Registration Dossier

Administrative data

Description of key information

NOAEL in male and female rats = 80 mg/kg bw/day (based on the OECD 422 study on OB 3a-DSA)

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
Remarks:
OECD 422
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From February 26 to November 30, 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29th July 2016
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
SPF quality
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River SPF breeding, supplied via VELAZ s.r.o., Czech Republic, RČH CZ 11760500
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: DRFE: 11 weeks at arrival, Main study: 9-10 weeks on arrival,
- Fasting period before study: the animals were without feed two hours before application and two
hours after application of the test item.
- Housing: main study: SPF conditions according to internal SOP No.12.
- Bedding: sterilized soft wood fibers Lignocel
- Animal per cage: 2 rats of the same sex in one cage in pre-mating period, during mating period – one male and one female in one cage, pregnant females – individually, offspring – with mother satellite animals - 2 rats of the same sex in one cage
- Diet (e.g. ad libitum): maintenance pelleted diet for rats and mice - Altromin for rats/mice, Manufacturer: Altromin Spezialfutter GmbH & Co. KG, Germany
- Water (e.g. ad libitum): drinking water ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): relative humidity 30-70 %
- Photoperiod (hrs dark / hrs light): 12 hour light / 12 hour dark
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
Aqua pro iniectione
Details on oral exposure:
- Concentration in vehicle: 150 mg, 300 mg and 600 mg in 10 ml
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical Method
Stability and homogeneity were determined by means of measuring of a peak area of the test item by a high-performance liquid chromatography based on a method developed at the test facility.
The Preparation of Application Form
The application form for analysis was prepared in the same manner as for application to animals – i.e. solution in aqua pro injection.
Concentration Level 10 mg/10 mL
Ca 0.10 g of the test item was weighed into a 150mL glass beaker calibrated to 100 mL and the beaker was slowly replenished by the vehicle. The test item was dissolved in ultrasonic bath for 5 min. The solution was stirred by magnetic stirrer at 350 rpm for 5 minutes.
The beaker with test item was during dissolving and homogenisation covered by aluminium foil due to possible light instability of test item.
Concentration Level 1000 mg/10 mL
Ca 10.0 g of the test item was weighed into a 150mL glass beaker calibrated to 100 mL. The beaker was slowly replenished by the vehicle (ca. 80 % of total volume) together with occasional mixing by glass rod. The test item was dissolved in ultrasonic bath together with occasional mixing by glass rod for 10 minutes. After this the glass beaker was diluted to mark by vehicle and the application form was stirred by magnetic stirrer at 350 rpm for 10 min.
The beaker with test item was during dissolving and homogenisation covered by aluminium foil due to possible light instability of test item.
The Stability of the Application Form
The samples were taken from the middle of the beaker content at required time intervals (0, 30, 60, 90 and 120 minutes) for the determination of stability of both application forms. Two samples were taken at all time intervals.
Conc. level 10 mg/ 10 mL: Time interval 0 min represents for this concentration the time after 5 minutes of ultrasonication and 5 minutes of mixing by magnetic stirrer at 350 rpm.
Conc. level 1000 mg/ 10 mL: Time interval 0 min represents for this concentration the time after 10 minutes of ultrasonication together with occasional mixing with glass rod and 10 minutes of mixing by magnetic stirrer at 350 rpm.
The Homogeneity of the Application Form
Conc. level 10 mg/ 10 mL: The samples were taken after 5 minutes in ultrasonic bath and 5 minutes of mixing by magnetic stirrer (350 rpm) from 3 given places - the bottom, the middle and the surface of the beaker content. Two samples were taken from each place.
Conc. level 1000 mg/ 10 mL: The samples were taken after 10 minutes in ultrasonic bath together with occasional mixing with glass rod and 10 minutes of mixing by magnetic stirrer (350 rpm) from 3 g.
Analytical Method
Stability and homogeneity were determined by means of measuring of a peak area of the test item by a high-performance liquid chromatography based on a method developed at the test facility.
The Preparation of Application Form
The application form for analysis was prepared in the same manner as for application to animals – i.e. solution in aqua pro injection.
Concentration Level 10 mg/10 mL
Ca 0.10 g of the test item was weighed into a 150 mL glass beaker calibrated to 100 mL and the beak er was slowly replenished by the vehicle. The test item was dissolved in ultrasonic bath for 5 min. The solution was stirred by magnetic stirrer at 350 rpm for 5 minutes.
The beaker with test item was during dissolving and homogenisation covered by aluminium foil due to possible light instability of test item.
Concentration Level 1000 mg/10 mL
Ca 10.0 g of the test item was weighed into a 150mL glass beaker calibrated to 100 mL. The beaker was slowly replenished by the vehicle (ca. 80 % of total volume) together with occasional mixing by glass rod. The test item was dissolved in ultrasonic bath together with occasional mixing by glass rod for 10 minutes. After this the glass beaker was diluted to mark by vehicle and the application form was stirred by magnetic stirrer at 350 rpm for 10 min.
The beaker with test item was during dissolving and homogenisation covered by aluminium foil due to possible light instability of test item.
The Stability of the Application Form
The samples were taken from the middle of the beaker content at required time intervals (0, 30, 60, 90 and 120 minutes) for the determination of stability of both application forms. Two samples were taken at all time intervals.
Conc. level 10 mg/ 10 mL: Time interval 0 min represents for this concentration the time after 5 minutes of ultrasonication and 5 minutes of mixing by magnetic stirrer at 350 rpm.
Conc. level 1000 mg/ 10 mL: Time interval 0 min represents for this concentration the time after 10 minutes of ultrasonication together with occasional mixing with glass rod and 10 minutes of mixing by magnetic stirrer at 350 rpm.
The Homogeneity of the Application Form
Conc. level 10 mg/ 10 mL: The samples were taken after 5 minutes in ultrasonic bath and 5 minutes of mixing by magnetic stirrer (350 rpm) from 3 given places - the bottom, the middle and the surface of the beaker content. Two samples were taken from each place.
Conc. level 1000 mg/ 10 mL: The samples were taken after 10 minutes in ultrasonic bath together with occasional mixing with glass rod and 10 minutes of mixing by magnetic stirrer (350 rpm) from 3 g.
Duration of treatment / exposure:
Parental males totally 49 days of administration
Satellite males totally 49 days of administration + 14 days of observation
Parental females 42 days + gestation → lactation → day 12 post-partum
Satellite females totally 49 days of administration + 14 days of observation
Non-pregnant females without evidence of copulation 42 days → 25th day after the end of mating period
Non-pregnant females with evidence of copulation 42 days → 25th day after confirmed mating
Frequency of treatment:
Daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control of the basic group and satellite
Dose / conc.:
150 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
600 mg/kg bw/day (nominal)
Remarks:
Control of the basic group and satellite
No. of animals per sex per dose:
Basic group: 12 males and 12 females per group
Satellite groups: 6 males and 6 females per group
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale:
Based on the result of the DRF study on the substance, the selected highest tested dose for the main study should have been 1000 mg/kg bw/day, since no significant toxicological effects have been observed at this dose and it could have been considered as limit dose. However, it should be noticed that this study has been performed not only to investigate the toxicological properties of the substance in itself, but also and mainly to compare the effects within the members of the category; therefore the highest tested dose has been selected as below reported, taking into account the existing following studies:
• 1-DSA – Subchronic toxicity study – Relevant toxicological effects on kidney at 750 mg/kg bw/day
• 1-DSA – Subchronic toxicity study – Relevant toxicological effects on testes at 750 mg/kg bw/day
• 3a-MSA – Subacute toxicity study – Effects to be confirmed on haematological parameters, liver and kidney starting from 200 mg/Kg bw/day
• 3a-MSA – Two generation toxicity study - Relevant toxicological effects mainly in terms of maternal toxicity at 1000 mg/kg bw/day and minor effects at 300 mg/kg bw/day

Indeed, in this respect all systemic studies concerning repeated dose, reproductive and developmental toxicity (OECD 422, 408 and 414) have been set on similar dose level in order to be able to compare all members of the category on the specific organ effects.
The lowest and the medium doses have been selected in order to have a coherent spacing of doses respect to the highest selected one, also taking into account all the available data on the repeated dose toxicity ( 3a-MSA, 1-DSA).
- Fasting period before blood sampling for clinical biochemistry: The animals were fasted approxim
ately for 18 hours before blood collection but they were supplied drinking water ad libitum
Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before the first application and then weekly (except the mating period)

BODY WEIGHT: Yes
- Time schedule for examinations: males and satellite animals - the first day of administration and then weekly,
females - the first day of administration and then weekly,
during pregnancy: 0., 7th, 14th, 20th day,
during lactation: 1st, 4th day, 12th day and 13th day
pups (litters) - 1st, 4th day, 12th day and 13th day
pups – individually – 4th day of lactation

FOOD CONSUMPTION:
- Food consumption: Yes
weekly and on the same days as body weight (except the mating period); satellite males and females – weekly

WATER CONSUMPTION: Yes
- Time schedule for examinations: satellite males and females – three times a week

URINALYSIS: Yes. This examination was performed only in 6 males of each group and in satellite males. In females this examination was not performed (dams should not be removed from the pups for prolonged periods).The rats were kept in the metabolic cages for the collection of urine for two hours. Immediately before entering metabolic cages the animals were administered 2 mL of drinking water per 100 g of body weight by gavage to the stomach. The following parameters were determined by analyser PocketChem PU-4010 (Arkray, Inc., Japan).

MORTALITY CONTROL: twice daily

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera

HISTOPATHOLOGY / ORGAN WEIGTHS
Absolute weights of liver, kidneys, adrenals, testes or ovaries, epididymis/epididymides or uterus, prostate gland + seminal vesicles, thymus, spleen, brain, pituitary gland and heart were recorded. Afterwards the somatic indexes - SI (= relative weight of organ) were computed according to the following formula: SI = weight of organ x 100/ body weight.
From all adult males and females thyroid glands were preserved in fixation medium. The thyroid weight was determined after fixation.
Full histopathology of the preserved reproductive organs and tissues was performed for all high dose and control animals.
Other examinations:
Postmortem examinations (parental animals)
During the necropsy a revision of the external surface of the body, of all orifices and the cranial, thoracic and abdominal cavities were carried out. Organs for consequent histopathological examination were taken out and stored in containers with fixative (buffered 4 % formaldehyde). Testes and epididymides were fixed in modified Davidson’s fixative.
Statistics:
For statistical evaluation the software Statgraphic ® Centurion (version XVII, USA) was used.
Males/females from control group were compared with males/females from three treated groups. Satellite males/females from control group were compared with satellite males/females from treated group.
As the first step the test for normality (Shapiro-Wilk test) was used. If the data were not normally distributed than the transformation of data was performed (Box-Cox transformation). If still the normal distributed distribution was not achieved than non-parametric tests (Kruskal-Wallis Test, Mann-Whitney test) were used.
For normally distributed data have at first the variance check has been performed (Levene´s test) to verify if standard deviations within each group are equal. One-Way ANOVA (probability level p ≤ 0.05) was used to detect whether there are any significant differences amongst the means. In case of significant differences, the post hoc statistical testing was performed (Fisher's least significant difference - LSD test). The non-parametric tests were used for statistical evaluation of
• selected reproduction parameters with non-continuous distribution (number of live born pups, number of pups, number of implantations)
• selected haematology parameters with non-continuous distribution (total erythrocyte count, total leucocyte count, total platelet count)
The Kruskal-Wallis test was used for the comparison of the measured effect in all treatment groups with the vehicle control group (as global test) and the two-groups Mann-Whitney test (probability level p ≤0.05).
Clinical signs:
no effects observed
Description (incidence and severity):
Males
No signs of disease, clinical changes and clinical signs of intoxication were recorded during the application period in all treated males. One male from the group of 150 mg/kg/day had overgrown teeth which were adjusted during the study (as needed).
Satellite males
No signs of disease, clinical changes and clinical signs of intoxication were recorded during the application period in all treated males. During the observation period (recovery; weeks 8 and 9) no changes of health status were noted in satellite treated males.
Females
No signs of disease, clinical changes and clinical signs of intoxication were recorded during the application period in treated females.
Female No.124 of the dose level 150 mg/kg/daywas found dead on day 25 of application (this female was in a group designated for the reproduction part of study). Piloerection and apathy were recorded during the observation a day before the death. The cause of death was unknown.
Satellite females
No signs of disease, clinical changes and clinical signs of intoxication were recorded during the application period in all treated females. During the observation period (recovery; weeks 8 and 9) no changes of health status were noted in satellite treated females.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Males:
There were no unscheduled deaths during the study.
Females:
Female No.124 of the dose level 150 mg/kg/day was found dead on day 25 of application. There were no other unscheduled deaths during the study.
Description (incidence and severity):
Males:
At the beginning of study, mean body weight of all groups of animals was similar. Slightly lower body weight was recorded in animals at the dose level 600 mg/kg/day in comparison with the control group during the whole application period. Statistically significant differences in necropsy body weight were not found in treated males.
Satellite males:
Body weights of satellite treated males were lower in comparison with the satellite control males during the whole study. Statistically significant differences in necropsy body weight were not found in satellite treated males.
Females:
Before the mating period, body weights of females in all treated groups was similar to the control group. During the pregnancy and lactation periods, body weights of treated females in all groups was comparable with the control females. Statistically significant differences in necropsy body weight were not found in treated females.
Satellite females:
Body weight of satellite treated females was slightly lower in comparison with the control group from the second week of administration. Statistically significant differences in necropsy body weight were not found in satellite treated females.

Mean Body Weight Increment
Males:
Weight increments of treated males were not adversely influenced by the test item treatment.
Satellite males:
Weight increments of satellite treated males in application and recovery period were variable and not adversely influenced by the test item administration.
Females:
Weight increments in treated females were variable in comparison with the control females and not affected by the test item treatment.
Satellite females:
Weight increments in treated females were variable and not affected by the test item treatment.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Males:
The food consumption of treated males was similar or slightly higher in comparison with the control males during the whole application period of study.
Satellite males:
The food consumption of satellite treated males was comparable to the control group during application and recovery period.
Females:
During the pre-mating period, pregnancy and lactation period the food consumption of treated females was similar or higher in comparison with the control females.
Satellite females:
The food consumption of satellite treated females was similar in comparison with the control group during the entire application and recovery periods.
Food efficiency:
no effects observed
Description (incidence and severity):
Food Conversion:
Males:
The food conversion of treated males was comparable with the control males in the pre-mating period and variable but not negatively influenced by the test item treatment during the period after mating.
Satellite males:
The food conversion of satellite treated males was similar or slightly lower compared to the satellite control.
Females:
The food conversion of treated females in the pre-mating period was variable but not adversely influenced by the test item treatment. During the pregnancy period, the food conversion of treated females was similar with the control group. During the lactation period, the food conversion of treated females at all dose levels was similar in comparison with the control females.
Satellite females:
The food conversion of satellite treated females was variable compared to satellite control females, but not adversely influenced by the test item treatment.
Description (incidence and severity):
Satellite males:
The water consumption of satellite treated males was slightly lower compared to the satellite control group till the 6th week of study, then the water consumption of treated satellite animals was similar to the control animals.
Satellite females:
The water consumption of satellite treated males was similar or higher compared to the satellite control group during the entire application and recovery period.
Description (incidence and severity):
Males:
Changes in the values of white blood components were recorded in treated groups of males. The WBC value of treated groups of males was comparable to the control group and was not affected by the test item treatment. Some differences were observed in the five-population differential of white blood cells. Statistically significantly increased value of lymphocytes (p ≤0.05) and decreased values of neutrophils (p ≤ 0.05) were recorded in dose groups 150 and 300 mg/kg/day. Statistically significantly decreased values of eosinophils (p ≤0.05) was recorded in dose groups 300 mg/kg/day.
The red blood components were not affected by the test item treatment. The values of RBC, Hgb, Hct, MCV and Plt were similar in treated groups of males in comparison with the control group of males. The values of haemocoagulation parameters were not significantly affected by the test item treatment except the value of fibrinogen concentration (p ≤ 0.05) in males of the dose level 600 mg/kg/day. This value was significantly increased in comparison with the control group of males.
All values were in a historical control range, without dose-dependency and reversible (not observed in satellite animals).
Satellite males:
No significantly changed parameters were recorded except the value of reticulocytes in satellite treated males. All values were in a range of historical control.
Females:
The white blood components were not affected by the test item treatment. No significantly changed values of total leucocyte count (WBC) and five-population differential of white blood cells in treated groups of females were recorded in comparison with the control group.
The changes in values of red blood components were recorded in treated groups of females. Statistically significantly increased value of RBC (p ≤0.05) associated with increased value of Hct (p ≤0.05) and decreased MCV (p ≤0.05) were recorded in females at the dose level 600 mg/kg/day in comparison with the control group of females.
The values of haemocoagulation parameters were not significantly affected by the test item treatment. All values were in a historical control range.
Satellite females:
A decreased value of RBC (p ≤0.05) and associated decreased value of Hct (p ≤0.05) and Hgb (p ≤0.05) were recorded in satellite treated females in comparison with the satellite control females. Other values were similar with satellite control females and in a range of historical control.
Description (incidence and severity):
Males
Significantly changed values (p ≤0.05) as ALT and BUN – decreased in all dosed groups of males, GLU decreased in males at the dose level 300 and 600 mg/kg/day and Ca in males at the dose level 150 and 300 mg/kg/day were recorded in comparison with the control group of males. Significantly changed value (p ≤0.05) of Crea – increased in males at the dose level 600 mg/kg/day was recorded.
Decreasing of ALT value was irreversible (recorded also in satellite treated group). Increased value of Crea in high dosed males persisted in satellite treated group of males. Values of other biochemical parameters of treated males were not significantly altered in comparison with the controls. All values are in a historical control range.
Satellite males
A statistically significant increased value (p ≤0.05) of Crea and Cl were recorded in satellite treated males compared to control males. Significantly decreased value (p ≤0.05) of ALT and CHE were recorded in satellite treated males.
Values of other biochemical parameters of satellite treated males were similar to the satellite control group. All values are in a historical control range.
Females
Significantly changed biochemical values were recorded only sporadically in treated females in comparison with the control females.
Significantly (p ≤0.05) increased value of T-Chol was reported in females at the dose level 150 and 600 mg/kg/day/day. Significantly (p ≤0.05) increased value of TG was recorded in females at the dose level 150 mg/kg/day/day. Significantly (p ≤0.05) decreased value of GLU and Na was recorded in females at the dose level 150 mg/kg/day/day only.
Increased value of Triglycerides (TG) were out of historical control range in all dosed groups of females.
Although the value of Crea was not increased statistically significantly, dose dependence and elevated values in treated groups of females compared to control were recorded. This trend was confirmed by a statistically significantly increased value of creatinine in satellite treated females.
Values of other biochemical parameters of treated females were comparable to the control group. All values except the value of TG are in a historical control range.
Satellite females
Statistically significantly (p ≤0.05) increased values of Crea and Cl and decreased (p ≤0.05) value of ALT were noted in satellite treated females in comparison with the satellite control females.
Values of other biochemical parameters of treated satellite females were comparable to the control group. All values are in a historical control range.
Description (incidence and severity):
Males
A statistically significantly increased pH of urine was detected in males at the dose level 150 mg/kg/day/day.
The presence of proteins was recorded sporadically - only in males at the dose level 150 mg/kg/day. Specific gravity was increased in males at the dose levels 300 and 600 mg/kg/day in comparison with the control group of males. Also presence of blood was recorded in males at the dose level 300 and 600 mg/kg/day.
The presence of leucocytes was recorded in treated males as well as in control males. In males at the dose level 300 and 600 was presence of leucocytes in the urine more frequent.
Satellite males
The volume and pH of urine was statistically significantly decreased in treated males. The presence of leucocytes was recorded in satellite treated males as well as in satellite control
males. The blood in urine was recorded in one satellite treated male only.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Males
Reactions to touch, noise, pain and pupillary reflex of treated males were the same as in the control group. The number of upstanding in treated males was comparable to the control. Emiction and defecation in treated males was similar with the control males. The values of grip strength of pectoral legs and pelvic legs did not show any significant differences between control and treated males.
Satellite males
No significant differences were detected in examined parameters.
Females
Reactions to touch, noise, pain and pupillary reflex of treated females were the same as in the control females. The number of upstanding in treated females was similar with the control females. The values of grip strength of pectoral and pelvic legs were without significant differences between control and treated females.
Satellite females
No significant differences were detected in examined parameters
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Absolute Organ Weight:
Males:
Statistically significantly (p ≤0.05) decreased absolute weight of spleen was recorded in males at the dose levels 150 and 600 mg/kg/day/day in comparison with the control males.
Absolute weight of liver was insignificantly decreased in all treated groups of males compared to control males.
Absolute weights of other organs were in dosed males comparable with the control males.
Satellite males:
No statistically significantly changed absolute weight of organs were recorded in dosed males in comparison with the satellite control males. Absolute weight of liver was insignificantly decreased in treated group of satellite males compared to control satellite males.
Females:
Statistically significantly (p ≤0.05) increased absolute weight of kidneys was recorded in females at all dose levels in comparison with the control females (irreversible). Statistically significantly (p ≤0.05) increased absolute weight of liver was recorded in females at dose levels 150 and 300 mg/kg/day in comparison with the control females. A significant (p ≤0.05) decrease in the weight of the ovaries was recorded in females at the dose level 600 mg/kg/day/day in comparison with the control group of females. Absolute weights of other organs were in dosed females comparable with the control females.
Satellite females:
Statistically significant differences were recorded in the absolute weight of kidneys (p ≤0.05) in satellite females. Absolute weight of kidneys was increased in treated females in comparison with the control females.
Relative Organ Weight:
Males:
Statistically significantly (p ≤0.05) decreased relative weight of spleen and liver was recorded. Relative weight of spleen was decreased in males at the dose level 150 and 600 mg/kg/day. Relative weight of liver was significantly decreased in males at all dose levels in comparison with the control males.
Relative weight of epididymis was significantly (p ≤0.05) increased in males at the dose level 150 mg/kg/day.
Relative weight of kidneys was insignificantly increased in males at the dose level 600 mg/kg/day.
Relative weights of other organs were in dosed males comparable with the control males.
Satellite males:
A statistically significant changes in relative weight of organs were not recorded in satellite treated males compared to satellite control males. Insignificantly increased weight of kidneys was noted in satellite treated males.
Females:
A statistically significantly changed relative weight of organs were recorded in dosed females in comparison with the control females. Significantly (p ≤0.05) increased relative weight of kidneys and liver was recorded in all dosed groups of females.
Satellite females:
Statistically significant difference was recorded in the relative weight of kidneys (p ≤0.05) in satellite females. Relative weight of kidneys was increased in treated satellite females in comparison with the control satellite females.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Males
Control: no macroscopic findings were recorded in all 6 males.
150 mg/kg/day: no macroscopic findings were recorded in all 6 males.
300 mg/kg/day: no macroscopic findings were recorded in all 6 males.
600 mg/kg/day: no macroscopic findings were recorded in 1 male. Very light colour of kidneys was recorded in 5 males.
Satellite males:
Control satellite: no macroscopic findings were recorded in all 6 males.
600 mg/kg/day satellite: no macroscopic findings were recorded in 1 male. Very light colour of kidneys was recorded in 5 males.
Females:
Control: no macroscopic findings were recorded in all 6 females.
150 mg/kg/day: no macroscopic findings were recorded in all 6 females.
300 mg/kg/day: no macroscopic findings were recorded in 2 females. Very light colour of kidneys was recorded in 4 females.
600 mg/kg/day: Very light colour of kidneys was recorded in all 6 females.
Satellite females:
Control satellite: no macroscopic pathological findings were recorded in all 6 females (dilatation of uterus – non-pathological finding was recorded in two of six females).
600 mg/kg/day satellite: no macroscopic pathological findings were recorded in 2 females. Very light colour of kidneys was recorded in 4 females. (dilatation of uterus – non-pathological finding was recorded in one of six females).
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Males:
For examination of the repeated toxicity of the test item, the full histopathology was performed for the first six males of control (No 1-6) and high dose (No 61-66). Histological examination of kidneys only was also performed in males Nos. 21-26 (150 mg/kg/day) an d 41-46 (300 mg/kg/day).
Kidneys were probably target organ. Only sporadic findings on other examined organs were recorded in the control group of males as well as in dosed group of males.
Hydronephrosis in kidneys was detected in 2-4-1-2 males. Tubular necrosis in kidneys was recorded in 0-0-2-6 males.
The incidence of other microscopical findings was very sporadic and these findings are mentioned in individual tables. Mild to marked hydronephrosis was revealed as a spontaneous lesion.
No treatment-related changes were found in male genital tract.
Satellite males:
The satellite animals (control - No. 81-86 and high dosed – No.91-96) as a part of the repeated dose toxicity study were histopathologically examined. The full histopathology was performed.
Histological findings on kidneys - tubular necrosis was found out in all satellite treated males 0-6 males).
Mild to marked hydronephrosis was revealed as a spontaneous lesion. The incidence of other microscopical findings was very sporadic and these findings are mentioned in individual tables.
Histological examination revealed minimal to marked signs of tubular necrosis in kidneys in all high dose males as a direct effect of the test item administered. This lesion was histologically characterized by a variety of findings: dilatation of cortical tubules lined by flattened epithelium, sometimes was epithelium of basophilic color as a sign of reparation. Further, amorphous eosinophilic material or cellular debris were present in the lumen of some tubules, and in some cases chronic interstitial inflammation was found. This basic pattern was accompanied also by tubular vacuolation and presence of hyaline casts. Subsequent examination of animals from the middle dose group found this lesion in minimal extent, mainly vacuolation of tubular epithelium in two males. All recovered high dose males showed also this lesion.
Females:
For examination of the repeated toxicity of the test item, the full histopathology was performed for the first six females delivered pups from control (Nos. 103,105,106,109,111,112) and high dose group (Nos. 161,162,165,166,167,171).
Histological examination of kidneys as probably target organ was also performed in females Nos.121,122,123,126,128,131 (150 mg/kg/day) and 141,143,146,147,149,150 (300 mg/kg/day).
Only sporadic findings were recorded in the control group of females as well as in dosed group of females.
Kidneys were probably target organ. Only sporadic findings on other examined organs were recorded in the control group of males as well as in dosed group of females.
Hydronephrosis in kidneys was detected in 0-3-1-0 females. Mild to marked hydronephrosis was revealed as a spontaneous lesion. Tubular necrosis in kidneys was recorded in 0-5-5-6 females.
The changes related to pregnancy were found in both control and high treated females: focal accumulation of lipophages and siderophages in mesometrium of uterus in 6-/-/-4 females, hemosiderin in mucosa in 6-/-/-6 females and lobular hyperplasia of mammary gland in 6-/-/-6 females. The incidence of other microscopical findings was very sporadic and these findings are mentioned in individual tables No treatment-related changes were found in female genital tract.
Satellite females:
The satellite animals (control - No. 181-186 and high dosed – No.191-196) as a part of the repeated dose toxicity study were also histopathologically examined. The full histopathology was performed.
Histological findings on kidneys - tubular necrosis was found out in all satellite treated females (0-6 females).
The incidence of other microscopical findings was very sporadic and these findings are mentioned in individual tables.
Histological examination revealed minimal to marked signs of tubular necrosis in kidneys in all high dose females as a direct effect of the test item administered. This lesion was histologically
characterized by a variety of findings: dilatation of cortical tubules lined by flattened epithelium, sometimes was epithelium of basophilic color as a sign of reparation. Further, amorphous eosinophilic material or cellular debris were present in the lumen of some tubules, and in some cases chronic interstitial inflammation was found. This basic pattern was accompanied also by tubular vacuolation and presence of hyaline casts. Subsequent examination of animals from the middle dose group found this lesion in minimal extent, mainly vacuolation of tubular epithelium in five females and also in the low dose in five females. All recovered high dose females showed also this lesion.
Description (incidence and severity):
Thyroid hormones
Blood (serum) samples from all adult parental males were assessed for thyroid hormones thyroxine (T4 total) and rat thyroid stimulating hormone (TSH).
Mean concentrations of T4 and TSH hormones at all dosed groups were not significantly changed in comparison with the control group of males except statistically significantly decreased value in T4 con centration in males at the dose level 150 mg/kg/day. No dose-dependence was observed, therefore this decreasing was probably not due to application of the test item.
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
gross pathology
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Effect level:
< 150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
gross pathology
histopathology: non-neoplastic
Critical effects observed:
yes
Lowest effective dose / conc.:
150 mg/kg bw/day (nominal)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes

Results of the main study

                                                                                                                 

dose (mg/kg day)

notes

0

100

300

1000

Body Weight

No effects

No effects

No effects

insignificantly

decreased during the whole study

Results given as comparison to control

group

Mortality

No effects

No effects

No effects

No effects

 

Signs of toxicity

No effects

No effects

No effects

No effects

 

Haematological tests with relevant baseline values

No effects

No effects

No effects

No effects

 

Necropsy findings

No effects

No effects

Changed colour of kidneys - very light colour, in 1F

Changed colour of kidneys - very light colour, in 6F

 

Histopathological findings (out of 6 animals)

not

examined

not

examined

minimal presence of brown pigment – very light colour of kidneys was in 1F.
other changes

found (chronic pyelitis, hydronephrosis) were most probably of spontaneous origin.

marked vacuolation of the tubular epithelial cells accompanied by presence of small amount of fine brown pigment in 6F

Vacuolation of renal epithelial cells could be caused by accumulation of some substances – lipid substances, glycogen, or water, which could be distinguished by special histological staining. This tubular vacuolation was in direct relation to the test item administered.
The cause of deposition of small amount of brown pigment in renal epithelial cells is unclear. Its relation to the test item given could not be excluded. The other changes found (hyaline casts, chronic pyelitis, hydronephrosis) were most probably of spontaneous origin
Findings per animal (300 mg/jkg bw/day)
- Kidneys: no findings;
- Kidneys: fine brown pigment (minimal);
- Kidneys: chronic pyelitis (unilateral, mild);
- Kidneys: no findings;
- Kidneys: hydronephrosis (unilateral,mild);
 
- Kidneys: hydronephrosis (bilateral, minimal);
 
Findings per animal (1000 mg/jkg bw/day)
- Kidneys: tubular vacuolation (marked); fine brown pigment (mild); hyaline casts (solitary);
- Kidneys: tubular vacuolation (marked); fine brown pigment (mild); chronic pyelitis (bilateral, minimal);
- Kidneys: tubular vacuolation (marked); fine brown pigment (mild); hydronephrosis (bilateral, mild); chronic pyelitis (unilateral, minimal);
- Kidneys: tubular vacuolation (marked); fine brown pigment (mild); hydronephrosis
 (unilateral, mild);
- Kidneys: tubular vacuolation (marked); fine brown pigment (mild);
- Kidneys: tubular vacuolation (marked); fine brown pigment (mild);

Reproductive parameters

Number of implantations

No effects

No effects

No effects

statistically significantly decreased

Results given as comparison to control group

18.60 ± 1.14

18.00 ± 1.22

17.83 ± 2.14

14.67 ± 4.93*

Number of resorptions

No effects

No effects

No effects

No effects

Results given as comparison to control group

0.40 ± 0.55

1.00 ± 1.22

1.33 ± 1.51

0.67 ± 0.82

Number of corpora lutea

No effects

No effects

No effects

statistically significantly decreased

Results given as comparison to control group

20.40 ± 0.89

19.40 ± 1.82

18.50 ± 2.17

17.33 ± 3.39*

Number of live foetuses

No effects

slightly decreased

No effects

slightly decreased

 

90

85

99

84

 

Average number of foetuses per litter

One dead foetus was recorded in one litter

No effects

No effects

statistically significantly decreased

 

Number of live foetuses

18.00 ± 0.71

17.00 ± 1.87

16.50 ± 2.17

14.00 ± 4.56

 

Number of dead foetuses

0.20 ± 0.45

0.00 ± 0.00

0.00 ± 0.00

0.00 ± 0.00

 

External, soft tissue and skeletal malformations and other relevant alterations

No effects

No effects

No effects

No effects

 

Results of DRF study

                                                                                                                 

dose (mg/kg day)

notes

0

100

300

1000

Body Weight

No effects

No effects

No effects

insignificantly decreased during the whole study

Results given as comparison to control group

Mortality

No effects

No effects

No effects

No effects

 

Signs of toxicity

No effects

No effects

No effects

No effects

 

Haematological tests with relevant baseline values

No effects

No effects

No effects

No effects

 

Necropsy findings

No effects

No effects

Changed colour of kidneys - very light colour, in 1F

Changed colour of kidneys - very light colour, in 6F

 

Histopathological findings (out of 6 animals)

not examined

not examined

minimal presence of brown pigment – very light colour of kidneys was in 1F.
other changes found (chronic pyelitis, hydronephrosis) were most probably of spontaneous origin.

marked vacuolation of the tubular epithelial cells accompanied by presence of small amount of fine brown pigment in 6F

Vacuolation of renal epithelial cells could be caused by accumulation of some substances – lipid substances, glycogen, or water, which could be distinguished by special histological staining. This tubular vacuolation was in direct relation to the test item administered.
The cause of deposition of small amount of brown pigment in renal epithelial cells is unclear. Its relation to the test item given could not be excluded. The other changes found (hyaline casts, chronic pyelitis, hydronephrosis) were most probably of spontaneous origin
Findings per animal (300 mg/jkg bw/day)
- Kidneys: no findings;
- Kidneys: fine brown pigment (minimal);
- Kidneys: chronic pyelitis (unilateral, mild);
- Kidneys: no findings;
- Kidneys: hydronephrosis (unilateral, mild);
 
- Kidneys: hydronephrosis (bilateral, minimal);
 
Findings per animal (1000 mg/jkg bw/day)
- Kidneys: tubular vacuolation
(marked); fine brown pigment (mild); hyaline casts (solitary);
- Kidneys: tubular vacuolation (marked); fine brown pigment (mild); chronic pyelitis (bilateral, minimal);
- Kidneys: tubular vacuolation (marked); fine brown pigment (mild); hydronephrosis (bilateral, mild); chronic pyelitis (unilateral, minimal);
- Kidneys: tubular vacuolation (marked); fine brown pigment (mild); hydronephrosis
 (unilateral, mild);
- Kidneys: tubular vacuolation (marked); fine brown pigment (mild);
- Kidneys: tubular vacuolation (marked); fine brown pigment (mild);

Reproductive parameters

Number of implantations

No effects

No effects

No effects

statistically significantly decreased

Results given as comparison to control group

18.60 ± 1.14

18.00 ± 1.22

17.83 ± 2.14

14.67 ± 4.93*

Number of resorptions

No effects

No effects

No effects

No effects

Results given as comparison to control group

0.40 ± 0.55

1.00 ± 1.22

1.33 ± 1.51

0.67 ± 0.82

Number of corpora lutea

No effects

No effects

No effects

statistically significantly decreased

Results given as comparison to control group

20.40 ± 0.89

19.40 ± 1.82

18.50 ± 2.17

17.33 ± 3.39*

Number of live foetuses

No effects

slightly decreased

No effects

slightly decreased

 

90

85

99

84

 

Average number of foetuses per litter

One dead foetus was recorded in one litter

No effects

No effects

statistically significantly decreased

 

Number of live foetuses

18.00 ± 0.71

17.00 ± 1.87

16.50 ± 2.17

14.00 ± 4.56

 

Number of dead foetuses

0.20 ± 0.45

0.00 ± 0.00

0.00 ± 0.00

0.00 ± 0.00

 

External, soft tissue and skeletal malformations and other relevant alterations

No effects

No effects

No effects

No effects

 

Conclusions:
NOAEL (No Observed Adverse Effect Level) value for REPEATED DOSE TOXICITY in MALES was established as 150 mg/kg/day and in FEMALES was established to be less than 150 mg/kg/day
Executive summary:

The test item was tested for effects on reproduction and subacute toxicity using the OECD Test Guideline No. 422


Before the start of study with laboratory animals the stability and homogeneity of application form were determined at the test facility. A dose-range finding experiment was performed to determine the dose levels for the main study.


 


Wistar rats (SPF quality) were used for testing. The test item was administered in the form of a solution in water for injection. Oral application by stomach tube was performed daily.The animals were without feed two hours before application and two hours after application of the test item. The main study includes four main groups -3 treated groups (doses 150, 300, 600 mg/kg/day) and one control group (vehicle only) and two satellite groups of animals (one control group (vehicle only) and one treated group (600 mg/kg/day). Each main group consisted of 12 males and 12 females; each satellite group consisted of 6 males and 6 females.


 


The first six males and six mothers who delivered pups per group and a satellite groups of animals (control and treated) are part of therepeated dose toxicity studyand examined with respect to toxicity of the test item. Satellite animals were used for observation of reversibility, persistence or delayed occurrence of systemic toxicity effects up to 14 days post treatment. All twelve males and females per group are a part of thereproduction studyand examined with respect to reproduction parameters.


 


The treated groups were administered daily. During the study, clinical observation and health status controls were performed daily. The body weight and food consumption were measured weekly or at the specified time intervals. Detailed clinical observation was carried out weekly. Functional observations were performed at the end of the application and observation periods. Vaginal smears were prepared daily, 2 weeks before start of the administration period (oestrous cycle monitoring), during the mating period (until the presence of spermatozoa) and at necropsy. Reproduction parameters relevant to pups (number of pups, weight of litters and weight of pups, sex and vitality of pups, measurement of anogenital distance, nipple retention, serum levels of thyroid hormones (T4 and TSH in pups) were also recorded. The study was completed with urinalysis, haematological and biochemical analysis and gross necropsy of animals. In all males of the main groups, the sperm parameters, sperm motility and sperm morphology were examined. Selected organs from adult animals and pups were removed for weighing and histopathological examination.


 


Results


Repeated oral administration of the test item did not cause the death of animals except one female from the dose level 150 mg/kg/day/day, who was found dead on day 25 of application; the cause of death was not determined due to partial autolysis of organs. This death was accidental and not treatment-related.


Test item treatment did not produce clinical changes in health status of animals, did not affect the normal growth of males and females.


 


Repeated Dose Toxicity part of study


The haematological examination did not reveal toxic effect of the test item on administered animals.


The biochemical examination of treated animals showed a toxicologically significant effect of the test item on the treated animals. Changes of Creatinine concentration in serum (irreversibly increased values in males and females) and increased concentration of chloride ions in satellite animals can be associated with the findings reported during the histological examination of kidneys.


The examination of urine parameters showed the presence of blood and leucocytes in urine of animals from middle and highest dose level.


The test item had toxicology significant effect on weight of organs of treated animals. The weight of kidneys (absolute and relative) was significantly increased in all dose groups of females and this increase was irreversible. Insignificant increase of relative weight of kidneys was also reported in males. This was confirmed by pathological and histopathological examination, where the findings would indicate a toxic effect of the test item on kidneys of dosed animals.


During the macroscopic examination, findings related to the test item treatment were found out. Changed colour of kidneys in the mid and high dose groups. 


Histological examinationrevealedminimal to marked signs of tubular necrosis in kidneys of rats administered by highest dose of test item (600 mg/kg).Thislesion was subsequently revealed in some rats from the middle and low dose groups (300, respective 150 mg/kg). This lesion was irreversible during recovery period. All recovered high dose males and females showed also this lesion.


 


CONCLUSION


According to the study results the NOAEL (No Observed Adverse Effect Level) value for REPEATED DOSE TOXICITY in MALES was established as 150mg/kg/day and in FEMALES was established to be less than 150 mg/kg/day.


This value is based on toxicologically significant changes recorded in treated males and females. Damage of kidneysconfirmed by the histological examination, evaluation of biochemical parameters and biometry of organs was observed. Histopathological finding -tubular necrosis in the kidneys –was reported in a middle and high dose level in males and in all dose levels of females. This finding was irreversible. All satellite high dose males and females showed also this lesion at the end of recovery period.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
80 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

The substance under registration OB 2-DSA belongs to the category of Stilbene Fluorescent Whitening Agents. The repeated dose toxicity of this category of substances was extensively explored and data on the toxic effects, after oral repeated exposure, are available on the substance as such and on similar substances belonging to the same category. Details on the Read Across approach for category are reported in a document attached in IUCLID section 13.


As for the standard requirements of this registration, the repeated dose toxicity potential is assessed through Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening (OECD 422).


Repeated oral administration at doses of 150, 300 and 600 mg/kg bw/day to rats by gavage did not cause the death of animals. Test item treatment did not produce clinical changes in health status of animals, did not affect the normal growth of males and females.


The haematological examination did not reveal toxic effect on administered animals. The isolated findings were found in all dose levels. These findings were not associated with any pathological and/or histopathological findings of haematogenous organs. Haematological examination in males did not reveal changes of the red blood components in dosed groups in comparison with the control group. Only one statistically significant difference in haemocoagulation parameter - increased value of fibrinogen at the dose level 600 mg/kg/day was recorded. Sporadic changes in the values of white blood components were recorded in treated groups of males. Statistically significantly increased value of lymphocytes and decreased values of neutrophils were recorded in dose groups 150 and 300 mg/kg/day. Decreased value of eosinophils was recorded in males at the dose level 300 mg/kg. All changes were reversible. No statistically significantly differences were recorded in satellite treated group of males compared to satellite control group. Haematological examination in females showed the changes in values of red blood components in treated groups of females. Statistically significantly increased value of RBC associated with increased value of Hct and decreased MCV were recorded in females at the dose level 600 mg/kg/day in comparison with the control group of females. The values of haemocoagulation parameters were not significantly affected by the test item treatment. The white blood components were not affected by the test item treatment. On the contrary, a decreased value of RBC and associated decreased value of Hct and Hgb were recorded in satellite treated females in comparison with the satellite control females. However, all haematological values of treated animals (males and females) were in a historical control range. The biochemical examination of treated animals showed a toxicologically significant effects on creatinine concentration in serum (irreversibly increased values in males and females) and increased concentration of chloride ions in satellite animals. These finding can be associated with the findings reported during the histological examination of kidneys. Moreover, during the biochemical examination of males, significantly irreversibly changed values (decreased) of Alanine aminotranferase (ALT) and reversibly decreased Urea (BUN) in all dosed groups of males were recorded. Decreased value of Glucose (GLU) in males at the dose level 300 and 600 mg/kg/day and decreased concentration of Calcium ions (Ca) in males at the dose levels 150 and 300 mg/kg/day were recorded in comparison with the control group of males. The value of Creatinine (Crea) was irreversibly increased in males at the dose level 600 mg/kg/day. In satellite males a statistically significant increased value of Creatinine (Crea) and concentration of Chloride (Cl) ions were recorded in satellite treated males compared to control males. Significantly decreased value of Alanine aminotranferase (ALT) and Cholinesterase (CHE) were also recorded in satellite treated males.


As for females, statistically significantly changed biochemical values were recorded only sporadically. Increased value of Total Cholesterol (T-Chol) was reported in females at the dose level 150 and 600 mg/kg/day and increased value of Triglycerides (TG) was recorded in females at the dose level 150 mg/kg/day only. Decreased value of Glucose (GLU) and concentration of sodium ions (Na) were recorded in females at the dose level 150 mg/kg/day only. In satellite treated females, increased values of Creatinine (Crea) and Chloride (Cl) ions and decreased value of Alanine aminotranferase (ALT) were noted in satellite treated females in comparison with the satellite control females. Although the value of Creatinine (Crea) was not increased statistically significantly in females, dose dependence and elevated values in treated groups compared to control were recorded. This trend was confirmed by a statistically significantly increased value of creatinine reported in a satellite treated females. All values except the value of Triglycerides were in a historical control range. Increased value of Triglycerides (TG) were out of historical control range in all dosed groups of females. The examination of urine parameters showed the presence of blood and leucocytes in urine. An increased pH of urine (statistically significantly) was detected in males at the dose level 150 mg/kg/day. Specific gravity was increased in males at the dose levels 300 and 600 mg/kg/day in comparison with the control group of males. Also presence of blood was recorded in males at the dose level 300 and 600 mg/kg/day. The presence of leucocytes was recorded in treated males as well as in control males, but in males at the dose level 300 and 600 was presence of leucocytes in the urine more frequent. In satellite males the volume and pH of urine was statistically significantly decreased in treated males. The blood in urine was recorded in one satellite treated male only.


The test item had toxicology significant effect on weight of organs of treated animals. The weight of kidneys (absolute and relative) was significantly increased in all dose groups of females and this increase was irreversible. This was confirmed by pathological and histopathological examination, where the findings would indicate a toxic effect of the test item on kidneys of dosed animals. In males, statistically significantly decreased absolute and relative weight of spleen were recorded at the dose levels 150 and 600 mg/kg/day. Absolute weight of liver was decreased insignificantly, but relative weight of liver was decreased significantly in males at all dose levels. Increased absolute weight of liver was recorded in dosed females. Statistically significant increasing was recorded at the dose levels 150 and 300 mg/kg/day and insignificantly increased value at the dose level 600 mg/kg/day. Relative weight of liver was significantly increased in females at all dose groups. In satellite treated females, a statistically significant changes in absolute and relative weight of kidneys were recorded in treated animals in comparison with the control satellite animals. During the macroscopic examination, findings related to the test item treatment were found out. Changed colour of kidneys.


Histological examination revealed minimal to marked signs of tubular necrosis in kidneys of rats administered by highest dose of test item (600 mg/kg). This lesion was subsequently revealed in some rats from the middle and low dose groups (300, respective 150 mg/kg). This lesion was irreversible during recovery period and was histologically characterized by a variety of findings: dilatation of cortical tubules lined by flattened epithelium, sometimes was epithelium of basophilic color as a sign of reparation. Further, amorphous eosinophilic material or cellular debris were present in the lumen of some tubules, and in some cases chronic interstitial inflammation was found. This basic pattern was accompanied also by tubular vacuolation and presence of hyaline casts. Histological examination of kidneys was expanded in the case of macroscopic findings in kidneys (very light colour) of test animals. Tubular necrosis in kidneys was also reported in 2 male of the middle group and in 7 females of the low and middle group. Accordingly, the NOAEL value for repeated dose toxicity in males was established as 150 mg/kg/day and in females was established to be less than 150 mg/kg/day.


 


A further test carried out according to the OECD guideline 422 and under GLP on the analogue substance, OB 3a-DSA, supports these outcomes in terms of target organs and was used in weight of evidence within a read-across approach. This derivative was tested at doses of 80, 250 and 750 mg/kg/day. Decreases of body weight were reported and were marked in the highest dose level in both sexes and can be considered as biologically significant. No effect of the test item on the food consumption was recorded during the study. Slight changes of these parameters were without toxicological importance. No clinical findings revealed influence of the test item on clinical status of treated animals and satellite treated animals were recorded. Statistically significant differences were registered during the haematological examination in red blood components – irreversible increased value of platelet count and delayed decreased value of mean corpuscular volume in males, delayed decreased value of total erythrocyte count, accompanied with decreased values of haemoglobin and haematocrite in satellite treated females. Statistically significantly increased value of fibrinogen in males at the middle and highest dose levels was also recorded. In white blood components, statistically significantly increased total leucocyte count in males in the highest dose level and in females in all treated groups was recorded. Decreased percentage portion of monocytes in males in all treated group (without dose dependence) and percentage portion of eosinophils in males in the highest dose level and in satellite treated males were observed. The values of total leucocyte count (WBC) and fibrinogen in males were out of the historical control limits. Increased values of WBC may be related to the organism´s reaction to the test item administration. This effect was reversible, no significant change of value of WBC was observed in satellite treated animals. Biochemical examination showed following statistically significant differences in males: increased value of creatinine (dose-dependent, irreversible) in the middle and highest dose level; in males in the highest dose level this increasing was out of historical control limits, irreversible increased activity of AST, decreased activity of ALP in all treated dose levels, decreased value of bilirubin total in the middle and highest dose levels, decreased value of protein total, albumin and bile acid in the highest dose level and delayed increased value of cholinesterase. In females the values of cholesterol total, triglycerides and inorganic phosphorus (dose dependently) were statistically significantly increased in all treated groups. Increased concentration of inorganic phosphorus was recorded also in satellite treated females; this value was out of historical control limits. Statistically significantly increased value of glucose in middle and highest dose levels was detected (dose dependently). Activity of AST was statistically significantly increased in females in the highest dose level. Concentration of potassium ions in females in the lowest dose level (80 mg/kg/day) was statistically significantly decreased. Delayed increasing of values of calcium ions and bile acid were detected in satellite treated females. Irreversible increased concentration of creatinine in males and irreversible increased concentration of inorganic phosphorus in females can be considered as biologically significant and can be related to the histopathological findings of kidneys (tubular necrosis), that were found out during the histopathological examination. The examination of urine parameters in males showed statistically significantly decreased urine volume in lowest and highest dose levels and in satellite treated males. Moreover, presence of protein in highest dose level and leucocyte in all treated groups were recorded. The presence of blood was recorded in males of middle and highest dose levels. Changed urine parameters can be related to histopathological findings of kidneys that were found out during the histopathological examination.


Biometry of organs showed statistically significantly delayed increased absolute weight of spleen in both sexes, statistically significantly increased absolute weight of kidneys in females at the middle and highest dose levels and in satellite treated animals of both sexes. Relative weight of kidneys was increased in males in the highest dose level and in satellite treated males and also in females in the middle, highest dose levels and in satellite treated females. This increasing of kidneys was irreversible. In spleen, relative weight was irreversibly statistically significantly increased in satellite treated males and dose-dependently, irreversibly in females at all treated groups. Relative weight of liver was increased in females in the middle and highest dose levels (irreversibly). Irreversible increased relative weight of kidneys in males and females can be related to histopathological findings of kidneys that were found out during the histopathological examination.


Histopathological examination showed minimal to marked signs of tubular necrosis in kidneys in two males in the middle dose level, six males in the highest dose level and in six males in the satellite treated males. The same finding was found out in three females in the middle dose level, six females in the highest dose level and two satellite treated females. This lesion was histologically characterized by variety of findings: dilatation of cortical tubules lined by flattened epithelium, sometimes was epithelium of basophilic colour as a sign of reparation. Further, amorphous eosinophilic material or cellular debris were present in the lumen of tubules, and in some cases chronic interstitial inflammation was found. This basic pattern was accompanied also by tubular vacuolation and presence of hyaline casts. Histological examination of kidneys was expanded in the case of macroscopic findings in kidneys (very light colour) of test animals. Totally the tubular necrosis on kidneys was recorded in four males in the middle, twelve males in the highest dose level, in seven females in the middle dose levels and eight females in the highest dose level. Based on these findings, the NOAEL for repeated dose toxicity in males and females was established as 80 mg/kg body weight/day.


 


In conclusion, the NOAEL values found in all the studies above mentioned are in line with the one obtained for the target substance, confirming an analogous behavior for the disulfonated subcategory after prolonged repeated oral exposure. In this respect, a conservative value was selected from the study carried out on OB 3a-DSA, according to OECD TG 422, were the NOAEL was established to be 80 mg/kg bw/day.

Justification for classification or non-classification

According to the CLP Regulation (EC 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 judgement, 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.

Classification in Category 2 is applicable, when significant toxic effects observed in a 90-day repeated-dose study conducted in experimental animals are seen to occur within the guidance value ranges as:

- oral (rat): 10 < C ≤ 100 mg/kg bw/day

The No Observed Adverse Effect Level was established at 80 mg/kg bw/day, on the basis of the results from an OECD 422 study on rats. The LOAEL is therefore extrapolated as 250 mg/kg bw/day. The duration of the treatment was 49 days for males and 54 for females.

As reported in the CLP guidance, Annex I: 3.9.2.9.5. "The guidance values refer to effects seen in a standard 90-day toxicity study conducted in rats. They can be used as a basis to extrapolate equivalent guidance values for toxicity studies of greater or lesser duration, using dose/exposure time extrapolation similar to Haber’s rule for inhalation, which states essentially that the effective dose is directly proportional to the exposure concentration and the duration of exposure. The assessment shall be done on a case-by-case basis; for a 28-day study the guidance values below is increased by a factor of three."

For this reason, taken into account the duration of treatment of males, an assessment of 1.8 was applied and the extrapolated limit values for classification were adapted as follows:oral (rat): 18 < C ≤ 180 mg/kg bw/day.

In conclusion, as the LOAEL value falls above the upper limit of the range of classification, the substance is not classified for repeated dose toxicity according to the CLP Regulation (EC 1272/2008).