3-dodecyl-1-(2,2,6,6-tetramethyl-4-piperidyl)pyrrolidine-2,5-dione

Endpoint summary

Description of key information

In a 28 day oral repeated dose toxicity study rats were exposed to 0, 25, 100 and 300 mg/kg bw/d. Adverse effects (mortality and histiocytosis in small intestines, lungs, adrenals, thymus and mesenteric / axillary lymph nodes, vacuolation in liver, adrenals and kidneys, forestomach foci) were observed in the mid and high dose group. The NOAEL was derived at 25 mg/kg bw/d.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

February 04, 2016 to June 06, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Identification Hostavin 3055
Appearance Clear, medium viscous, slightly yellowish liquid
Chemical Name [3-dodecyl-1-(2,2,6,6-tetramethyl-4-piperidyl)pyrrolidin-2,5- dione]
Formulation Type 98.3 % 3-dodecyl-1-(2,2,6,6-tetramethyl-4-piperidyl)pyrrolidine 2,5- dione
Source Clariant India Limited
Manufacturer Clariant India Limited
Reliable Tech Park, Gut No. 31,
Village Elthan, Thane-Belapur Road,
Airoli, Navi Mumbai 400 708, India
Batch number DEF2101115
CAS No. 79720-19-7
Purity (%) 98.3% (w/w)
Molecular weight 406 g/mol
Molecular formula C25H46N2O2
Manufacture Date July 08, 2013
Expiry Date July 07, 2020
Stability of test item Stable at room temperature
Storage conditions Room Temperature (20 to 30°C)

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Rat is chosen as the test system because this species is commonly used for repeated dose Oral toxicity testing and it meets the regulatory requirement of most of the regulatory agencies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: RCC Laboratories India Private Limited,
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 6 - 8 Weeks
- Weight at study initiation: (Male -130.8to 138.2 g, Female 121.4 to 130.4 g)

- Housing: In groups of three/ two of same sex per cage in Polycarbonate cages (Approximate internal dimensions of 365 mm x 202 mm x 180 mm height) with paddy husk bedding.
Diet -Teklad Certified Global 14% Protein Rodent Maintenance Diet from ENVIGO were provided ad libitum.
Water - Aquaguard filtered tap water was provided ad libitum.
Acclimation period:Main study 6 days for Step I and 9 days for Step II animals of Allocation A and B under laboratory conditions, after veterinary examination.

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.5°C to 22.7°C
- Humidity (%): 52 to 66%
- Air changes (per hr): Above 10 air changes per hour
- Photoperiod (hrs light)/ (hrs dark) : light cycle of 12 hours light and 12 hours dark.

IN-LIFE DATES: From: April 07, 2016 To: May 28, 2016

Route of administration:

oral: gavage

Details on route of administration:

Oral route is the intended route of administration in humans

Vehicle:

other: Refined groundnut oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The dose levels were in terms of test item as supplied by the sponsor.
The test item was formulated in vehicle.
The dose formulations were prepared shortly before each dosing.
The test item was weighed in a pre-weighed glass beaker on sartorius weighing balance. This was then transferred to a volumetric flask. Sufficient quantity of vehicle was added to make up the required volume of formulation. This was transferred back to beaker.
Homogeneity of the test item in the vehicle was maintained by using magnetic stirrer during test item administration.



VEHICLE
- Justification for use and choice of vehicle (if other than water): Refined groundnut oil was selected as vehicle based on preliminary solubility testing which was performed at the test facility

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

• The analytical method was validated and the detector response was found to be linear (r2 = 1.000) in the range of 50 to 250 mg/L concentration.
• The active ingredient was found homogeneously distributed in the suspension at concentrations 2.5 mg/mL, 10 mg/mL and 30 mg/mL for the test item respectively. (i.e. % coefficient of variation (RSD) ranged in between 0.06 to 0.4).
• The A.I. content obtained in all the dose groups were in agreement with the target concentration (i.e. ranged between 99.32 % to 99.63 %)

Duration of treatment / exposure:

The test item was administered by oral gavage to the animals once daily for 28 consecutive days.
Duration of recovery 14 days

Frequency of treatment:

daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

25 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Five animals per sex

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Doses were selected based on results of a dose range finding study (RCC Study No. 101).
Dose levels Group 1 : 0 mg/kg body weight (Control)
Group 2 : 25 mg/kg body weight ( Low )
Group 3 : 100 mg/kg body weight (Intermediate)
Group 4 : 300 mg/kg body weight (High)
Group 1R : 0 mg/kg body weight (Control recovery)
Group 4R : 300 mg/kg body weight (High dose recovery)

- Rationale for selecting satellite groups: The reversibility of treatment-related changes was assessed after a treatment free 14 day recovery period.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily cage-side clinical observations


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once during acclimatization, week 1, week 4 and week 6

BODY WEIGHT: Yes
- Time schedule : Once Weekly

FEED Consumption
- Time schedule : Once Weekly

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Once during acclimatization (In 20% of total population), at the end of week 4 in groups 1 to 4.
- Dose groups that were examined: vehicle control and high dose group.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week 4 Group 1 to 4 (Allocation A) and Week 6 Group 1R and 4R (Allocation B)

- Anaesthetic used for blood collection: Yes
- Animals fasted: Yes
- How many animals: surviving animals


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
Week 4 Group 1 to 4 (Allocation A)
Week 6 Group 1R and 4R (Allocation B)
- Animals fasted: Yes
- How many animals: surviving animals

URINALYSIS: Yes
- Time schedule for collection of urine:
Week 4 Group 1 to 4 (Allocation A)
Week 6 Group 1R and 4R (Allocation B)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Last week of treatment
- Dose groups that were examined: (Control , low and Intermediate)
- Battery of functions tested: sensory activity / grip strength / motor activity Yes



OTHER:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Other examinations:

Hormone analysis parameters (T3, T4 and TSH)

Statistics:

The following statistical methods were used to analyze the body weight, feed consumption, organ weights as well as clinical pathology data.
• Data was summarized in tabular form. Statistical analysis was performed using statplus program.
• All the data was checked for normality with Shapiro-Wilk W test
• Data for each group of animals was subjected to analysis of variance (ANOVA). Values were given as mean ± standard deviation (SD).
• t-test was used to compare the difference between treated and control groups. Statistical significances of differences were calculated with one-way analyses of variance.
P≤0.05 (5% level of significance) was considered to represent the significance in the respective parameters

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At least once prior to the first exposure twice daily on day 1, 2 & 3 and once a week thereafter, in addition, detailed clinical observations were also made in all survived animals at acclimatization, week1, week 4 and week 6. These observations were made outside the home cage, preferably in a standard arena and at similar times on each occasion. All the observations were carefully recorded. Signs noted include, but not be limited to, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (lacrimation, piloerection, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes were examined.
Animals of group 1 and 1R (vehicle control) did not show any clinical signs of toxicity throughout observation period. Similarly, animals of group 2 (treated with 25 mg/kg b.w.) did not show any clinical signs of toxicity throughout observation period.
Male animals of group 3 (treated with 100 mg/kg b.w.) had clinical signs of burrowing in all animals and dullness in one male and two female during observation period, these observations were not considered related to treatment.
Animals of group 4 and 4R, (treated with 300 mg/kg b.w.) had clinical signs of piloerection, polyuria, mild salivation, burrowing, dullness and chromodacryorrhea. These observations were considered related to treatment.

Mortality:

mortality observed, treatment-related

Description (incidence):

No mortality was observed in control, low and intermediate dose groups throughout the experimental period, whereas three female animals and one male of high dose group (G4) and two females & one male of high dose recovery (G4R) were found dead during treatment period.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In males, the body weights were significantly decreased in high dose group when compared with control, low dose and intermediate dose group animals on days 8, 15, 22 and 28. Day 15 male bodyweights showed significant decrease in intermediate dose when compared with low dose group and Day 22 there was significant decrease in intermediate compared to control and low dose group.
In females, the body weights were significantly decreased in the high dose group when compared with control group, low dose group and intermediate dose group animals on days 8, 15, 22 and 28. On Days 15, 22 and 28, body weights were significantly decreased in the intermediate dose group when compared with low dose group.
In recovery group (G4R) both treated male and female animals showed significant decrease in body weight on day 8, 15, 22, 29, 36 and 42 as compared to control (G1R) group. These observations were considered related to treatment.
In males, the body weight gain was significantly decreased in the high dose group when compared with control group, low dose group and intermediate dose group animals on days 8, 15, 22 and 28. On Days 8 body weight gain were significantly increased in the low dose group when compared with control dose group. On Days 15 body weight gain were significantly decreased in the intermediate dose group when compared with low dose group. On Days 22 body weight gain were significantly decreased in the intermediate dose group when compared with low dose group and control group.
In females, the body weight gain was significantly decreased in the high dose group when compared with control group, low dose group and intermediate dose group animals on days 8, 15, 22 and 28. On Days 15, 22 and 28, body weight gain were significantly decreased in the intermediate dose group when compared with low dose group.
In recovery group (G4R) both treated male and female animals showed significantly decrease in body weight gain on day 8, 15, 22, 29, 36 and 42 as compared to control (G1R) group. These observations were considered related to treatment.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

No treatment-related changes were observed in food consumptionat dose levels oflow dose G2 (25 mg/kg) and intermediate dose G3 (100 mg/kg) groups. However, the high dose animals (G4 and G4R - 300 mg/kg) showed severely decreased food consumption, this effect could be considered to be related to the test item administration.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Hematology analysis of surviving animals was performed for main group at the end of treatment period and at experiment period for recovery group. In Mean corpuscular volume males, showed significant decrease in high dose (G4) group compared to G2 and G3 group. Significant increase of Reticulocytes in high dose (G4) group was observed when compared with dose G1 and G2 group. Significant increased in Monocytes levels were observed in G4 when compared with G1, G2 and G3.
In female animals, significant increase in Reticulocytes was observed in high dose (G4) group compared with (G1), low (G2) and mid dose (G3) group. Increase in Reticulocytes was noticed in mid dose (G3) when compared with (G1) and low dose (G2) group. Whereas marginal increase in neutrophils and decrease in lymphocyte were observed in treated (G4) group female as compared to (G2) group. Significant increase in Monocytes were noticed in high dose (G4) as compared to control dose G1 and G2 low dose group animals. However in female animals, Prothrombin time levels were marginally increased in high dose (G4) group compared to dose (G3) group.
In male recovery, there was significant decrease in red blood corpuscular and significant increase in mean corpuscular volume, and Platelet (thrombocyte) counts were noticed in high dose (G4R) group animals compared to dose (G1R) group.
In high dose recovery females (G4R), there was significant decrease in Hb and mean corpuscular hemoglobin concentration were observed when compared with the control G1R group.
The rest of the hematological parameters evaluated showed no significant variations when compared with vehicle control group.
The changes observed in the hematological parameters are marginal, not clinically significant and could not be attributed to the test item administartion as these values were within biological variation except G4 and G4R.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Biochemistry analysis of surviving animals was performed for main group at the end of treatment period and at experiment period for recovery group. In males, there was increase in glucose level in intermediate dose when compared with control G1. In high dose G4 there was significant decrease in creatinine, Albumin, Globulin and total protein when compared with G1, G2 and G3. In high dose (G4) there was significant increase in A/G ratio noticed when compared with control and increase of Alanine aminotransferase was also noted in high dose group when compared with intermediate dose and control group.
In treated females, increase in glucose level and Triglycerides in intermediate dose was observed when compared with control G1 and increase in Aspartate aminotransferase was observed in high dose group as compared with G1, G2 and G3. Increase in Alkaline phosphatase in high dose was noticed when compared with control group and increase of potassium was also observed in intermediate dose when compared with low dose group. Decrease in sodium in high dose was noticed when compared with control and low dose. Significant decrease in total protein was noticed in high dose (G4) group animals compared to the groups G1, G2 & G3. However, significant decrease in total protein in intermediate dose (G3) group was observed when compared with (G1) group. Decrease in albumin in high, intermediate and low dose group was noticed when compared with control. Decrease in albumin in high dose group was noticed when compared with intermediate and low dose group.
In high dose recovery group (G4R) males, there was significant decrease in Creatinine and increase in alkaline phosphatase when compared with the respective control group.
In high dose recovery group (G4R) females, there was significant decrease in total protein, albumin and increase in Aspartate aminotransferase and Alkaline phosphatase were observed when compared with the respective control group.
The rest of the biochemistry parameters evaluated showed no significant variations when compared with vehicle control group.
The changes observed in the biochemistry parameters are marginal, not clinically significant and could not be attributed to the test item administration as these values were within biological variation except G4 and G4R.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Urine analysis parameters (volume, specific gravity, colour, crystal, clarity, pH, Leukocytes, urobillinogen, bilirubin, ketone bodies, proteins and glucose and microscopic examination) did not reveal any test item related changes in both sex during treatment and recovery period, with exception of increased erythrocytes in high dose females when compared with (G1), (G2) and (G3), whereas epithelial cells were increased in G4, G3, G2 in males as compared to G1 and epithelial cells was increased of (G4R) in females when compared with that of control females (G1R). The variations were considered not treatment related.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

The home cage, hand-held, open field, stimulus response, neuromuscular measurements (grip strength) and rota rod of low dose (G2 - 25 mg/kg body weight), Mid Dose (G3 - 100 mg/kg body weight) group male and female animals were observed to be comparable with vehicle control animals during fourth week. Since High dose group animals (G4 and G4R) exhibited clinical signs, these measurements were not extended to High dose (G4 and G4R) - 300 mg/kg body weight), and recovery groups (G4R). Neurological evaluation for individual animals, as applicable, was recorded in the form of scores. Group (G1R) of FOB data was also recorded

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

In treatment groups there was significant decrease in absolute weights of kidneys, liver, thymus, epididymides, prostate and seminal vesicles in male animals of group G4 (300 mg/kg) when compared with that of control group G1 (0 mg/kg), G2 (25 mg/kg) and G3 (100 mg/kg). Also absolute weights of spleen and testes of male animals from group G4 (300 mg/kg) were having significant lower weights when compared with control (0 mg/kg) and G3 (100 mg/kg).
Absolute weights of kidneys of female animals from group G4 (300 mg/kg) were having significant lower weights to that of group G2 (25 mg/kg) female animals.
In contrast to absolute organ weights in male animals of group G4 (300 mg/kg) relative organ weights of adrenals, kidneys, liver, heart, spleen, testes, epididymides, brain were having significantly higher values than that of control (0 mg/kg), G2 (25 mg/kg) and G3 (100 mg/kg). Likewise relative weights of testes from G3 (100 mg/kg) animals was higher than that of G2 (25 mg/kg). However both relative and absolute weight of prostate and seminal vesicle of group G4 males was significantly lower when compared with G3 (100 mg/kg) male animals.
Relative organ weights of kidneys, liver, heart, spleen, ovaries and brain of female animals from group G4 (300 mg/kg) were increased when compared to that of G1 (0 mg/kg), G2 (25 mg/kg) and G3 (100 mg/kg) females. Also relative organ weights of adrenals from G4 (300 mg/kg) females was increased to that of G3 (100 mg/kg) group female animals.
In recovery group animals absolute organ weights of heart in group G4R (300 mg/kg) male animals was significantly lower when compared to G1R (0 mg/kg). Whereas there was no significance observed in absolute organ weights in female animals from recovery groups G1R (0 mg/kg) and G4R (300 mg/kg).
There was a significant higher relative organ weight of kidneys, liver, heart, thymus, spleen, brain in both male and female animals from group G4R (300 mg/kg) when compared to that of G1R (0 mg/kg). In addition to this relative organ weights of adrenals, testes, epididymides in male and ovaries of female animals from group G4R (300 mg/kg) were significantly higher with respect to control group G1R.
The absolute decrease in organ weights of thymus, spleen and seminal vesicle, prostate correlated with microscopic examination in these organs. While the changes observed in other organs weight may be related as secondary to loss in body weight and biological variations.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

There were seven mortalities occurred in the study before scheduled necropsy from group G4 (300 mg/kg) and G4R (300 mg/kg) and the animals found dead were having emaciation, wet mouth and perineum, reddish discharge from both eyes as external findings. Internally there was complete involution of thymus, and increase in size of adrenals. Slight decrease in size of spleen, marked decrease in axillary lymph nodes, slight decrease in mesenteric lymph node size, small sized uterus was observed. In gastrointestinal system raised whitish foci in forestomach of one animal and yellowish white thick content in intestine was observed.
Cannibalism was observed in two animals hence gonads, accessory sex organs, left thigh muscle, urinary bladder in one animal and small intestine in another animals cannot be obtained and processed for histological examination. Also autolytic changes were set in all dead animals pertaining to the time of death.
Necropsy performed at the end of the treatment period of high dose G4 (300 mg/kg) animals revealed emaciation in all male and female animals. Seminal vesicle and prostate of all male animals were moderately small in size, two males were having small sized spleen and mesenteric lymph node, slightly raised whitish foci in forestomach of 2 males was observed in addition one animal was having slightly reddened mucosa of glandular stomach as well. Unilateral hydronephrosis was observed in one male from G4 and G1R each. One female from group G4 had reddish foci in thymus. Following recovery period, gross examination revealed no abnormality in male and female animals of control recovery G1R (0 mg/kg) and no treatment related findings in high dose recovery G4R (300 mg/kg) group.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Animals found dead on day 15, 16, 17 and 19 of study from G4 (300 mg/kg) and G4R (300 mg/kg) were having similar microscopic findings as that of the high dose treated live animals from G4 (300 mg/kg). Stomach was having polymorphonuclear cells infiltrate, acanthosis, hyperplasia of squamous epithelium and necrosis in forestomach, while hemorrhage in glandular stomach. Foamy histiocytosis was observed in small intestines along with atrophy of Payer's patch. In liver microscopically bridging vacuolation, rarefaction and sinusoidal dilatation was observed. Kidneys of all male and female animals had tubular vacuolation and tubular dilatation and basophilic tubules in males only. Foamy histiocytosis was a major microscopic change in lungs along with it adrenals, thymus, mesenteric and axillary lymph nodes and intestines were also having foamy histiocytosis. Adrenals were having vacuolation and/or hemorrhage and/or necrosis. Thymus, mesenteric and axillary lymph nodes were having apoptotic necrosis and atrophy as common change. Spleen, bone marrow of all animals, seminal vesicle prostate of all males and uterus of all females had atrophy as a common microscopic finding.
In group G3 (100 mg/kg) liver of 1 male had vacuolation; in kidney tubular vacuolation in 1 female and tubular dilatation, proteinaceous casts and dilated pelvis in another female; in lungs foamy histiocytosis and mononuclear cell infiltrate of one female was observed. However stomach of only one female animal was having acanthosis and polymorphonuclear infiltrate in forestomach. Rest of the animals from G3 were not having any microscopic finding in their organs.
Gross findings observed in stomach, thymus, spleen, lymph nodes, seminal vesicles, prostate correlated with the relevant microscopic finding in these organs.
No microscopic changes were observed in low dose (25 mg/kg) group animals.
All other histopathological findings observed in various tissues during evaluation of test item group animals were comparable with control group and were considered incidental. These changes observed can usually be considered to be species, age, gender, congenital, physiological or mode of death related and are covered in background historical data of pathology.
All microscopic findings are listed in the “Summary of incidence of microscopic findings” and macroscopic and microscopic findings are presented in the “Text of gross and microscopic findings”.

Key result

Dose descriptor:

NOAEL

Effect level:

25 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

gross pathology

haematology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day (nominal)

System:

respiratory system: lower respiratory tract

Organ:

lungs

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day (nominal)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Conclusions:

HOSTAVIN 3055 was administered once daily by oral route to Wistar rats for a period of 28 days at doses of 25, 100 and 300 mg/kg body weight/day. Test item related changes in the body weights, haematology, biochemistry parameters, organ weight, clinical signs and mortality were observed in (high dose - G4 and G4R). Macroscopic lesions and Microscopic lesions were noted in G4 and G3. These changes and abnormalities are attributable to the treatment at the intermediate (100 mg/kg body weight/day) and highest dose (300 mg/kg body weight/day). Hence, under the conditions of the experiment and based on the findings of the present study entitled “Repeated dose28-Day Oral Toxicity Study of Hostavin 3055 in Wistar Rats” the NOAEL (No Observed Adverse Effect Level) was found to be the lowest dose level employed i.e., 25 mg/kg body weight/day.

Executive summary:

Repeated Dose 28 Days Oral Toxicity Study with Hostavin 3055 in Rats

This study was conducted based on the following guideline

• "Repeated Dose 28-Day Oral Toxicity Study in Rodents", OECD Guidelines for the Testing of Chemicals, Section 4, Health Effects, Number 407, 3^rdOctober, 2008. 

The test item, formulated in Refined groundnut oil was administered once daily for 28-days to Wistar rats at dose levels of 0 (G1), 25 (G2), 100 (G3) and 300 (G4) mg/kg body weight/day. In total 4 groups, each group consisting of five males and five females were used for the study. In addition, recovery groups were included, each consisting of five males and five females were allocated to control (G1R) and high dose recovery group (G4R). 

The following observations were performed: Mortality/ Viability (Twice daily), Ophthalmoscopy (Before start of treatment and towards end of the treatment period), Clinical signs (daily), Body weights, Functional Observational Battery (FOB- during fourth week), feed consumption (once weekly), Haematology, Clinical Biochemistry and Urine Analysis at the end of treatment (after 4 weeks) and recovery (6 weeks).

At the end of the treatment and recovery period all animals were weighed, sacrificed and necropsied. Histological examination was performed on all organs and tissues of control G1 (0 mg/kg) and high dose G4 (300 mg/kg) group and preterminal dead animals as per the study plan and target organs of low dose G2 (25 mg/kg) and intermediate dose G3 (100 mg/kg) groups. Urine samples were collected for analysis and blood was sampled for clinical pathology from all the survived animals.

Statistical analysis was performed using Statplus software to compare the significant difference between treatment and control group of animals (t-test/ANOVA). Data is summarized in tabular form. 

Analysis of homogeneity and dose concentration of the prepared dose formulations revealed acceptable levels.

Clinical signs

No clinical signs of toxicity were observed in control (G1) and low dose group (G2) animals during the study. Burrowing was observed in all animals of G3 and dullness was observed in one male and two female animals. In addition to dullness and burrowing, piloerection, polyuria, mild salivation and chromodacryorrheawas observed in all the male and female animals of high dose group (G4). These clinical signs were observed in recovery high dose group also during treatment period and gradually animals recovered from these signs after the treatment withdrawal. This indicates that these clincal signs may attribute to treatment. The test item triggered autonomic system mostly affected in the high dose animals.

Mortality

Mortality was observed in 1/5 males and 3/5 females in G4 (300 mg/kg); 1/5 males and 2/5 females in Group 4R (300 mg/kg). In the remaining groups, no mortality was observed in (G1, G2 and G3) throughout experimental period. The majority of the deaths observed in Groups (G4) and (G4R) were attributed to test item administration

Functional Observational Battery

No treatment-related neuromuscular and physiological abnormalities were observed in the animals of treated groups (G2 and G3). However, Dullness, burrowing, piloerection, polyuria, mild salivation and chromodacryorrheawas observed in all the male and female animals of high dose (G4) and high dose recovery (G4R). Therefore Functional Observational Batterytest was not performed in High dose groups.

Ophthalmological examination

No treatment-related abnormalities were observed in the ophthalmological examination conducted during acclimatization and at the end of treatment in vehicle control and high dose group.

Body weights and Body weight gain

No treatment-related changes were observed in Body weights and Body weightgain at dose levels oflow dose G2 (25 mg/kg) and intermediate dose G3 (100 mg/kg) groups. However, high dose G4 and high dose recovery animals G4R animals ( 300 mg/kg) showed severely decreased body weight gain and emaciation, This effect could be considered to be related to the test item administration as well as it is likely attributable to the decrease in food consumption.

Food Consumption

No treatment-related changes were observed in food consumptionat dose levels oflow dose G2 (25 mg/kg) and intermediate dose G3 (100 mg/kg) groups. However, the high dose animals (G4 and G4R - 300 mg/kg) showed severely decreased food consumption, this effect could be considered to be related to the test item administration.

Haematology

No toxicologically significant effects were detected in the measured hematological parameters of treatment groups at dose levels of low dose G2 (25 mg/kg) and intermediate dose G3 (100 mg/kg) compared with respective control groups. However, there was a marginal difference observed in Haematology parameters in high dose (G4) and high dose recovery (G4R) (300 mg/kg) as compared to respective control. None of these Haematology findings is directly attributable to administration of the compound.

Biochemistry

No toxicologically significant effects were detected in the measured biochemistry parameters of treatment groups at dose levels of low dose G2 (25 mg/kg) and intermediate dose G3 (100 mg/kg) compared with respective control groups. However, some biochemistry parameters minor differences were observed between the treated high dose groups as compared to respective vehicle control group at 4^thweek and 6^th week respectively. This effect caused by the compound.

Hormone analysis parameters (T3, T4 and TSH)

No toxicologically significant effects were detected in the Hormone analysis parameters (T3,T4 and TSH) of treatment groups compared with respective control groups.

Urinalysis

No treatment-related effect was observed either in the group mean values or in the incidence of semi quantitative observations made across different dose groups.

Organ Weights

The group mean values of the absolute and relative organ weights oflow dose G2 (25 mg/kg) and intermediate dose G3 (100 mg/kg) compared with respective control groupsdid not reveal any treatment-related effect. However, Compound-related weight changes were noted only in the kidney, liver, thymus, spleen, testis, epididymides, prostate and seminical vessel, heart, brain and ovaries of the 300 mg/kg/day animals. These organ weight variations correlated with microscopic findings. Following the recovery period, significant difference was still present in the (G4R) animals.

Macroscopic Findings

There were no treatment related macroscopic findings in treated group of both sexes except G4 and G4R.

Compound-related effects consisted of the following (adverse findings are inbold italics -G4 and G4R):

·        Forestomach: Raised white foci

·        Glandular : stomach reddened

·        Intestine: Yellowish white thick content

·        Thymus: Completely involuted

·        Thymus: Reddish foci

·        Thymus: Small in size

·        Spleen: Small in size

·        spleen: Pale colored

·        Axillary Lymph Node: Small in size

·        Mesenteric Lymph Node: Small in size

·        Adrenals: Enlarged

·        Lungs: Reddened

·        Prostate and seminal vesicle : Small in size

·        Urinar Bladder: Distended

·        Kidney: Hydronephrosis

·        Uterus: Small in size

·        Uterus: Distended with watery content

Microscopic examination

There were no treatment related microscopic findings in low dose (25 mg/kg) group animals. However, The treatment related lesions was observed in Liver, kidneys, adrenals, spleen, thymus, lymph nodes, lungs, stomach, intestines and bone marrow in high dose group (G4) and high dose recovery (G4R) , Whereas intermediate dose (100 mg/kg) Hostavin 3055 produced microscopic changes in liver, kidneys, lungs and stomach.

Changes observed in forestomach viz. polymorphonuclear infiltrate, necrosis, acanthosis and hyperplasia of squamous epithelium, and hemorrhage in glandular stomach may be due to local irritant effect of the test item.

Foamy histiocytosis observed in lungs, adrenals, thymus, spleen, mesenteric/axillary lymph nodes, Payer's patches and intestines; in liver bridging vacuolation, rarefaction and sinusoidal dilatation; in kidneys tubular vacuolation and tubular dilatation and basophilic tubules; while vacuolation, hemorrhage and/or necrosis in adrenals and atrophy of spleen, bone marrow may be attributed to systemic effect of the test item.

Atrophy seminal vesicle, prostate and uterus may be due to secondary to emaciation and decreased body weight of animals.

All other histopathological findings observed in various tissues during evaluation of test item group animals were comparable with control group and were considered incidental. These changes observed can usually be considered to be species, age, gender, congenital, physiological or mode of death related and are covered in background historical data of pathology.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

25 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

reliable without restriction

System:

immune system

Organ:

adrenal glands

kidney

liver

lymph node

mesenteric lymph node

thymus

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

In the absence of any evidence for species specific effects or modes of action the effects observed in animals are regarded as relevant for humans.

Additional information

An OECD 407 study was performed with the test item. Rats were dosed with the substances at doses of 0, 25, 100 and 300 mg/kg bw/d daily for 28 days. Recovery groups were included.

The most distinctive findings were mortality and histiocytosis in small intestines, lungs, adrenals, thymus and mesenteric / axillary lymph nodes at 300 mg/kg bw/d and in lungs at 100 mg/kg bw/d.

Furthermore, vacuolation in liver, adrenals and kidneys were observed at 300 mg/kg bw/d and vacuolation in liver and kidneys at 100 mg/kg bw/d.

Reversible foci in the forestomach were reported for animals dosed with 300 mg/kg bw/d.

The NOAEL was derived at 25 mg/kg bw/d.

Justification for classification or non-classification

Due to the LOAEL of 100 mg/kg bw/day in an OECD 407 repeated dose toxicity study in rats the substance has to be labelled with STOT (RE) Cat. 2 regarding systemic and target organ toxicity after repeated exposure according to the criteria laid down in the Regulation (EC) No 1272/2008.