3,5-bis(2,4-dimethylcyclohex-3-en-1-yl)polyheterocycle

Endpoint summary

Description of key information

REACH_NOAEL = 1000 mg/kg bw/d | rat (male/female) | OECD 407 | #key study#

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

repeated dose toxicity: oral, other

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-02-28 to 2019-03-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

see below "Any other information on results"

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

yes

Remarks:

see below "Any other information on results"

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Preparation of the Animals
Prior to the start of the treatment period a detailed clinical observation outside the home cage was made. Only healthy animals were used for this study.
Before the first administration all animals used for the study were weighed and assigned to the experimental groups with achieving a most homogenous variation
in body weight throughout the groups of males and females, respectively (randomisation was performed with IDBS Workbook 10.1.2 software).

Test System
Species/strain: healthy Wistar rats, Crl: WI(Han) (Full Barrier)
Source: Charles River, 97633 Sulzfeld, Germany
Sex: male and female; the female animals were non-pregnant and nulliparous.
Age at the start of the treatment period: approximately 7-8 weeks old
Body weight at the allocation of the animals to the experimental groups:
males: 158 - 193 g (mean: 176.35 g, ± 20 % = 141.08 – 211.62 g)
females: 134 - 171 g (mean: 150.35 g, ± 20 % = 120.28 – 180.42 g)

The animals were derived from a controlled full-barrier maintained breeding system (SPF). According to the German Act on Animal Welfare the animals were bred for experimental purposes.
This study was performed in an AAALAC-accredited laboratory. According to German animal protection law, the study type has been reviewed and accepted by local authorities.
Furthermore, the study has been subjected to Ethical Review Process and was authorised by the Bavarian animal welfare administration.

Housing and Feeding Conditions
- Full barrier in an air-conditioned room
- Temperature: 22 ± 3 °C
- Relative humidity: 55 ± 10 %
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice
- Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- The animals were kept in groups of 5 animals / sex / group / cage in IVC cages (type IV, polysulphone cages) on Altromin saw fibre bedding
- Certificates of food, water and bedding are filed for two years at BSL Munich and afterwards archived at Eurofins Munich
- Adequate acclimatisation period (at least 5 days) under laboratory conditions

Route of administration:

oral: gavage

Vehicle:

corn oil

Remarks:

Manufacturer: Sigma-Aldrich Batch No.: MKCC0462 / MKCC9871 Physical State: liquid Storage Conditions: room temperature Expiry Date: 15 May 2018 (MKCC0462), 23 May 2018 (MKCC9871)

Details on oral exposure:

Experimental Groups and Doses
According to the results of a previous dose range finding study (BSL Munich Study No. 178329, non GLP) and in consultation with the sponsor the following doses
were selected for the 3 dose groups (LD = low dose, MD = medium dose, HD = high dose).

C 0 mg/kg bw
LD 100 mg/kg bw
MD 300 mg/kg bw
HD 1000 mg/kg bw

The highest dose level was chosen with the aim of inducing toxic effects, but no death or severe suffering.
Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dose related response and NOAEL.
The animals in the control group were handled in an identical manner to the test group subjects and received the vehicle using the same volume as used for the high dose group.


Administration of Doses
The test item formulation or vehicle was administered at a single dose to the animals by oral gavage. The application volume for all groups was 4 mL/kg body weight.
For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Before beginning of the treatment period, formulation samples were prepared and analysed in order to obtain knowledge about stability
and homogeneity of the test item in the selected vehicle at Eurofins Munich as part of a separate GLP study (Eurofins Munich Study No. 178332).
Prestart homogeneity investigation was included on the samples collected from various levels (top, middle and bottom) of high dose and low dose groups.
As the test item was shown to be homogenous according to Eurofins Study No. 178332 (after 30 min without stirring), samples were not collected
during the study for the investigation of homogeneity and only samples were taken for substance concentration in study week 1 and 3 of the
treatment for all doses (8 samples in total).
Each sample taken during the study was retained in duplicate (sample A, sample B, each of at least 5 mL).
The A-samples were analysed at Eurofins Munich (Eurofins Munich Study Phase No. 178333) and until then stored under appropriate conditions
based on available stability data. The B-samples will be retained at -15 to -35 °C at BSL Munich (test facility) and discarded after completion
of the final study report.

Duration of treatment / exposure:

The animals were treated with the test article or vehicle for a period of 28 days.

Frequency of treatment:

daily

Dose / conc.:

0 other: mg/kg bw/day

Dose / conc.:

100 other: mg/kg bw/day

Dose / conc.:

300 other: mg/kg bw/day

Dose / conc.:

1 000 other: mg/kg bw/day

No. of animals per sex per dose:

40 animals (20 males and 20 females) were included in the study (5 male and 5 female animals per group).

Control animals:

yes, concurrent vehicle

Details on study design:

- Rationale for animal assignment: random
- Section schedule rationale: alle male animals together and all female animals together

Observations and examinations performed and frequency:

Body Weight and Food Consumption
The body weight was recorded once before the assignment to the experimental groups, on the first day of administration and weekly during the treatment period.
Food consumption was measured weekly during the treatment period.

Clinical Observations
All animals were observed for clinical signs during the entire treatment period of 28 days.
General clinical observations were made at least once a day, preferably at the same time each day and considering the peak period of anticipated effects after dosing.
The health condition of the animals was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.
Detailed cage side observations considering spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in skin and fur,
eyes and mucous membranes (salivation, discharge), piloerection and pupil size were made outside the home cage in a standard arena once before the first administration and at least once a week thereafter.
Ophthalmological examinations, using an ophthalmoscope was made before the first administration and in the last week of the treatment period.
These examinations were conducted in all animals with exception of animals in the female MD and HD group in week 4.

 
Functional Observations
Once before the first exposure and once in the fourth week of exposure multiple detailed behavioural observations were made outside the home cage using a functional observational battery of tests.
These tests were conducted in all animals with exception of animals in the female MD and HD group in week 4.

Sacrifice and pathology:

Haematology
Haematological parameters were examined at the end of the treatment prior to or as part of the sacrifice of the animals.
After overnight fasting, blood from the abdominal aorta of the animals was collected in EDTA-coated tubes.
The following haematological parameters were examined: haematocrit value, haemoglobin content, red blood cell count, mean corpuscular volume, mean corpuscular haemoglobin,
mean corpuscular haemoglobin concentration, reticulocytes, platelet count, white blood cells, neutrophils, lymphocytes, monocytes, eosinophils, basophils and large unstained cells.

Blood Coagulation
Coagulation parameters were examined at the end of the treatment prior to or as part of the sacrifice of the animals.
After overnight fasting, blood from the abdominal aorta of the animals was collected in citrate tubes.
The following coagulation parameters were examined: prothrombin time and activated partial thromboplastin time.

Clinical Biochemistry
Parameters of clinical biochemistry were examined at the end of the treatment prior to or as part of the sacrifice of the animals.
After overnight fasting, blood from the abdominal aorta of the animals was collected in serum separator tubes.
The following parameters of clinical biochemistry were examined: alanine aminotransferase, aspartate-aminotransferase, alkaline phosphatase, creatinine, total protein, albumin, urea,
total bilirubin, total bile acids, total cholesterol, glucose, sodium and potassium.
Additionally, at necropsy serum samples of all animals were retained at the end of the treatment period and stored at -20° C for a possible evaluation of test item-related
effects on the pituitary-thyroid axis and thyroid hormones. The samples will be discarded at finalisation of the study report.

Urinalysis
A urinalysis was performed with samples collected from all animals prior to or as part of the sacrifice of the animals.
The following parameters were measured using qualitative indicators (Henry Schein Urine Stripes URI 10SL): specific gravity, nitritwe, pH-value, protein, glucose,
ketone bodies, urobilinogen, bilirubin, erythroctes and leukocytes.Additionally, urine colour/ appearance were recorded. 


Pathology
One day after the last administration (study day 29) all surviving animals of the treatment period were sacrificed using anesthesia (ketamine and xylazine) and subjected to a
detailed gross necropsy which includes careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.

Organ Weight
The wet weight of the following organs of all sacrificed animals was recorded as soon as possible: liver, uterus with cervix, kidneys, thymus, adrenals, thyroid/ parathyroid glands, testes, spleen, epididymides, brain, prostate, seminal vesicles and coagulating glands, pituitary gland, ovaries and heart. Paired organs were weighed together.
The following tissues from all animals were preserved in 4 % neutral-buffered formaldehyde except eyes, testes and epididymides which were fixed in Modified Davidson’s fixative for approximately 24 hours before they were transferred to 70 % ethanol:
adrenal glands, all gross lesions, aorta, brain (incl. medulla/pons, cerebellar and cerebral cortex), caecum, colon, duodenum, epididymides, eyes with optic nerve and Harderian gland, femur with knee joint,
heart, ileum (including Peyer´s patches), jejunum, kidneys, liver, lungs, lymph nodes (mandibular), lymph nodes (mesenteric and axillary), mammary gland area (male and female), oesophagus, ovaries,
oviducts, pancreas, parathyroid glands, pituitary, prostate and seminal vesicles with coagulating glands as a whole, rectum, salivary glands (sublingual, submandibular), sciatic nerve, skeletal muscle,
skin, spinal cord (cervical, thoracic and lumbar segments), spleen, sternum (with bone marrow), stomach, testes, thymus, thyroid gland, tongue, trachea, ureters, urinary bladder, uterus with cervix and vagina

Histopathology
The afore-listed organs (Table 8) were examined histopathologically after preparation of paraffin sections and haematoxylin-eosin staining for the animals of the groups 1 and 4 sacrificed at the end of the treatment period and any animal found dead or euthanised before the planned day of sacrifice:
adrenal glands, all gross lesions, aorta, brain (incl. medulla/pons, cerebellar and cerebral cortex), caecum, colon, duodenum, epididymides, eyes with optic nerve and Harderian gland, femur with knee joint,
heart, ileum (including Peyer´s patches), jejunum, kidneys, liver, lungs, lymph nodes (mandibular), lymph nodes (mesenteric and axillary), mammary gland area (male and female), oesophagus, ovaries,
oviducts, pancreas, parathyroid glands, pituitary, prostate and seminal vesicles with coagulating glands as a whole, rectum, salivary glands (sublingual, submandibular), sciatic nerve, skeletal muscle,
skin, spinal cord (cervical, thoracic and lumbar segments), spleen, sternum (with bone marrow), stomach, testes, thymus, thyroid gland, tongue, trachea, ureters, urinary bladder, uterus with cervix and vagina

These examinations were not extended to animals of all other dosage groups for treatment-related changes that were observed in the high dose group.
Any gross lesion macroscopically identified was examined microscopically in all animals. Discoloration possibly due to the test item was evaluated in the organs of all dose groups.

The histological processing of tissues to microscope slides was performed at the GLP-certified contract laboratory AnaPath Services, Hammerstrasse 49, 4410 Liestal, Switzerland (test site for tissue processing). The histopathological evaluation was performed at the GLP-certified contract laboratory AnaPath GmbH, Buchsweg 56, 4625 Oberbuchsiten, Switzerland (test site for histopathology).
The study phase from test site 1 and 2 was performed in compliance with the Swiss Ordinance relating to Good Laboratory Practice adopted 18 May 2005 [SR 813.112.1]
(Status as of 01 December 2012. Blocking, embedding, cutting, H&E staining and scientific slide evaluation were performed according to the corresponding SOP’s of the test sites.
The principal investigator for histopathological tissue processing sent all raw data (including blocks, slides, paper raw data, statement of compliance and quality assurance statement) to the study director.
The principal investigator for histopathological evaluation provided the histopathology results to the study director by e-mail and sent a pathology phase report to the study director upon the completion of the study.

Statistics:

A statistical assessment of the results of the body weight, food consumption, parameters of haematology, blood coagulation and clinical biochemistry
and absolute and relative organ weights were performed for each gender by comparing values of dosed with control animals using either a parametric
one-way ANOVA and a post-hoc Dunnett Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity
and normality tests. These statistics were performed with Ascentos 1.1.3 software or GraphPad Prism V.6.01 software (p<0.05 is considered
as statistically significant).

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

see Details on results

Mortality:

no mortality observed

Description (incidence):

see Details on results

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

see Details on results

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

see Details on results

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

see Details on results

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

see Details on results

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

see Details on results

Endocrine findings:

not examined

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

see Details on results

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

see Details on results

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

see Details on results

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

see Details on results

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

see Details on results

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

see Details on results

Other effects:

not examined

Details on results:

Mortality
No mortality occurred during the treatment period of this study in all groups.

Clinical Observations
No clinical findings were observed in both control and LD groups during the treatment period.
Moving the bedding was seen in all males and females on several days during the observation period for MD and on most days for the HD group.
Additionally, slight to moderate salivation was recorded for nearly all male and female animals in the MD and HD group on several days.
Moving the bedding or salivation were considered to be a sign of a local reaction to the test item rather than a systemic adverse effect and has
no toxicological relevance. There were no clinical signs of toxicity in this study. No abnormalities were seen during weekly detailed clinical observations.
Statistical significance for decreased mean values of animal sleeps and increase for animal moving in cage for all male dose groups in one single week
(week 1) when compared to control is not considered to be of biological or toxicological relevance.

Functional Observation Battery
In males and females, no relevant effects were observed in any of the parameters of the functional observation battery before and
at the end of the treatment period when compared with the controls.
There were no biologically relevant differences in body temperature between the groups.
Statistically significant mean group values in all female dose groups for urination in the first week before treatment is not considered to
be biologically relevant. As this finding was seen before the first application a relation to test item treatment can be excluded.

Body Weight Development
There was no effect on body weight development in this study. The statistical significance for a decreased mean body weight gain of 27%
compared to control in the male HD group during the first week of treatment is not considered to be of toxicological relevance.
All animals showed weight gain between study day 1 and day 28 and no dose dependency was found.
Furthermore, there were no considerable differences in body weights between dose and control groups.
 
Food Consumption
The test item had no effect on food consumption in this study. Mean food consumption calculated from day 1 to day 28
showed a slight tendency to decrease in male and female dose groups compared to control but no dose dependency occurred.
During the overall treatment period no further considerable differences in food intake were observed between dose group and control group.

Haematology and Blood Coagulation
The test item had no effect on haematological parameters and coagulation parameters determined at the end of the treatment period.
No statistical significances were found for haematological and coagulation parameters in all male and female groups with exception of a severely
and statistical significant increased mean value for eosinophils in the female MD group, which was seen without dose dependency.
The increase is based on high individual values of 4 MD females compared to control. In the male HD group eosinophiles was moderately
but not statistically significantly increased when compared to control. No dose dependency was seen. Therefore, the statistical significance
for eosinophils is considered to be without toxicological relevance and considered to be incidental.

Clinical Biochemistry
The test item had no effect on parameters of clinical biochemistry determined at the end of the treatment period.
In the male groups statistical significance was found for slightly decreased mean values for parameters Crea in the MD and HD group,
for ASAT in the LD and MD group, for Chol in the HD group and Urea in the MD and HD group. Glucose was slightly and statistically significant
decreased for female MD and HD group animals when compared to control and a moderate statistical significant increase without dose
dependency was seen for TBA in female HD group.
All mean values for these parameters were within the range of historical control data for males and females and as there were no
histopathological findings that correlate to statistical significant parameters, a toxicological relevance is not considered.

Urinalysis
At the end of the treatment period no conspicuous differences were noted in urinary parameters between dose groups and control group of this study.
Positive nitrite measurement in the male HD group is considered to be of no toxicological relevance as in all female dose groups and in the control
group nitrite was found at urinalysis.
 
Pathology
At necropsy, the thymus was enlarged for animal no. 7 (male LD), for animal no. 20 (male HD) the thymus was brown coloured and axillary
and mesenterial lymph nodes were additionally coloured red in animal no. 20
Black foci were seen at the lung of male control animal no. 5 and rec foci on thymus of control animal no. 4.
Urinary bladder was red coloured in control male no. 2.
In females, thymus showed red foci in MD group animal no. 35 and uterus was seen dilated in one LD (animal no. 27) and MD animal (animal no. 33).
Considering the results at histopathological evaluation for the observed gross lesions, no relation to the treatment with test item can be concluded.

Organ Weight
Absolute mean brain weight was slightly and statistically significant increased in male MD group. As no dose dependency was seen and
histopathological evaluation showed no findings in the examined animals a relation to test item treatment is not considered.
In the male and female HD group mean relative weight to body weight was slightly statistically significant increased (males: 19.06% above control,
females: 30.06% above control) for liver. No findings were found at histopathological assessment and the significance is therefore considered
to be not test item related.
Mean relative organ weight to body weight for kidney was statistically significant and slightly increased in male MD (13.61 % above control)
and HD (16.67 % above control). No statistical significance was seen in female animals. Histopathologically in kidneys in kidneys,
a minimal increase of hyaline inclusions in tubular epithelia in male HD animals was found. This finding was not associated with any further lesions.

Histopathology
In kidneys, there was a minimal increase of hyaline inclusions in tubular epithelia in males at 1000 mg/kg.
The finding was not associated with any further lesions. Histopathologically, it is considered to represent the rat male specific deposition
of α2-microglobulin. The alteration was not associated with a further adverse lesion in the kidneys.
There was no finding that distinguished controls from test item-treated animals.
Therefore, based on the pathology evaluation, the NOAEL may be established at 1000 mg/kg bw/day.

Dose Formulation Analysis
Concentration analysis of formulation samples was determined at three concentrations, 25 mg/mL, 75 mg/mL and 250 mg/mL in study weeks 1 and 3.
The mean recoveries observed for the LD dose group was 95.5 % and 91.2 % of the nominal value, 90.9 % and 90.4 % for the MD dose group and 91.2% and 101.7 % of the nominal value for HD dose group.
The mean recoveries observed in the low dose (LD), medium dose (MD) and high dose (HD) groups were 93.4 %, 90.6 %, and 96.4 % of the nominal concentration, respectively.
Nominal concentrations were confirmed for all dose groups, as measured concentrations were within acceptance criterion of 10 %.

 

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: Under the conditions of this study, the test item Sa 57 did not cause indicators of toxicity. The NOAEL may be established at 1000 mg/kg bw/day.

Key result

Critical effects observed:

no

Dose Formulation Analysis
Concentration analysis of formulation samples was determined at three concentrations, 25 mg/mL, 75 mg/mL and 250 mg/mL in study weeks 1 and 3. The mean recoveries

observed for the LD dose group was 95.5 % and 91.2 % of the nominal value, 90.9 % and 90.4 % for the MD dose group and 91.2% and 101.7 % of the nominal value for HD

dose group. The mean recoveries observed in the low dose (LD), medium dose (MD) and high dose (HD) groups were 93.4 %, 90.6 %, and 96.4 % of the nominal

concentration, respectively. Nominal concentrations were confirmed for all dose groups, as measured concentrations were within acceptance criterion of 10 %.

Conclusions:

On the basis of this 28-Day Repeated Dose Oral Toxicity study with Sa 57 in male and female Wistar rats with dose levels of 100, 300, and 1000 mg/kg body weight/day, the following conclusions can be made:
Under the conditions of this study, the test item Sa 57 did not cause indicators of toxicity. The NOAEL may be established at >= 1000 mg/kg bw/day.

Executive summary:

The aim of this study was to assess the possible health hazards which could arise from repeated exposure of Sa 57 via oral administration to rats over a period of 28 days.

The test item was administered daily in graduated doses to 3 groups of test animals, one dose level per group for a treatment period of 28 days.

Animals of an additional control group were handled identically as the dose groups but received corn oil, the vehicle used in this study.

The 4 groups comprised of 5 male and 5 female Wistar rats.

The following doses were evaluated:

Control:             0          mg/kg body weight

Low Dose:        100     mg/kg body weight

Medium Dose:  300    mg/kg body weight

High Dose:        1000 mg/kg body weight

 

The test item formulation was prepared at least every 10 days. The test item was emulsified in corn oil and administered daily during a 28-day treatment period to male and female animals.

Dose volumes were adjusted individually based on weekly body weight measurements.

During the period of administration, the animals were observed precisely each day for signs of toxicity. At the conclusion of the test, surviving animals were sacrificed and observed macroscopically.

Body weight and food consumption were measured weekly.

At the conclusion of the treatment period, all animals were sacrificed and subjected to necropsy. The wet weight of a subset of tissues was taken and a set of organs/tissues was preserved.

A full histopathological evaluation of the tissues was performed on high dose and control animals.Any gross lesion macroscopically identified was examined microscopically in all animals.

 

Summary Results

No mortality occurred during the treatment period of this study in all groups.

No clinical findings were observed in both control and LD groups during the treatment period. Moving the bedding was seen in all males and females on several days during the observation period for MD and on most days for the HD group. Additionally, slight to moderate salivation was recorded for nearly all male and female animals in the MD and HD group on several days. Moving the bedding or salivation were considered to be a sign of a local reaction to the test item rather than a systemic adverse effect and has no toxicological relevance. There were no clinical signs of toxicity in this study. No abnormalities were seen during weekly detailed clinical observations.

In males and females, no relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period when compared with the controls. There were no biologically relevant differences in body temperature between the groups.

There was no effect on body weight development in this study. The statistical significance for a decreased mean body weight gain of 27 % compared to control in the male HD group during the first week of treatment is not considered to be of toxicological relevance. All animals showed weight gain between study day 1 and day 28 and no dose dependency was found.

The test item had no effect on food consumption in this study. During the overall treatment period no relevant differences in food intake were observed between dose group and control group.

The test item had no effect on haematological parameters and coagulation parameters determined at the end of the treatment period. Severely and statistical significant increased mean value foreosinophilsin the female MD group, which was seen without dose dependency is considered to be without toxicological relevance and considered to be incidental.

The test item had no effect on parameters of clinical biochemistry determined at the end of the treatment period. In the male groups statistical significance was found for slightly decreased mean values for parameters Crea in the MD and HD group, for ASAT in the LD and MD group, for Chol in the HD group and Urea in the MD and HD group. Glucose was slightly and statistically significant decreased for female MD and HD group animals when compared to control and a moderate statistical significant increase without dose dependency was seen for TBA in female HD group. All mean values for these parameters were within the range of historical control data for males and females and as there were no histopathological findings that correlate to statistical significant parameters, a toxicological relevance is not considered.

At the end of the treatment period no conspicuous differences were noted in urinary parameters between dose groups and control group of this study.

At necropsy, the thymus was enlarged for animal no. 7 (male LD), for animal no. 20 (male HD) the thymus was brown coloured and axillary and mesenterial lymph nodes were additionally coloured red in animal no. 20. In females, thymus showed red foci in MD group animal no. 35 and uterus was seen dilated in one LD (animal no. 27) and MD animal (animal no. 33). Considering the results at histopathological evaluation for the observed gross lesions, no relation to the treatment with test item can be concluded. 

In the male and female HD group mean relative weight to body weight was slightly statistically significant increased (males: 19.06 % above control, females: 30.06 % above control) for liver.

No findings were found at histopathological assessment and the significance is therefore considered to be not test item related.

Absolute mean brain weight was slightly and statistically significant increased in male MD group. As no dose dependency was seen and histopathological evaluation showed no findings in the examined animals a relation to test item treatment is not considered.

Mean relative organ weight to body weight for kidney was statistically significant and slightly increased in male MD (13.61 % above control) and HD (16.67 % above control). No statistical significance was seen in female animals. Histopathologically, in kidneys, there was a minimal increase of hyaline inclusions in tubular epithelia in males at 1000 mg/kg.The finding was not associated with any further lesions.Histopathologically, it is considered to represent the rat male specific deposition ofα2-microglobulin. The alteration was not associated with a further adverse lesion in the kidneys. There was no finding that distinguished controls from test item-treated animals. Therefore, based on the pathology evaluation, the NOAEL may be established at >= 1000 mg/kg bw/day.

Concentration analysis of formulation samples confirmed nominal concentrations for all dose groups, as measured concentrations were within acceptance criterion of 10 %.

 

Conclusion

On the basis of this 28-Day Repeated Dose Oral Toxicity study with Sa 57 in male and femaleWistarrats with dose levels of 100, 300, and 1000 mg/kgbody weight/day,

the following conclusions can be made:

Under the conditions of this study, the test item Sa 57 did not cause indicators of toxicity. The NOAEL may be established at 1000 mg/kg bw/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

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

Justification for classification or non-classification

Since no substance related adverse effects were observed at the highest dosage group, the substance does not need to be classified for repeated dose toxicity according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.