Reaction product of 2-hydroxybenzoic acid, styrene and oxozinc

Endpoint summary

Description of key information

NOAEL: 5 mg/kg bw/day for male animals

NOAEL: 5 mg/kg bw/day for female animals

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Name: Reaction Product of 2-Hydroxybenzoic Acid, Styrene and Oxozinc
Batch No. 2011121802
EC No. 700-321-2 (provisional EC number)
Aggregate State at room temperature:Solid
Color:Amber color
Odor: Slight odor
Molecular Formula: C30H26O6Zn, C46H42O6Zn, C62H58O6Zn,
Molecular Weight: 547.93 to 964.53 g/mol
Purity: 100 % (UVCB-Substance)
Date of Certificate of Analysis: June 04, 2013
Analysis (Method): HPLC/MS
Date of Manufacture: December 18, 2011
Stability: Stable under storage conditions as indicated
Storage conditions:0 – 50℃
Expiry Date: December 04, 2013
Safety precautions:Routine safety precautions will be applied (lab coat, mask, gloves, safety glasses)
Identification: The test item of a suitable chemical purity was supplied by the Sponsor. All precautions required in the handling and disposal of the test item, Certificate of Analysis, MSDS and Test Item Characterisation Sheet were supplied by the Sponsor and archived with the raw data.

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Rat, Hsd.Brl.Han: of Wistar origin

Sex:

male/female

Details on test animals or test system and environmental conditions:

Source: Toxi-Coop Zrt. Cserkesz u. 90. H-1103 Budapest, Hungary
Hygienic level at arrival: SPF
Hygienic level during the study: Good conventional
Justification of strain: The rat is regarded as suitable species for toxicity studies and the test guideline is designed to use the rat. The Wistar rat was selected due to the large experience accumulated with this strain in toxicity studies.
Number of animals: 20 males, 20 females 5 animals/sex in the control and dose groups
Sex: Males and nulliparous, non-pregnant females
Age of animals at starting: Male animals: 54 – 59 days old Female animals: 54 – 59 days old
Body weight range at starting: Male animals: 247 – 267 g Female animals: 160 – 179 g
Acclimatization time: 19 days

Route of administration:

oral: gavage

Vehicle:

other: 0.5 % aqueous methylcellulose

Details on oral exposure:

VEHICLE
The test item was not soluble in water therefore 0.5 % aqueous methylcellulose was used for preparing formulations appropriate for oral administration. 0.5 % aqueous methylcellulose was a suitable vehicle to facilitate formulation analysis for the test item.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Reaction product of 2-hydroxybenzoic acid, styrene and oxozinc content was determined in 0.5% methylcellulose formulations using the previously validated reverse phase HPLC method with UV detection on a 125-4 LiChrospher 100RP-18 column.

Duration of treatment / exposure:

28 days

Frequency of treatment:

The test item was administered in a single dose by oral gavage on a 7 days/week basis, every day at a similar time (+/- 2 hours). Concurrent control animals were treated with vehicle only and handled in an identical manner to the test groups. Animals were not treated on the day of gross pathology.
Dosing of both sexes began after the acclimatization (Day 0) and was continued up to and including the day before the necropsy.

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

20 mg/kg bw/day (nominal)

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

5 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose setting is based on findings obtained in a previous 14-Day oral gavage dose range finding study with Reaction Product of 2-Hydroxybenzoic Acid, Styrene and Oxozinc in rats (Toxi-Coop study no. 722.400.4163) and in agreement with the Sponsor. The high dose was chosen with the aim of inducing toxic effects but no mortality or severe suffering of animals.The low dose was chosen to induce no toxic effect.

Positive control:

no

Observations and examinations performed and frequency:

Clinical Examination
Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each day). General clinical cage side observations were made once a day, after the administration at approximately the same time. Detailed clinical observations were made on all animals outside the home cage in a standard arena once, prior to the first exposure and once weekly thereafter.
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.
Sensory reactivity to different types of stimuli (e.g. auditory, visual and proprioceptive), grip strength and motor activity were assessed in all animals during the last exposure week. General physical condition and behavior of animals were tested.

Weight Assessment
All animals were weighed in the treatment period with an accuracy of 1 g and body weight values were evaluated on Days 0, 7, 14, 21 and 27. Individual body weight changes were calculated.
Fasted body weight was measured on day of necropsy (Day 28).

Food Consumption
The food consumption was determined in the treatment phase with an accuracy of 1 g on Days 7, 14, 21 and 27 by re-weighing the non-consumed diet.

Examination of Estrous Cycle
Prior to necropsy on Day 28 , the estrus cycle of all females was determined by taking vaginal smears, which were prepared and stained with 1% aqueous methylene blue solution. The smear was examined with a light microscope, in order to provide information regarding the stage of oestrus cycle at the time of sacrifice and assist in histological evaluation of estrogen sensitive tissues.

Clinical Pathology
Clinical pathology examinations including hematology and clinical chemistry were conducted one day after the last treatment (Day 28).
Animals were food deprived for approximately 16 hours 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 determination of blood clotting times (for APTT and PT; 1.0 mL 9NC Microtube, 3.8 %), one for hematology (MiniCollect® K3EDTA tubes, spray-dried, 0.25 mL), and the third one (VACUETTE® Z Serum Tube, 2.5 mL) to obtain serum samples for clinical chemistry. Each tube was manufactured by Greiner Bio-One International AG.
Tubes for hematology and coagulation were filled up to the final volume (marked on the tubes) and at least 1.0 mL blood was collected, if possible into clinical chemistry tubes.

Hematology
The hematology parameters were measured in all animals by SYSMEX XT-2000iV.

Blood Coagulation
The blood coagulation parameters were determined by AMAX Destiny Plus.

Clinical Chemistry
The clinical chemistry measurement was performed by Konelab 60i in all animals.

Pathology
Gross necropsy was performed on each animal one day after the last treatment. Animals were euthanized by exsanguination after verification of deep narcosis by Isoflurane CP® .After examination of the external appearance, the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed, and any abnormality was recorded with details of the location, color, shape and size. The following organs/tissues were preserved in 4 % buffered formaldehyde solution except testes and epididymides, which were fixed in modified Davidson solution.

Organ Weight
The following organ weights were determined and recorded prior to preservation (wet weight):
With precision of 0.01g: liver, kidneys, testes, epididymides, uterus, thymus, spleen, brain and heart, prostate and seminal vesicles with coagulating glands as a whole;
With precision of 0.001g: adrenals, ovaries
Paired organs were measured together.

Histopathology
Full histopathology was performed on the preserved organs or tissues of the animals of the control (group 1) and high dose (group 4) groups. The testes, epididymides, prostate and seminal vesicles with coagulating glands of male animal of 5 and 20 mg/kg bw/day groups, as well as kidneys of one animals of 20 mg/kg bw/day group were also processed histologically due to the necropsy findings.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes

Statistics:

Statistical analysis was done with SPSS PC+ software package for the following data:
- body weight
- food consumption
- clinical pathology (hematology, blood coagulation, clinical chemistry)
- organ weight
The heterogeneity 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) was carried out. If the obtained result was significant Duncan Multiple Range test was used to access the significance of inter-group differences.
Where significant heterogeneity was found, we examined the normal distribution of data by Kolmogorov-Smirnov test. In case of not normal distribution, we applied the non-parametric method of Kruskal-Wallis One-Way analysis of variance. If there was a positive result, the inter-group comparisons were performed using Mann-Whitney U-test.
The frequency of clinical signs, pathology and histopathology findings were calculated.
Results were evaluated in comparison with values of control group (i.e. control value). Parameters indicated with statistical significances were listed as deviations from control value in paragraph “Results”.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no toxic signs related to the test item effect at the daily clinical observations. The behavior and physical condition of animals were considered to be normal at each dose level (50, 20 and 5 mg/kg bw/day) during the entire observation period.
Alopecia was noted on the right side of the neck of one female animal (1/5) in the 20 mg/kg bw/day group between Days 8 and 22. Alopecia is a common findings in this strain of experimental rats, occurred in single animal of the mid dose group therefore was not considered to be toxicologically relevant.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred in the control, 50, 20 or 5 mg/kg bw/day dose groups during the course of the treatment period.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

A test item influence on the body weight development was noted for male and female animals of 50 and 20 mg/kg bw/day groups. The reduced body weight gain resulted in a statistically significantly less body weight at 50 and 20 mg/kg bw/day from Day 7 and 14, respectively, in male animals and from Day 7 in female animals up to the end of the treatment period.
The mean body weight gain was statistically significantly less than in the control group in the male animals at 50 mg/kg bw/day during the entire study and between Days 0 and 27.
Thus, the mean body weight remained below the control value with statistical significances form Day 7 up to and including Day 27. The mean body weight gain was also less in the male animals at 20 mg/kg bw/day with respect to the control during the entire study and with statistical significances on weeks 1 and 2, and the total body weight gain (between Days 0 and 27) remained below the control value, too. Body weight of male animals at 20 mg/kg bw/day was below the control on Days 14, 21 and 27.
In female animals, body weight was statistically significantly reduced at the 50 and 20 mg/kg bw/day dose levels as compared to the control group during the entire observation period (Days 7, 14, 21 and 27). Body weight gain was also reduced in female animals of 50 and 20 mg/kg bw/day groups during the entire observation period but reaching statistical significance on week 1 (50 and 20 mg/kg bw/day groups) and on week 4 (50 mg/kg bw/day group) and between Days 0 and 27 (50 and 20 mg/kg bw/day groups) with respect to the relevant control.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The daily mean food consumption was slightly reduced in male and female animals at 50 mg/kg bw/day and in female animals of 20 mg/kg bw/day.
The mean daily food intake was slightly less than in the control group in male animals administered with 50 mg/kg bw/day during the entire observation period but statistical significances were only detected on weeks 1, 2 and 3.
Similarly, slight changes were observed in the mean daily food consumption in female animals of 50 and 20 mg/kg bw/day groups during the entire study, but statistically significant differences with respect to the control group were only indicated on weeks 2 and 3 for both groups.
The daily mean food consumption of female animals of 5 mg/kg bw/day group was slightly but statistically significantly higher with respect to the control on week 3.

Food efficiency:

not examined

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

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Hematological investigations did not reveal any test item related changes in the examined parameters (male and female, 50, 20 or 5 mg/kg bw/day).
In male animals, small but statistically significant differences were observed in the less mean percentage of neutrophil granulocytes (NEU) at 50 mg/kg bw/day, in the higher mean percentage of lymphocytes (LYM) at 50 mg/kg bw/day and in the reticulocytes (RET) at 50 and 20 mg/kg bw/day with respect to the relevant control. The hemoglobin concentration (HGB) and mean corpuscular (erythrocyte) hemoglobin content (MCH) were less in 50 and 20 mg/kg bw/day groups than in the control group. The mean corpuscular hemoglobin concentration (MCHC) was below (50 mg/kg bw/day), the mean protrombin time (PT) was above the value of the control group in male animals of 50 and 20 mg/kg bw/day group.
In the female animals, statistical significance was indicated for a slightly less mean hemoglobin concentration, hematocrit value (HCT), a higher mean percentage of reticulocytes and for a higher mean prothrombin time at 50 mg/kg bw/day with respect to the appropriate control value. The prothrombin time (PT) was slightly longer than in the control at 20 mg/kg bw/day.
Alterations in the parameters listed above (NEU, LYM, RET, MCH, MCHC, HGB, HCT and PT) reached statistical significances with respect to the appropriate control value however values remained within the historical control ranges or were independent from administered doses. Therefore, the changes noted in haematology parameters were not considered to be toxicologically relevant.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No pathologic test item effects were detected at the evaluation of the clinical chemistry parameters (male and female, 50, 20 or 5 mg/kg bw/day).
Statistically significant differences were observed between the control and 50 mg/kg bw/day treated male animals regarding the activity of aspartate aminotransferase (AST), and concentrations of creatinine (CREA), inorganic phosphorous (Pi), sodium (Na+), potassium (K+) and chloride (Cl-). The mean activity of aspartate aminotransferase of male animals of 20 mg/kg bw/day group was slightly but statistically significantly less than in the control group. The mean concentration of calcium (Ca2+) was slightly but statistically significantly higher than in the control group in male animals of (20 and 5 mg/kg bw/day). All values of these parameters (AST, CREA, Na+, K+, Cl- and Ca2+) remained well within the historical control ranges, or were marginal to the historical control (Cl-).The mean concentration of phosphorous was slightly below the lower limit of the historical control range, there were no related effects on other clinical pathology parameters and it was also not correlated with any histological findings. Therefore these findings in the clinical chemistry parameters were not considered to be of toxicological relevance.
In female animals, statistically significant differences were detected between the control and test item treated groups in the mean activity of aspartate aminotransferase (50 and 20 mg/kg bw/day), in the mean concentrations of cholesterol (CHOL; 50, 20 and 5 mg/kg bw/day), bile acids (BAC; 50 mg/kg bw/day) and calcium (5 mg/kg bw/day). These sporadic statistical differences were considered to be of little or no biological importance. Although the mentioned differences between the control and test item treated groups were statistically significant, all values remained within the historical control ranges for these parameters except cholesterol. The mean cholesterol concentrations slightly remained below the historical control range in the female animals of 50 mg/kg bw/day group however in the lack of any related pathological alterations it was considered to be indicative of biological variation. Therefore these findings were not considered to be of toxicological relevance.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Test item induced changes in the weights of male genital organs and accessory sex organs were observed in the 50 mg/kg bw/day group.
In male animals of the 50 mg/kg bw/day group, the mean weights of the testes (absolute and relative to brain weight), epididymides and seminal vesicles with prostate (absolute and relative to body and brain weights) were less than in the control group. The adrenal gland weights (absolute and relative to body and brain weights) exceed the control value in male animals of 50 mg/kg bw/day group however in the lack of any supporting histopathological changes therefore it was considered to be an adaptive change to the altered demand.
Statistical significances with respect to control in the absolute or relative weights of some organs (liver, kidneys, heart and thymus) were considered to be the consequence of the body weight changes of male animals of 50 or 20 mg/kg bw/day groups. The thymus weight was slightly below the control value in male animals of the 5 mg/kg bw/day.
Statistically significant differences with respect to controls noted for the higher mean weights of testes and epididymides (absolute) at 5 mg/kg bw/day, testes weights relative to body and brain weights at 20 and 5 mg/kg bw/day, prostate and seminal vesicle (absolute and relative to brain weight) at 20 mg/kg bw/day, were not considered to be of toxicological relevance due to the low degree and in the lack of related histopathological changes.
In the female animals of 50 and 20 mg/kg bw/day group, the absolute mean weight of kidneys and heart, as well as kidneys weight relative to brain weight were slightly but statistically significantly less than in the control group. Similarly, the mean heart and thymus weights relative to brain weight were also less than the control values in female animals the 50 mg/kg bw/day. The thymus weight was slightly less than in the control group in female animals of 50 mg/kg bw/day.
The mean brain weight relative to body weight (50 and 20 mg/kg bw/day), the mean liver weight and the mean adrenal weight relative to body weight (50 mg/kg bw/day) exceeded the control value. All these statistically significant differences in the mean weights of brain, liver, kidneys, heart, thymus and adrenal glands with respect to the appropriate control value were considered to be the consequence of the significant body weight changes of the female animals or indicative of biological variation and no test item influence was supposed.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Gross necropsy findings of gential organs and accessory sex organs were indicative of the test item influence in male animals at 50 mg/kg bw/day. Smaller than normal testes (3/5), epididymides (3/5), prostate (5/5) and seminal vesicles (5/5)] were observed in animals of the high dose group.
Pyelectasia was detected in one male animal at 20 mg/kg bw/day (1/5, one side) and in one control male animal (1/5; both sides).
Moderate hydrometra was noted for one female animal of 5 mg/kg bw/day (1/5).
Renal pyelectasia is a common observation of this strain of experimental rats and it was only present in single male animals of the mid dose and control group. Hydrometra, related to the female sexual cycle is a frequent observation in experimental rats and it was only present in one female of the low dose group. Both of these findings were considered to be without any toxicological relevance in this study.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histological examination revealed sings of decreased secretion in the tubuli (less amount of secretary material and narrow diameter of tubuli) in the seminal vesicle and coagulating gland in all male animals of the 50 mg/kg bw/day group and decreased amount of secrete in the tubuli of prostate (4/5) was observed. In addition, in three animals (3/5) decreased intensity of spermatogenesis was detected in a proportion (30-40 %) of seminiferous tubuli in the testes. In the affected tubuli, the lack of mature spermatozoa and spermatids was observed (2/5), however the Sertoli-cells, the spermatogonia and spermatocytes, were intact. In the other 60-70% of the seminiferous tubuli the histological picture of active spermatogenesis was seen. Lack of mature spermatozoa in the ductuli of epididymides was seen in three animals (3/5). These findings were not accompanied with inflammation, degeneration or necrosis in the affected organs and seemed to be functional and reversible in character.
The various spermatogenic cells (spermatogonia, spermatocytes, spermatids and spermatozoa), representing different phases in the development and differentiation of the spermatozoons, and the interstitial cells were normal in all animals of 20 and 5 mg/kg bw/day and control groups. The histological picture of prostate, seminal vesicle and coagulating gland was normal in all animals of 20 and 5 mg/kg bw/day and control groups.
The structure and cellular morphology of endocrine glands including the pituitary, the adrenal glands, the thyroid glands and the interstitial cells in the testes was the same in the control and treated animals.
In the lungs, alveolar emphysema (1/5 control female), acute hemorrhage (1/5 control male) and hyperplasia of bronchus associated lymphoid tissue (BALT) were observed (1/5 male and 1/5 female at 50 mg/kg bw/day; 1/5 male and 2/5 female in control group). The alveolar emphysema and the acute hemorrhage were related to the hypoxia, dyspnea and circulatory disturbance, developed during the exsanguination. The hyperplasia of BALT is a physiological immune-morphological phenomenon.
In two male animals (1/1 at 20 mg/kg bw/day; 1/5 in the control group) pyelectasia was seen. This finding (without degenerative, inflammatory or fibrotic lesion) is an individual disorder without toxicological significance.
No morphological evidence of acute or subacute injury (degeneration, inflammation, necrosis etc.) of the liver, the alimentary system, the immune system, the hemopoietic system, the skeleton, the muscular system, or the central and peripheral nervous system was observed.

Dose descriptor:

NOAEL

Effect level:

5 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

Critical effects observed:

yes

Lowest effective dose / conc.:

50 mg/kg bw/day (nominal)

System:

male reproductive system

Organ:

coagulating gland

seminal vesicle

other: Prostate

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

Under the conditions of the present study, Reaction Product of 2-Hydroxybenzoic Acid, Styrene and Oxozinc caused reduction in body weight development and food consumption (male and female), macroscopic and weight changes accompanied by histological alterations in male genital organs and accessory sex organs in Hsd.Brl.Han: Wistar rats after 28-days oral (gavage) administration at 50 mg/kg bw/day.
At 20 mg/kg bw/day, test item influence was observed in the reduced body weight and body weight gain in male and female animals, as well as in the reduced food consumption of female animals.
In the 5 mg/kg bw/day group adverse alterations were observed neither on body weight and food consumption nor on clinical and histopathological parameters.
Based on the observations made in the study the No Observed Adverse Effect Level (NOAEL) was determined as follows.
NOAEL: 5 mg/kg bw/day for male animals
NOAEL: 5 mg/kg bw/day for female animals

Executive summary:

In an 28-day toxicity study conducted under GLP compliance according to OECD TG 407,the test item was administered orally (by gavage) to Hsd.Brl.Han: Wistar rats (n=5/sex/group) once a day at 0, 50, 20 and 5 mg/kg bw/day doses corresponding to concentrations of 0, 10, 4 and 1 mg/mL, applied in a dose volume of 5 mL/kg bw for 28 days.

The suitability of the chosen vehicle (0.5 % Methylcellulose) for the test item at the intended concentrations was analytically verified up front. Reaction Product of 2-Hydroxybenzoic Acid, Styrene and Oxozinc was stable in concentrations ca. 1 and 200 mg/mL for 24 hours at room temperature and for three days in the refrigerator.

Animals were observed for mortality twice a day in the course of the study. Daily general clinical observations and weekly detailed clinical observations were performed. A functional observation battery was conducted in the last week of the treatment period. The body weight and food consumption were evaluated weekly. Clinical pathology examinations including hematology and clinical chemistry were conducted one day after the last treatment. Gross pathology was performed on animals on the day following the last treatment. The absolute and relative weights of selected organs were determined. A full histopathology was performed on the preserved organs or tissues of the animals of the control and high dose groups. The testes, epididymides, prostate and seminal vesicles with coagulating gland also were processed and evaluated histologically in 20 and 5 mg/kg bw/day groups due to the macroscopic findings at the necropsy.
The results of the study were interpreted comparing test item treated groups with respect to controls, which were administered concurrently with vehicle (0.5 % methylcellulose) only.

 

Results:

Mortality
No mortality occurred during the observation period.
Clinical observations
Toxic signs related to the test item were not found at any dose level at the daily and detailed weekly clinical observations and in the course of functional observation battery.
Body weight and body weight gain
A test item influence on the body weight development was noted for male and female animals of 50 and 20 mg/kg bw/day groups. The reduced body weight gain resulted in a less body weight at 50 and 20 mg/kg bw/day from Day 7 and 14, respectively, in male animals and from Day 7 in female animals up to the end of the treatment period.

Food consumption
The daily mean food consumption was slightly reduced in male and female animals at 50 mg/kg bw/day and in female animals at 20 mg/kg bw/day.
Clinical pathology
Clinical pathology examinations did not reveal any pathologic changes in the examined hematology, blood coagulation or clinical chemistry parameters.
Necropsy
Test item influence on the male genital organs was detected at the necropsy. The testes, epididymides, prostate and seminal vesicles with coagulating gland were smaller than normal in male animals of 50 mg/kg bw/day group.
Organ weight
Test item induced macroscopic changes in the male genital organs and accessory sex organs were supported by the organ weight evaluation. The weights of testes, epididymides, prostate and seminal vesicles with coagulating gland (as a whole) were statistically significantly less with respect to the control at 50 mg/kg bw/day dose level.
Histopathology
Histological examination – in full compliance with the results of the necropsy and organ weight examinations – revealed decreased secretion activity in the ductuli of seminal vesicles and prostate, decreased intensity of spermatogenesis in the testes, lack of the mature spermatozoa in the testes and epididymides in male animals of the 50 mg/kg bw/day group.

Conclusion
Under the conditions of the present study, Reaction Product of 2-Hydroxybenzoic Acid, Styrene and Oxozinc caused reduction in body weight development and food consumption (male and female), macroscopic and weight changes accompanied by histological alterations in male genital organs and accessory sex organs in Hsd.Brl.Han: Wistar rats after 28-days oral (gavage) administration at 50 mg/kg bw/day.
At 20 mg/kg bw/day, test item influence was observed in the reduced body weight and body weight gain in male and female animals, as well as in the reduced food consumption of female animals.
In the 5 mg/kg bw/day group adverse alterations were observed neither on body weight and food consumption nor on clinical and histopathological parameters.
Based on the observations made in the study the No Observed Adverse Effect Level (NOAEL) was determined as follows.
NOAEL: 5 mg/kg bw/day for male animals
NOAEL: 5 mg/kg bw/day for female animals

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

5 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

A Klimisch score of 1 has been assigned . The overall quality of the database is high

System:

male reproductive system

Organ:

coagulating gland

seminal vesicle

other: Prostate

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 an 28-day toxicity study conducted under GLP compliance according to OECD TG 407,the test item was administered orally (by gavage) to Hsd.Brl.Han: Wistar rats (n=5/sex/group) once a day at 0, 50, 20 and 5 mg/kg bw/day doses corresponding to concentrations of 0, 10, 4 and 1 mg/mL, applied in a dose volume of 5 mL/kg bw for 28 days.

No mortality occurred during the observation period. Toxic signs related to the test item were not found at any dose level at the daily and detailed weekly clinical observations and in the course of functional observation battery.A test item influence on the body weight development was noted for male and female animals of 50 and 20 mg/kg bw/day groups. The reduced body weight gain resulted in a less body weight at 50 and 20 mg/kg bw/day from Day 7 and 14, respectively, in male animals and from Day 7 in female animals up to the end of the treatment period. The daily mean food consumption was slightly reduced in male and female animals at 50 mg/kg bw/day and in female animals at 20 mg/kg bw/day.Clinical pathology examinations did not reveal any pathologic changes in the examined hematology, blood coagulation or clinical chemistry parameters. Test item influence on the male genital organs was detected at the necropsy. The testes, epididymides, prostate and seminal vesicles with coagulating gland were smaller than normal in male animals of 50 mg/kg bw/day group. Test item induced macroscopic changes in the male genital organs and accessory sex organs were supported by the organ weight evaluation. The weights of testes, epididymides, prostate and seminal vesicles with coagulating gland (as a whole) were statistically significantly less with respect to the control at 50 mg/kg bw/day dose level.Histological examination – in full compliance with the results of the necropsy and organ weight examinations – revealed decreased secretion activity in the ductuli of seminal vesicles and prostate, decreased intensity of spermatogenesis in the testes, lack of the mature spermatozoa in the testes and epididymides in male animals of the 50 mg/kg bw/day group.

In conclusion, Reaction Product of 2-Hydroxybenzoic Acid, Styrene and Oxozinc caused reduction in body weight development and food consumption (male and female), macroscopic and weight changes accompanied by histological alterations in male genital organs and accessory sex organs in Hsd.Brl.Han: Wistar rats after 28-days oral (gavage) administration at 50 mg/kg bw/day.At 20 mg/kg bw/day, test item influence was observed in the reduced body weight and body weight gain in male and female animals, as well as in the reduced food consumption of female animals.In the 5 mg/kg bw/day group adverse alterations were observed neither on body weight and food consumption nor on clinical and histopathological parameters.

Based on the findings of this study,the No Observed Adverse Effect Level (NOAEL) was determined as 5 mg/kg bw/day for male and female animals.

Justification for classification or non-classification

According to the CLP Regulation (Annex I of 1272/2008/EC), the substance Reaction product of 2-hydroxybenzoic acid, styrene and oxozinc  is classified as:  Repeated toxicity Category 2. In the 28-day repeated oral toxicity study, animals exposed to 5 mg/kg bw of the substance did not show any toxic effects and significant toxic effects were observed on 50 mg/kg bw/day dose level.The guidance value to assist in Category 1 classification was less than 10 mg/kg bw/day for a 90-day repeated-dose study. Therefore for a 28-day study , the guidance value should be increased by a factor of three, which should be less than 30 mg/kg bw/day.Similarly, the guidance value for Category 2 should be between 30-300 mg/kg bw/day.Therefore, the substance should be classified as Category 2.