kg bw/d

 

The oral toxicity of the test substance when given by daily administration to rats, has been investigated over a period of 13 consecutive weeks and recovery from any potential treatment-related effects over a period of 4 consecutive weeks.

No mortality occurred and no significant clinical signs were observed during the in vivo phase of the study. The slight reductions in body weight and food consumption observed in the high dose animals were not considered to be of toxicological importance due to the low magnitude (<20%). The statistically significant changes observed in haematological parameters during dosing and/or recovery phase were considered to be of no toxicological significance, as they were of low severity and not dose-related. Minor fluctuations of some biochemical parameters were recorded in treated animals during the dosing and/or recovery phase. However, changes were of low magnitude and not in a direction to have pathological importance. In addition, there were no histopathological findings which could be related with the liver and kidney markers changes. Therefore they were considered to be of no toxicological importance. No changes of toxicological significance were observed in urine parameters. A slight but statistically significant decrease of the absolute and relative weight of the spleen was observed in the males dosed at 225 mg/kg/day. The toxicologically significance of this change was not supported by the microscopic examination of this organ. No changes of toxicological significance were found at post mortem macroscopic observation and microscopic examination performed at the end treatment and recovery periods. No abnormalities were noted at the evaluation in the seminiferous tubules of the stage in the spermatogenic cycle and in the integrity of the various cell types within the different stages

Based on the results of this study, the no-observed-adverse-effect-level (NOAEL) for the test item was considered to be 225 mg/kg body weight/day for males and 75 mg/kg bw/d for females (highest doses tested).

 

An additional OECD 407 28 day oral toxicity study was performed using dose levels of 0, 15, 62.5, 250 and 1000 mg/kg bw/d.

All animals dosed at 1000 mg/kg/day died or were humanely killed within 4 days of dosing. In addition, all of the females dosed at 250 mg/kg/day were humanely killed on Day 8 of the study. Clinical signs, observed in the early decedent animals of both groups on the days prior to death, included hunched posture, dispnoea, matted fur, skin/fur staining, decreased activity, ataxia, tremors, swollen abdomen and moribund appearance. Body weight and food consumption were significantly reduced in females dosed at 250 mg/kg/day prior to sacrifice on Day 8. A number of macroscopic and microscopic abnormalities, considered as related to treatment, were noted in these animals at post mortem observations. Only minor clinical signs were occasionally seen in males dosed at 250 mg/kg/day. Slight but statistically significant reductions in body weight and food consumption were noted in these animals. No significant variations were observed in haematological parameters, while a dose-related decrement of triglycerides was observed at clinical chemistry analyses. The relative and absolute weights of the liver and spleen were slightly but statistically significantly reduced. However, this change was not supported by any changes at macroscopic or microscpic observations.

 

A second 28 days oral toxicity study with the test substance followed by a 14 day recovery period was performed in groups of six female and male rats at 5 weeks of age. The high dose was set at 200 mg/kg bw/d, and together 4 doses including 100, 20 and 5 mg/kg bw/d were employed. Recovery groups were also set for the 200 and 100 mg/kg bw/d level. Body weight change, food intake, general condition, hematological and clinical parameters were observed as well as urinalysis, organ weight determination, gross pathology and histopathology were performed.

Based on the effects observed in this study the NOEL is set at 5 mg/kg bw/d and the NOAEL is considered to be 200 mg/kg bw/d.

For further details please refer to attached document "Preleminary assessement of the toxicoligical mode of action of Hostavin N 20"

Justification for classification or non-classification

Due to the NOAEL of 75 mg/kg bw/day in an OECD 408 repeated dose toxicity study in females rats (chosen as most conservative approach), the test substance has to be classified with STOT (RE), Cat. 2 / H373 (May cause damage to organs through prolonged or repeated exposure) regarding systemic and target organ toxicity according to the criteria laid down in the EU Classification Labelling and Packaging Regulation (1272/2008/EC).