Sodium 4-[4-[[2-methyl-4-[[(p-tolyl)sulphonyl]oxy]phenyl]azo]anilino]-3-nitrobenzenesulphonate

Endpoint summary

Description of key information

NOAEL (55d) (males and females): 1000 mg/kg bw/day.

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:

From January 11th to March 23rd, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 29 July 2016

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The rat is regarded as suitable species for reproduction studies and the test guideline is designed to use the rat. The Wistar rat was selected due to large experience with this strain of rat in reproduction toxicity studies and known fertility.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Toxi-Coop ZRT
- Females: nulliparous, non-pregnant females.
- Age at study initiation: males 90 – 92 days; females 99 – 102 days.
- Weight at study initiation: males 353 – 419 g ; females 212 – 248 g. The weight variation did not exceed ± 20 per cent of the mean weight.
- Fasting period before study:
- Housing: 2 animals of the same sex/cage before mating; 1 male and 1 female/cage during mating. Pregnant females were housed individually, while males were housed 2 animals/cage after mating. Recovery animals: 2 or 3 animals of the same sex/cage.
- Cages: type III polypropylene/polycarbonate; size of 22 x 32 x 19 cm (width x length x height).
- Diet: ad libitum, ssniff® SM R/M-Z+H complete diet for rats and mice. Food was changed at weekly intervals.
- Water: ad libitum, tap water. Fresh drinking water was given daily.
- Health: animals were SPF (Specific pathogen-free) at arrival and kept in good conventional environment during the study. Animals that failed to exhibit typical 4-5 days cycles were not included in the study if it was feasible.
- Acclimation period: 34 days.

DETAILS OF FOOD AND WATER QUALITY
The food was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. The supplier provided an analytical certificate of the standard diet for the batch used.
Animals received tap water from watering bottles. Water quality control analysis and microbiological assessment are performed once in every six months by Government Office of Capital Budapest Department of Public Health and Medical Officer Service.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30 - 70 %
- Air changes: above 10 air-exchanges/ hour by a central air-condition system.
- Photoperiod: artificial light, from 6 a.m. to 6 p.m.

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

distilled

Details on oral exposure:

- Doses: 137, 411 and 1370 mg/kg bw/day, corresponding to 100, 300 and 1000 mg/kg bw/day, calculated by the active ingredient content.
- Nominal concentration of formulation: 13.70, 41.10 and 136.99 mg/ml, corresponding to 10, 30 and 100 mg/ml, calculated by the active ingredient content.
- Dosing volume: 10 ml/kg bw.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of the test item in the dosing formulations administered to the animals was checked two times during the study. the substance concentrations in the dosing formulations varied in the range between 101 % and 106 % of the nominal values and confirming the proper preparation of the dosing formulations.

Duration of treatment / exposure:

Pre-mating period: 14 days, males and females
Mating period: 1-8 days and 23 days for one female
Post-mating males: 32-40 days
Gestation period: 21-23 days
Lactation period: 13-18 days

Frequency of treatment:

once daily

Dose / conc.:

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

Remarks:

calculated by the active ingredient content

Dose / conc.:

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

Remarks:

calculated by the active ingredient content

Dose / conc.:

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

Remarks:

calculated by the active ingredient content

No. of animals per sex per dose:

12 x sex x dose group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: dose levels were chosen on the basis of the results of a preliminary toxicity screening test conducted on rats, dosed for 14-day oral (by gavage) at 100, 300 or 1000 mg/kg bw/day.

Observations and examinations performed and frequency:

MORTALITY
Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each working day).

DETAILED CLINICAL OBSERVATIONS
General clinical observations were made on parental animals once a day, after the administration at approximately the same time. Detailed examinations were made at the times of weekly weighing, prior to and during the mating and until necropsy.
Observations were performed on the skin, fur, eyes and mucous membranes, autonomic activity (lachrymation, piloerection, pupil size, respiratory pattern, occurrence of secretions and excretions), circulatory and central nervous system, somatomotor activity and behavior pattern, changes in gait, posture and response to handling. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhea, lethargy, sleep and coma.

BODY WEIGHT
All parental animals were weighed with an accuracy of 1 g. Parental males were weighed on the first day of dosing (Day 0) and weekly thereafter and on the day of the necropsy.
Parental females were weighed on the first day of dosing (Day 0) then weekly, on gestation days 0, 7, 14 and 21 and on days 0 (within 24 hours after parturition), 4 and 13 post-partum. Body weight of the female animals was additionally weighed on gestational day 10 in order to give accurate treatment volumes, but these data were not valuated statistically. Body weight was measured on day of necropsy for female animals subjected to organ weighing (selected for further examinations).
Body weight data were reported individually for adult animals. Individual body weight change was calculated.

FOOD CONSUMPTION
The food consumption was determined weekly by reweighing the non-consumed diet with a precision of 1 g during the treatment period except mating phase (pre-mating days 7, 13, and post-mating days 20, 27, 34, 41, 48 and 53 for male animals, pre-mating days 7, 13, gestation days 0, 7, 14 and 21, lactation days 0, 4 and 13 for female animals).

CLINICAL PATHOLOGY
Clinical pathology examinations including hematology and clinical chemistry were conducted in five male and five female animals randomly selected from each group one day after the last treatment (i.e. on the day of necropsy).
Animals were food deprived for approximately 16 hours (overnight) prior to blood collection. Blood samples were harvested from the retro-orbital venous plexus under Isofluran anesthesia. Three samples were taken from each animal: one for hematology, one for determination of blood clotting times and the third one to obtain serum samples for clinical chemistry.
In addition, blood samples were collected for possible determination of serum levels of thyroid hormones (T4) from all parent male animals at termination on Day 54 or 55. All samples for T4 determination are stored at approximately -20 °C.

HEMATOLOGY
Blood samples for hematology measurements were collected in tubes containing K3EDTA (spray-dried) and tubes were filled up to the final volume (marked on the tubes). Blood were stored at 2-8 °C until analysis (not longer than for 24 hours) by SYSMEX XT-2000iV.
The following parameters were measured in all selected animals and in recovery animals: White Blood Cell (leukocyte) count (WBC), Red Blood Cell (erythrocyte) count (RBC), Hemoglobin concentration (HGB), Hematocrit (relative volume of erythrocytes - HCT), Mean Corpuscular (erythrocyte) Volume (MCV), Mean Corpuscular (erythrocyte) Hemoglobin (MCH), Mean Corpuscular (erythrocyte) Hemoglobin Concentration (MCHC), Platelet (thrombocyte) count (PLT), Reticulocytes (RET), Differential white blood cell count (i.e. percentage of neutrophil, lymphocyte, eosinophil, monocyte, basophil.

BLOOD COAGULATION
Blood samples for determination of blood clotting times (APTT and PT) were collected in tubes containing 9NC Coagulation 3.8 %. Tubes were filled up to the final volume (marked on the tubes). Blood were centrifuged at 2500 rpm for 15 minutes within 20 – 30 minutes after the sampling. Supernatant plasma samples were stored at 2-8 °C and measured.
The following blood coagulation parameters were determined in selected animals and in recovery animals: Activated partial Thromboplastin Time (APTT), Prothrombin Time (PT).

CLINICAL CHEMISTRY
Blood samples collected for clinical chemistry measurements were drawn in tubes Vacuette 2.5 ml. At least 1.0 ml blood was collected into clinical chemistry tubes. Samples were stored in a dark place at room temperature for 30-40 minutes and then centrifuged at 4000 rpm for 15 minutes. Serum samples were stored at 2-8 °C and measured. The following parameters were measured in all selected animals and in recovery animals:
Alanine Aminotransferase activity (ALT), Aspartate Aminotransferase activity (AST), Total Bilirubin concentration (TBIL), Creatinine concentration (CREA), Urea concentration (UREA), Glucose concentration (GLUC), Cholesterol concentration (CHOL), Bile acids (BAC), Sodium concentration (Na+), Potassium concentration (K+), Albumin concentration (ALB), Total Protein concentration (TPROT).

Sacrifice and pathology:

SACRIFICE
One male animal at 100 mg/kg bw/day was found dead and subjected to necropsy on Day 55. All other animals were euthanized by exsanguination after verification of deep narcosis by Isofluran CP® and were subjected to gross necropsy as follows: parental male animals: after the optionally extended post-mating period on Days 54 or 55; non-pregnant females on Day 54.

GROSS NECROPSY
Gross necropsy was performed on each animal.
After examination of the external appearance, the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed, and any abnormality was recorded including details of the location, color, shape and size.
At the time of termination, body weight, brain weight, weight of the testes, epididymides and prostate and seminal vesicles with coagulating glands as a whole of all male adult animals were determined. Absolute organ weight was recorded. Relative organ weight (to body and brain weight) were calculated and reported.
In addition, for five males and females randomly selected from each group, adrenals, brain, heart, kidneys, liver, spleen and thymus were weighed.
The thyroid weight will be determined – if relevant – after fixation.
Paired organs were weighed together.

HISTOPATHOLOGY
Detailed histological examination was performed on the ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland in all animals of control and high dose groups with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure and on the ovaries covering the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.
Histological examination was also performed on the ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland in not mated or non-pregnant females and males these females cohabited with in the low and middle dose groups
Full histopathology examinations were performed on the preserved organs and tissues of the randomly selected animals in the control and high dose (1000 mg/kg bw/day).
The fixed tissues were trimmed, processed, embedded in paraffin, sectioned with a microtome, placed on glass microscope slides, stained with hematoxylin and eosin and examined by light microscopy.

Statistics:

The statistical evaluation of appropriate data was performed with the statistical program package SPSS PC+4.0. The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test.
Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) is carried out. If the obtained result was significant Duncan Multiple Range test was used to access the significance of inter-group differences. Getting significant result at Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed if feasible.
For evaluation of data obtained during the recovery period, the homogeneity of variance between groups was checked by F-test. Depending on the result pooled or separate variance estimate of the Two-Sample t-test was performed.
Frequency of toxic response, pathological and histopathological findings by sex and dose was calculated.

Clinical signs:

no effects observed

Description (incidence and severity):

Clinical signs of systemic toxicity related to the test item were not detected at any dose level, neither at the daily nor at the detailed weekly clinical observations.
There were no clinical signs in any group, i.e. the parental animals (male and female) exhibited normal behavior and physical condition with no abnormalities in the control and at 100, 300 or 1000 mg/kg bw/day.
Yellowish color of stool was observed in the bedding material of animals administered with 1000 mg/kg bw/day (12/12 male and 12/12 female) from Day 6 up to the termination of the study. Yellowish color of the faeces was due to the elimination of test item or its metabolite by the gastrointestinal tract.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One male animal (No. 206) at 300 mg/kg bw/day was found dead on Day 55. There were no clinical signs but significant body weight loss was detected during the preceding week. Necropsy observation revealed yellowish fluid in the thoracic cavity and empty intestines indicative of para-gastric treatment (damage of the esophagus).
There was no test item related mortality at 300 or 1000 mg/kg bw/day groups during the course of study (male and female).

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The body weight development was not adversely affected by test item in survival male or female animals at 100, 300 or 1000 mg/kg bw/day during the entire treatment period.
The mean body weight was similar in the control and at 100, 300 and 1000 mg/kg bw/day groups in male animals. Only in the one male animal (No. 206) died after necropsy and dosed at 300 mg/kg bw/day, significant body weight loss was detected during the preceding week.
Although the mean body weight gain was slightly reduced in male animals at 1000 mg/kg bw/day (Days 0-7 and Days 20-27, and for study overall), this slight change did not result in significant changes in the mean body weight of this groups during the treatment period.
The mean body weight was similar in the control and at 100, 300 and 1000 mg/kg bw/day groups in male animals.
Statistical significance with respect to the control was detected in male animals at 300 mg/kg bw/day at the slightly lower mean body weight gain of on Days 20-27 and at the slightly higher mean body weight gain between Days 34-41 and 41 and 48.
The mean body weight gain was lower than in the control group in female animals at 100 and 1000 mg/kg bw/day during week 1 (between Days 0 and 7) of the pre-mating period. These slight changes were not related to doses and did not cause significant alterations in the mean body weight therefore were judged to be toxicologically not significant.
In the female animals, the body weight development was undisturbed during the gestation and lactation periods. There were no significant differences between the control and test item treated groups in the mean body weight or mean body weight gain (100, 300 and 1000 mg/kg bw/day).

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no test item or treatment related changes in the mean daily food consumption of male animals at 100, 300 or 1000 mg/kg bw/day.
Statistically or biologically significant differences were not seen in the mean daily food consumption of male or female animals at any dose level (100, 300 or 1000 mg/kg bw/day) with respect to their control group during the entire observation period (pre-mating and post mating periods in male animals; pre-mating, gestation and lactation periods in female animals).

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Hematological evaluation did not reveal adverse test item related changes in the examined parameters at 100, 300 or 1000 mg/kg bw/day.
Slight but statistically significant differences with respect to their controls were noted for some parameters as follows: lower mean corpuscular hemoglobin concentration (MCHC) at 1000 mg/kg bw/day and lower mean platelet count (PLT) at 300 mg/kg bw/day, both in male animals; higher mean percentage of eosinophil granulocytes (EOS) in female animals at 300 mg/kg bw/day.
These differences with respect to control were judged to be toxicologically not relevant due to the minor degree (PLT, EOS and MCHC) and in the lack of dose relevance (PLT, EOS). All values were within the historical control ranges are indicative of biological variation and not related to the test item.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

There were no test item related adverse effects on the examined clinical chemistry parameters at 100, 300 or 1000 mg/kg bw/day (male or female).
Slight but statistically significant differences were observed between the control and treated groups of male animals at the higher mean concentration of total bilirubin (TBIL; at 300 and 1000 mg/kg bw/day) as well as at the higher mean concentration of sodium (Na+; at 100, 300 and 1000 mg/kg bw/day) and albumin (ALB; at 300 mg/kg bw/day).
In the female animals, statistically significant difference with respect to the control was observed at 300 and 1000 mg/kg bw/day at the slightly mean cholesterol concentrations (CHOL).
These slight, but statistically significant differences with respect to their controls were considered to have little or no toxicological importance because all these changes were with low degree, values – mean and individual – remained within the historical control ranges (TBIL, Na+, ALB, CHOL) or there were no dose relevance (Na+, ALB, CHOL). These were considered to be indicative of biological variation and not related to the test item.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Functional observation battery did not demonstrate any treatment-related alterations in the behavior or in reactions to different type of stimuli at the end of the treatment period (selected male and female, 100, 300 or 1000 mg/kg bw/day groups, on Day 49).
There were no changes in the physical condition, behavior or in reactions to different types of stimuli in the male or female animals of control and test item treated groups in the examined parameters during the course of the functional observations.
Yellowish color of stool was also detected in the cages of high dose treated male and female animals at the functional (cage side) observations.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

No test item adverse effect was observed relating to the weight of the investigated organs. There were no toxicologically significant differences in the examined organ weights between the control and test item treated male or female animals at any dose level (100, 300 and 1000 mg/kg bw/day).
In the male animals at 100 mg/kg bw/day, the mean heart weights (absolute and relative to brain weight) were slightly below that of the control group.
In the male animals at 300 mg/kg bw/day, statistical significance was noted for the slightly lower mean brain weight for the higher mean liver weights (absolute and relative to body and brain weights) and for the lower mean heart weights (absolute and relative to brain weight) when compared to the control group.
In the male animals administered with 1000 mg/kg bw/day, lower mean brain weight, higher mean liver weights (absolute and relative to body and brain weights), lower mean heart weights (absolute and relative to brain weight) as well as lower mean testes weight were observed with respect to their controls.
These slight differences with respect to the control were judged to be toxicologically not relevant but indicative of individual variations.
There were no statistically or biologically significances between the control and 100, 300 or 1000 mg/kg bw/day Acid Orange 67 treated female animals in the examined organ weights at the end of the treatment period.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Specific macroscopic alterations indicative of test item effect were not observed in the organs or tissues at any dose levels 100, 300 or 1000 mg/kg bw/day at the necropsy. Presence of the test item or its metabolite was observed in the stomach or cecum of male animals at 1000 mg/kg bw/day.
In dead male animal (1/12 at 100 mg/kg bw/day), gross pathology examination revealed yellowish fluid content in the thoracic cavity and empty intestines indicative of para-gastric treatment (damage of the esophagus) of this animal.
There were no macroscopic findings in the organs or tissues in male animals in the control, 100 or 300 mg/kg bw/day groups (12/12, 11/11 and 12/12, respectively).
In male animals dosed at 1000 mg/kg bw/day, the stomach (11/12) or cecum (7/12) contained yellowish material and the thymus was smaller than normal and spotted by point-like hemorrhages (1/12).
In the female animals, slight hydrometra (1/12 control dam and 2/10 dams at 100 mg/kg bw/day) was observed at the end of the treatment (lactation) period.
Yellowish fluid content in the intestines (1/12 at 300 mg/kg bw/day) and solid formation in the liver (1/11 at 1000 mg/kg bw/day) were detected in dams.
In one of the non-pregnant females, slight hydrometra was observed (1/2 at 100 mg/kg bw/day). There were no macroscopic findings in the organs or tissues in the other non-pregnant female animals (1/2 at 100 mg/kg bw/day and 1/1 at 1000mg/kg bw/day).
Yellowish content in the gastrointestinal tract referred to the presence of the test item or its metabolite.
Solid formation in the liver and smaller than normal thymus were considered to be individual changes therefore were not related to the test item.
Hemorrhage in the thymus was present in one male animal at 1000 mg/kg bw/day due to circulatory disturbance developed during exsanguination procedure. Hydrometra, related to the female sexual cycle, is a frequent observation in experimental rats. In the lack of related histopathological alterations (inflammatory or other pathological lesion) these were judged to be toxicologically not relevant.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Histopathological examinations did not reveal any test item related alterations in the organs or tissues of selected male and female animals at the highest dose (1000 mg/kg/bw/day). There were no pathologic changes in the examined reproductive organs or tissues of male or female animals (control, 100 and 1000 mg/kg bw/day).
In the male animals belonging to the treated and control groups (12/12 control; 2/2 at 100 mg/kg bw/day; 12/12 at 1000 mg/kg bw/day), the investigated organs of reproductive system (testes, epididymides, prostate seminal vesicles, coagulating glands) were histologically normal and characteristic on the sexually mature organism in all cases of control and treated groups. The various spermatogenic cells (the spermatogonia, the spermatocytes, the spermatids and spermatozoa); representing different phases in the development and differentiation of the spermatozoons and the interstitial cells were the same in quantity and morphologically in the testes of investigated control and treated animals. The histological picture of epididymides, seminal vesicles, and coagulating glands was normal in all cases as well.
In the female animals belonging to the treated and control groups (12/12 control; 2/2 at 100 mg/kg bw/day; 12/12 at 1000 mg/kg bw/day), the ovaries, uterus, cervix and vagina had a normal structure characteristic of the species, age and phase of the active sexual cycle in all cases of control and treated groups. The cortical region of ovaries contained primary, secondary and tertiary follicles and corpora lutea, indicating the active maturation of oocytes, and ovulation. The epithelial capsule and ovarian stroma was normal in all cases as well.
In animals selected for full histological examinations, minimal alveolar emphysema in the lungs (2/5 control male, 1/5 male at 1000 mg/kg bw/day) and acute hemorrhage in the thymus (1/5 male at 1000 mg/kg bw/day) occurred sporadically and these were considered as a consequence of hypoxia, dyspnea and circulatory disturbance developed during exsanguinations.
Hyperplasia of bronchus associated lymphoid tissue (BALT) was observed in single male animals of control (1/5) and 1000 mg/kg bw/day (1/5) groups. Hyperplasia of BALT is a physiological immune-morphological phenomenon, occurring also in not treated rats and has no toxicological significance.
Chronic hematoma surrounded with fibrotic encapsulation was detected in the liver of one dam (1/11) at 1000 mg/kg bw/day, which was considered to be an individual lesion probably due to mechanical injury attributable to para-gastric treatment.
The histological structure and the cellularity of pituitary with special attention on the cytomorphology and proportion of acidophilic and basophilic cells in the adenohypophysis were the same in the control (5/5 male and 5/5 female) and treated male (5/5) and female (5/5) animals.
There was no morphological evidence of acute or subacute injury (degeneration, inflammation, necrosis etc.) of the stomach, the small and large intestines, the liver (except hematoma), the pancreas, the cardiovascular system, the immune system, the hematopoietic system, the skeleton, the muscular system, the male and female reproductive system or the central, or peripheral nervous system in surviving animals.
The quantity and quality of hemopoietic cells in the bone marrow, and the cyto-morphology of endocrine glands was the same in the control and treated animals.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Conclusions:

NOAEL (males and females): 1000 mg/kg bw/day.

Executive summary:

The objective of the study was to obtain initial information on the toxic potential of test item and on the possible effects of the test item on reproduction and development when repeatedly administered orally (by gavage), according to OECD 422.

Test item was administered orally (by gavage) once daily at 0 (vehicle only), 100, 300 and 1000 mg/kg body weight (mg/kg bw/day) doses to four groups of rats consisting of 12 animals per sex per group.

Test substance concentrations in the dosing formulations varied in the range between 101 % and 106 % of the nominal values and confirming the proper preparation of the dosing formulations.

All animals of the parent (P) generation were dosed prior to mating (14 days) and throughout mating. In addition, males received the test item or vehicle after mating up to the day before the necropsy (altogether for 54 or 55 days). Females were additionally exposed through the gestation period and up to lactation days 13 - 19, i.e. up to the day before necropsy (altogether for 54 or 55 days; one female animal at 1000 mg/kg bw/day was administered for 71 days due to the late mating).

There was no test item related mortality at any dose level; one male animal at 100 mg/kg bw/day was found dead on the day of scheduled necropsy (Day 55). Necropsy observation revealed sign of para-gastric treatment (yellowish fluid content in the thoracic cavity) and including damage caused by gavage.

Clinical signs of systemic toxicity related to the test item were not detected at any dose level, neither at the daily nor the detailed weekly clinical observations. The behavior and physical condition of the animals was not impaired at each dose level during the entire treatment period. Body weight or body weight gain were not affected.

Hematological evaluation did not reveal adverse test item related changes in the examined parameters at none of tested doses. There were no test item related adverse effects on the examined clinical chemistry parameters in both male and female.

Necropsy did not revealed any effect in male and female animals; furthermore, there were no test item related changes in the weights of brain, testes and epididymides of male animals at any dose level.

Histopathological examinations of the selected organs (ovaries, uterus, vagina, pituitary, testes, epididymides, prostate and seminal vesicles with coagulating gland) did not reveal any test item related changes.

 

Conclusion

Test item administered at 100, 300 or 1000 mg/kg bw/day by oral gavage did not cause signs of systemic toxicity in parental male and female rats.

Based on these observations the No Observed Adverse Effect Levels (NOAEL) for systemic toxicity of male/female rats was determined to be 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

Additional information

In order to obtain initial information on the toxic potential of test item and on the possible effects of the test item on reproduction and development, a test was performed according to OECD 422. Test item was administered orally (by gavage) once daily at 0 (vehicle only), 100, 300 and 1000 mg/kg body weight (mg/kg bw/day) doses to four groups of rats, consisting of 12 animals per sex per group. Test substance concentrations in the dosing formulations varied in the range between 101 % and 106 % of the nominal values and confirming the proper preparation of the dosing formulations. All animals of the parent (P) generation were dosed prior to mating (14 days) and throughout mating. In addition, males received the test item or vehicle after mating up to the day before the necropsy (altogether for 54 or 55 days). Females were additionally exposed through the gestation period and up to lactation days 13 - 19, i.e. up to the day before necropsy (altogether for 54 or 55 days; one female animal at 1000 mg/kg bw/day was administered for 71 days due to the late mating).

There was no test item related mortality at any dose level; one male animal at 100 mg/kg bw/day was found dead on the day of scheduled necropsy.

Clinical signs of systemic toxicity related to the test item were not detected at any dose level, neither at the daily nor the detailed weekly clinical observations. The behavior and physical condition of the animals was not impaired at each dose level during the entire treatment period. Body weight or body weight gain were not affected.

Hematological evaluation did not reveal adverse test item related changes in the examined parameters at none of tested doses. There were no test item related adverse effects on the examined clinical chemistry parameters in both male and female.

Necropsy did not revealed any effect in male and female animals; furthermore, there were no test item related changes in the weights of brain, testes and epididymides of male animals at any dose level. Histopathological examinations of the selected organs (ovaries, uterus, vagina, pituitary, testes, epididymides, prostate and seminal vesicles with coagulating gland) did not reveal any test item related changes.

Based on these observations, the No Observed Adverse Effect Levels (NOAEL) for systemic toxicity of male/female rats was determined to be 1000 mg/kg bw/day.

The main study outcomes agree with the results obtained into the 14 -days dose-range finding experiment.

In addition, an old experiment of low reliability is available; however, due to the fact that details on testing procedures and results are lacking, a reliability cannot be assigned. A group of ten male and ten female rats were given 14 daily oral doses by stomach tube of 1000 mg/kg as an aqueous solution. There were no toxic signs during the experimental period. Liver weights and body weight gains were comparable with control. Haematological examination of peripheral blood and bone marrow showed no abnormalities. Clotting function tests were normal. Plasma sodium and potassium ion concentrations and blood urea levels measured 24 hours after the last dose were normal and there were no changes in the protein, glucose, pH and bilirubin content of the final 18-hour urine sample. Histological examination of the usual ranges of tissues 24 hours and seven days after completion of dosing revealed no significant lesions.

Justification for classification or non-classification

According to the CLP Regulation (EC) No 1272/2008, 3.9 Specific target organ toxicity - repeated exposure section, substances are classified as specific target organ toxicants following repeated exposure by the use of expert judgment, on the basis of the weight of all evidence available, including the use of recommended guidance values, which take into account the duration of exposure and the dose/concentration, which produced the effect(s), and are placed in one of two categories, depending upon the nature and severity of the effect(s) observed.

In order to help reach a decision about whether a substance shall be classified or not, and to what degree it shall be classified (Category 1 or Category 2), dose/concentration ‘guidance values’ are provided for consideration of the dose/concentration which has been shown to produce significant health effects. The guidance values refer to effects seen in a standard 90-day toxicity study conducted in rats. Nevertheless, they can be used as a basis to extrapolate equivalent guidance values for toxicity studies of greater or lesser duration (the assessment shall be done on a case-by- case basis). For example, for 28-day study the guidance values are increased by a factor of three, thus the limit for sub-acute studies should be 300 mg/kg bw/day.

 

In the specific case, the No Observed Adverse Effect Level was established at 1000 mg/kg bw/day, on the basis of the results from the subacute study on rat; thus, the substance does not meet the criteria to be classified for repeated dose toxicity, according to the CLP Regulation (EC) No 1272/2008.