Zinc bis(benzenesulphinate)

Repeated dose toxicity: oral

Administrative data

Endpoint:

repeated dose toxicity: oral, other

Remarks:

combined repeated dose toxicity study with the reproduction/developmental toxicity screening test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From May 11, 2017 to Febraury 16, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2016

Deviations:

yes

Remarks:

short-term slight increase of relative humidity and temperature. Such microclimatic deviations did not impact on the study results.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Zinc bis(benzenesulphinate)

EC Number:

246-148-1

EC Name:

Zinc bis(benzenesulphinate)

Cas Number:

24308-84-7

Molecular formula:

C12H10O4S2Zn

IUPAC Name:

zinc bis(benzenesulphinate)

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Wistar CRL (SPF quality)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River via VELAZ s.r.o., Czech Republic
- Age at study initiation: 7-9 weeks on arrival (main study)
- Weight at study initiation: weight variation of animals in groups of each sex should not exceed ± 20 % of the mean weight
- Housing:
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: maintenance pelleted diet for rats and mice
- Water: ad libitum
- Acclimation period: 5 days for DRFE and 6 days for main study

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30 - 70 %
- Photoperiod: 12 hrs dark / 12 hrs light

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

Adiministration to stomach by gavage; animals treated 7 days per week at the same time (8.00 – 10.00 am).

Vehicle:

olive oil

Details on oral exposure:

Test substance was weighted into glass beaker and the beaker was replenished by olive oil. The test solution was stirred by magnetic stirrer (400 rpm) for 15 minutes. The concentrations of solution at all dose levels were adjusted to ensure the administration of 1 ml per 100 g of body weight. For each dose level concentration, the solution was prepared separately. The application forms were prepared daily just before administration. The administration of the test substance to animals was performed during one hour after preparation of application form. The stirring of solutions continued during administration.
Test substance was administered to the stomach by gavage. The animals were treated 7 days per week at the same time (7.00 – 10.00 am). The vehicle control group was administered by olive oil in the same volume.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Determination of the stability and homogeneity of the application form
1- Analytical method
Stability and homogeneity were determined by measuring the content of test substance by gravimetric method after extraction of vehicle by methyl ethyl ketone (MEK).

2- Preparation of application form
The application form for analysis was prepared in the same manner as for application to animals – i.e. solution in olive oil.

Concentration level 10 mg/10ml
600 mg of test substance was weighed into a 800 ml glass beaker calibrated to 600 ml and the beaker was replenished by the vehicle. The solution was stirred by magnetic stirrer (400 rpm) for 15 minutes.

Concentration level 1000 mg/10ml
40 g of test substance was weighed into a 800 ml glass beaker calibrated to 400 ml and the beaker was replenished by the vehicle. The solution was stirred by magnetic stirrer (400 rpm) for 15 minutes.

3- Stability of the application form
The samples were taken from the middle of the beaker content at required time intervals (0, 60 and 120 minutes) for the determination of stability of both application forms. Two samples were taken at each time interval.
Conc. levels 10 and 1000 mg/10 ml: time interval 0 min represents the time after 15 minutes of mixing by magnetic stirrer at 400 rpm.

4- Homogeneity of the application form
Conc. levels 10 and 1000 mg/10 ml: the samples were taken after 15 minutes of mixing by magnetic stirrer (400 rpm) from 3 given places - the bottom, the middle and the surface of the beaker content. Two samples were taken from each place.

5- Results of analysis
It follows from the results of analyses (homogeneity and stability) that both the application forms (10 mg and 1000 mg/10 ml) of test substance prepared at defined laboratory conditions (laboratory temperature, mixing, stirring) are homogenous and stable at least for 120 minutes from the finalisation of application form preparation.

Duration of treatment / exposure:

DRFE: 21 days

MAIN STUDY
Parental males (totally 49 days of administration):
1st day – 14th day (pre-exposure period) → 15th day - 28th day (pre-mating period, administration) → 29th day – 42nd day (mating, administration) → 43rd day – 63rd day (administration period) → 64th day (necropsy)

Satellite males (totally 49 days of administration + 14 days of observation):
1st day – 14th day (pre-exposure period) → 15th day - 63rd day (administration period) → 65th day - 77th day (observation period) → 78th day (necropsy)

Parental females:
1st day – 14th day (pre-exposure period) → 15th day - 28th day (pre-mating period, administration) → 29th day – 42nd day (mating, administration)→ gestation → lactation → day 12 post partum

Satellite females (totally 49 days of administration + 14 days of observation):
1st day – 14th day (pre-exposure period) → 15th day - 63rd day (administration period) → 65th day - 77th day (observation period) → 78th day (necropsy)

Non-pregnant females (without evidence of copulation):
1st day – 14th day (pre-exposure period) → 15th day - 28th day (pre-mating period, administration) → 29th day – 42nd day (mating, administration) → 25 days after the end of mating period

Non-pregnant females (with evidence of copulation):
1st day – 14th day (pre-exposure period) → 15th day - 28th day (pre-mating period, administration) → 29th day – 42nd day (mating, administration) → 25th day after confirmed mating

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

main study

Doses / concentrations 2

Dose / conc.:

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

Remarks:

main study

Doses / concentrations 3

Dose / conc.:

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

Remarks:

main study

No. of animals per sex per dose:

DRFE: 5/sex/dose at doses of 150, 300, 600 and 1000 mg/kg/day

Main study
Parental animals:
- control – vehicle: 12 nonpregnant females and 12 adult males
- low dose 50 mg/kg/day: 12 nonpregnant females and 12 adult males
- intermediate dose 200 mg/kg/day: 12 nonpregnant females and 12 adult males
- high dose 400 mg/kg/day: 12 nonpregnant females and 12 adult males

Satellite groups:
- control – vehicle: 6 males + 6 females (non-mated)
- high dose 400 mg/kg/day: 6 males + 6 females (non-mated)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: doses in DRFE, based on acute test result; doses in main study, based on DRFE.
- Post-exposure recovery period in satellite groups: 14 days

Positive control:

No

Examinations

Observations and examinations performed and frequency:

DRFE
- mortality: daily
- health conditions: daily
- clinical observation: twice a day
- body weight: before application and weekly
- haematological examination: day 22 of study
- pathological examination: day 22 of study

MAIN STUDY
- mortality/viability: twice daily

- body weight:
males and satellite animals: the first day of administration and then weekly;
females: the first day of administration and then weekly; during pregnancy on day 0, 7, 14, 20; during lactation on day 1, 4, 12 and 13;
pups (litters): day 1, 4, 12 and 13
pups (individually): day 4 of lactation

- anogenital distance measurement: pups, day 4 of lactation

- food consumption: weekly and on the same days as body weight (except the mating period); satellite males and females - weekly

- health condition control: daily, during the acclimatisation and the experimental part

- water consumption: satellite males and females - twice a week

- clinical observations of males and females: daily - during the administration period (after application), in natural conditions in their cages

- clinical observations of pups: as soon as possible after delivery and then daily; number and sex of pups, still births, live births and presence of gross anomalies is recorded. Changes of physical condition and behavioural abnormalities are recorded.

- detailed clinical observations: before the first application and then weekly (except the mating period); a part in cage, e.g. posture, position of eyelids, tonic or clonic movements, piloerection, stereotypic or bizarre behaviour, and a part during removal from cage, e.g. reaction to handling, elasticity of skin, colour of mucous membranes, salivation, lacrimation, cleanliness of fur around foramina.

- functional observations: at the end of administration/observation period; sensory reactivity on auditory, visual and proprioceptive stimuli, grip strength, motor activity, pupillary reflex.

- laboratory examinations: vaginal smears: daily between day 1 - 14; daily in mating period (max. 14 days); on necropsy day

- urinalysis: at the end of administratio/observation period; glucose, protein, bilirubin, urobilinogen, pH, specific weight, ketones, blood, nitrites and leucocytes and volume, cloudiness, colour and odour by examinator; only 6 males of each dose level and in satellite males.

- haematological examination: total erythrocyte count, mean corpuscolar volume, haematocrit, haemoglobin concentration, total leucocyte count, total platelets count, partial thromboplastin time, prothrombin time, fibrinogen, granulocytes, lymphocytes, monocytes, reticulocyte count
males: after the end of application period
parental females: on day 13 of lactation
satellite animals: after the end of observation period

- biochemical examination (main study only):
males and nonpregnant females: after the end of application period
2 pups per litter: on day 4 of lactation
parental females and pups: on day 13 of lactation
satellite animals: after the end of observation period
Blood samples from animals starved for 18 hours before blood collection, supported with drinking water ad libitum. Measurements of: total protein, total bilirubin, urea, creatinine, glucose, transaminases (AST, ALT), cholesterol, albumin, alkaline phosphatase (ALP), phosphorus and calcium, triglycerides, cholinesterase, bile acids; sodium, potassium and chloride ions.
Blood samples from the day 13 pups and the parental males: serum levels of thyroid hormone thyroxine (T4); further assessment of T4 in blood samples from the dams and day 4 pups, if relevant.

- observation of sperms: sperm motility and sperm morphology in parental males – during and after necropsy (only in main study, not performed in satellite males)

- nipples examination: in male pups, on day 13 of lactation

Note: functional observations, haematology, biochemistry for evaluation of repeated dose toxicity is performed only in 6 males + 6 females of each dose level and in satellite animals.

Sacrifice and pathology:

Pathological examination
Revision of the external surface of the body, of all orifices and the cranial, thoracic and abdominal cavities during necropsy. 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.

Biometry of organs
At the end of study the experimental animals were narcotised and sacrificed by cutting the neck spine and medulla. After gross necropsy of the cranial, thoracic and abdominal cavities, the organs for weighing and further histological examination were collected.
The 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 (repeated dose toxicity part of study – 6 males and females from each group + satellite groups); testes or ovaries, epididymis or uterus, prostate gland + seminal vesicles, pituitary gland (reproduction part of study – all animals). Afterwards the somatic indexes - SI (= relative weight of organ) were computed according to the following formula: SI = weight of organ × 100/ body weight.
From all adult males and females and one male and female day 13 pup from each litter, thyroid glands were preserved in fixation medium. The thyroid weight was determined after fixation.

Histopathological examination
In all males and females: pituitary gland, ovaries, uterus incl. cervix of uterus, vagina, epididymis/epididymides, prostate gland + seminal vescicles, testes, all gross lesions, thyroid gland.
In males and females of repeated dose toxicity part of study additionally: adrenal glands, aorta, brain (incl. cerebellum and med. oblongata), caecum, colon, duodenum, pancreas, rectum, salivary glands, sciatic nerve, skeletal muscle, skin, spinal cord - thoracic, spleen, stomach, thymus, thyroid gland, trachea, urinary bladder, female mammary gland area, femur, heart, ileum, jejunum, kidneys, liver, lungs, lymph nodes – mesenteric, paraaortal, oesophagus.

Tissues and organs were collected from all killed males and females at necropsy and fixed in buffered 4 % formaldehyde solution (v/v) for further histopathological evaluation. For histopathological processing the routine histopathological paraffin technique with haematoxylin-eosin staining was used.

In repeated dose toxicity part of study, the full histopathology of the preserved organs and tissues was performed for all high dose and control animals and satellite animals. Organs with macroscopical changes, liver and kidneys were examined at the lowest and middle dose level groups.
In reproductive toxicity part of study, the full histopathology of the preserved organs and tissues was performed for all high dose and control animals. Organs with macroscopical changes, thyroid gland, testes, epididymides and prostate gland were examined also at the lowest and middle dose level groups.
Detailed histological examination of testes was performed for all dose and control males from reproduction toxicity part of study (with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure).

Statistics:

Software: Statgraphic ® Centurion (version XV, USA)
Comparison between control group and 3 treated groups and satellite control group and satellite treated group.
Probability level of statistically significant result: p 0.05

Parametric tests for statistical evaluation of:
- body weight
- mean pups weight on the 1st ,4th and 13th day of lactation
- litter weight on the 1st ,4th and 13th day of lactation
- selected haematology parameters (haematocrit, haemoglobin, mean volume of erythrocyte, differential leucocyte count, values of blood coagulation)
- blood biochemistry
- urinalysis (volume, pH)
- biometry of organs (absolute, relative weight)

Test for normality: Shapiro-Wilk test
Box-Cox transformation for data not normally distributed
Levene's test for normally distributed data, to verify if standard deviations within each group are equal.
One-Way ANOVA (probability level 0.05) to detect whether there are any significant differences amongst the means.
In case of significant differences: Fisher's least significant difference - LSD test
In case of outliers: appropriate non-parametric test (Kruskal-Wallis Test, Mann-Whitney test)

Non-parametric tests 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)
- eventually histopathological findings

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

CLINICAL OBSERVATION
Males
No clinical changes were recorded in control and treated males during the application period.

Satellite males
No clinical changes were recorded in control and treated males in application period.

Females
At all control and treated females, no clinical changes were recorded during the whole study.

Satellite females
No clinical changes were recorded in control and treated females in application period.

DETAILED CLINICAL OBSERVATION
Males
The behaviour, clinical status and activity of all males of all treated groups were similar during the study and not different from males of the control group. No serious changes were found out during examination of skin, hair, eye, visible mucous membranes, respiration, poise, gait, tonic movements, clonic movements, reaction to handling and other activities of treated males.

Satellite males
The behaviour, clinical status and activity of satellite treated males were similar during the application and observation period of the study and not different from satellite control males. No serious changes were found out during examination of skin, hair, eye, visible mucous membranes, respiration, poise, gait, tonic movements, clonic movements, reaction to handling and other activities of satellite treated males.

Females
The behaviour, clinical status and activity of all females of all treated groups were similar during the study and not different from females of the control group. No serious changes were found out during examination of skin, hair, eye, visible mucous membranes, respiration, poise, gait, tonic movements, clonic movements, reaction to handling and other activities of treated females.

Satellite females
The behaviour, clinical status and activity of satellite treated females were similar during the application and observation period of the study and not different from satellite control females. No serious changes were found out during examination of skin, hair, eye, visible mucous membranes, respiration, poise, gait, tonic movements, clonic movements, reaction to handling and other activities of satellite treated females.

Mortality:

no mortality observed

Description (incidence):

No unscheduled deaths.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

MEAN BODY WEIGHT
Males
Different body weight on the 1st day of administration was caused by sequential including of animal groups to the study. Body weight of treated males of the dose levels 200 and 400 mg/kg/day at the end of the study was comparable with control group. Body weight of males of the dose level 50 mg/kg/day was increased compared to control males. Statistically significant differences in necropsy body weight were not found in treated males.

Satellite males
Different body weight on the 1st day of administration was caused by sequential including of satellite groups to the study. 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
Different body weight on the 1st day of administration was caused by sequential including of animal groups to the study.
Body weights of females at the dose level 50 mg/kg/day and control group were quite well balanced; body weights of females at the dose levels 200 and 400mg/kg/day were slightly decreased in comparison with the control group during pre-mating period.
At the end of pregnancy period, body weight of treated females of all groups was similar to body weight of females from the control group.
At the end of lactation period (on 12th day), the body weight of treated females was quite well balanced with control group of females.
Statistically significant differences in necropsy body weight were not found in treated females.

Satellite females
Body weight of satellite treated females was comparable with satellite control animals for the nearly whole time of application and recovery period. 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 substance treatment.

Satellite males
Weight increments of satellite treated males in application and recovery period were similar with satellite control males and were not adversely influenced by the test substance administration.

Females
Weight increments in treated females were quite balanced with the control females within the 1st and 2nd week of application. The evaluation of weight increments during pregnancy and lactation period is included in reproduction part of study.

Satellite females
No marked differences in weight increments were recorded between satellite treated females and satellite control females. Negative body weight increment was recorded in control as well as in treated animals.

Food efficiency:

effects observed, non-treatment-related

Description (incidence and severity):

Males
The food conversion of treated males at the dose level 400 mg/kg/day was comparable to control animals during the pre-mating period. In contrast the food conversion of treated males at the dose levels 50 and 200 was increased in comparison with the control group of males.
In the period after mating, the food conversion of males at the highest dose level 400 mg/kg/day was lower in comparison with the food conversion of control males (except the 5th week).

Satellite males
The food conversion of satellite treated males was quite similar to satellite control.

Females
The food conversion of treated females in the pre-mating period was variable but not adversely influenced by the test substance treatment.
In pregnancy period the food conversion of treated females was slightly increased against control group. At the end of lactation period the food conversion of treated females was higher in comparison with the food conversion of control females.

Satellite females
The food conversion of satellite treated females was variable compared to satellite control males, but not adversely influenced by the test substance treatment.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Males
White blood components in treated males were not significantly affected by the test substance treatment.
Statistically significant changes were found out in red blood components of treated males. The total value of erythrocytes (RBC) was dose dependently and statistically significantly decreased in treated males in comparison with the control males. This change was irreversible. This is related to dose dependent decrease of concentration of haemoglobin and value of haematocrite in treated males. Also these changes were statistically significant and irreversible. The value of reticulocytes was dose dependently and statistically significantly increased. This change was reversible.
The following changes of haemocoagulation parameters were recorded in treated males: statistically significantly decreased value of prothrombin time (PT) at the dose levels 50 and 400 mg/kg/day, and increase of fibrinogen concentration at the dose level 200 mg/kg/day.

Satellite males
White blood components of satellite treated males were not statistically significantly changed. The value of total leucocyte count (WBC) was non-significantly decreased in treated males in comparison with control males.
The following changes of red blood parameters were recorded in satellite treated males: statistically significant decrease of RBC, haemoglobin, haematocrite and value of mean corpuscular volume (MCV).

Females
No statistically significant differences were recorded in treated females.
No treatment related changes were observed in white blood component.
Non-significantly changed parameters of red blood components were recorded in treated females: dose dependently decreased value of haemoglobin and haematocrite. The value of reticulocytes in females at the dose level 400 mg/kg/day was increased compared to control value (it was caused by the extremely high finding in one female only).

Satellite females
Statistically significantly decreased value of haemoglobin was recorded in satellite treated females. Other values were similar with satellite control females. Parameters of white blood component were not significantly changed.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Males
Statistically significantly increased concentration of total protein (T-Pro) in males at the dose level 400 mg/kg/day, total cholesterol (T-Chol) in males at the dose level 200 and 400 mg/kg/day (dose dependent), albumin (ALB) in males at the dose level 400 mg/kg/day, concentration of calcium (Ca) in males at the dose level 400 mg/kg/day (dose dependent), phosphorus (IP) in males at the dose level 200 and 400 mg/kg/day (dose dependent) and cholinesterase (CHE) in males at the dose level 200 and 400 mg/kg/day.
Statistically significantly decreased concentration of total bilirubin (T-Bil) in males at the dose levels 50, 200 and 400 mg/kg/day (dose dependent), glucose (GLU) in males at the dose level 200 and 400 mg/kg/day (dose dependent) were recorded.
The values of alanine aminotranferase (ALT) were dose-dependently increased in treated males (without statistical significance).
Values of other biochemical parameters of treated males were quite well-balanced compared to control.

Satellite males
Statistically significant increase of urea (BUN) concentration and glucose (GLU) were recorded in satellite treated males.
Values of other biochemical parameters of satellite treated males were similar to the satellite control group.

Females
Statistically significantly increased concentration of total protein (T-Pro) in females at the dose level 400 mg/kg/day, total cholesterol (T-Chol) in females at the dose level 400 mg/kg/day and alanine aminotranferase (ALT) in females at the dose levels 50 and 200 mg/kg were recorded.
Statistically significantly decreased value of creatinine (Crea) in females at all dose levels (dose dependent, irreversible) was recorded.

Satellite females
The following statistically significant differences were found out in treated satellite females: increased value of alkaline phosphatase (ALP), triglycerides (TG), glucose (GLU); and decreased value of creatinine (Crea).

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Urinalysis was performed only in males (six of each group) during the last week of the study. Statistical evaluation was performed for pH and volume of urine.

Males
Statistically significant differences were not recorded in treated males. Presence of proteins, blood and leucocytes were recorded in treated males as well as in control males that is why these findings were not associated with the application of the test substance.

Satellite males
Statistically significantly decreased pH of urine in males at the dose level 400 mg/kg/day was recorded. Presence of proteins, blood and leucocytes were recorded in satellite treated males as well as in satellite control males.

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 activity – number of upstanding in treated males was quite well-balanced with control except slight decrease at the highest dose level. 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. The activity – number of upstanding in treated males was increased compared to control males.

Females
Reactions to touch, noise, pain and pupillary reflex of treated females were the same as in the control females. The activity – number of upstanding in treated females was quite well-balanced with control. 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.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

ABSOLUTE ORGAN WEIGHT
Males
Absolute weight of kidneys and liver was statistically significantly increased at all dose levels in comparison with control males (markedly and with dose dependence). Statistically significant increase of absolute weight of thyroid gland and decrease of absolute weight of brain were recorded in males at the dose level 200 mg/kg/day.
Insignificantly increased weight of adrenal glands (dose dependent) was recorded in treated groups of males compared to group of control males.

Satellite males
Statistically significant differences as increased absolute weight of kidneys and thyroid gland and decreased weight of epididymis were detected in satellite treated males. Absolute weight of adrenal glands and liver was insignificantly increased in treated satellite males.

Females
Statistically significant difference was observed only in absolute weight of kidneys: increase at the dose levels 200 and 400 mg/kg/day. Insignificant increase absolute weight of kidneys was also noted in kidneys at the dose level 50 mg/kg/day – so increase of absolute weight of kidneys was dose dependent.

Satellite females
Statistically significant differences were recorded in satellite females: decreased weight of brain and increased weight of liver and thyroid gland.

RELATIVE ORGAN WEIGHT
Males
Relative weight of kidneys and liver was statistically significantly increased in males at all dose levels (dose dependently) in comparison with the control males. Also relative weight of adrenal glands was statistically significantly increased in males at the dose levels 200 and 400 mg/kg/day. Increased weight of thyroid gland in males at the dose level 200 mg/kg/day (statist. significantly) and decreased weight of heart in males at the dose level 50 mg/kg/day were noted.

Satellite males
The following statistically significant differences were detected in satellite treated males compared to control males: increase of kidneys, liver and thyroid gland weight. Insignificantly increased weight of adrenal glands was also noted in satellite treated males.

Females
Statistically significant differences were recorded in treated females. Statistically significant increase relative weight of liver, kidneys (dose dependently) and pituitary gland was recorded.

Satellite females
Relative weight of liver and thyroid gland of treated females was statistically significantly increased. Insignificantly increased relative weight of kidneys was also noted in satellite treated females.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

DRFE
Males:
150 mg/kg/day: lighter colour of the kidneys was observed during necropsy in all treated animals.
300 mg/kg/day: change of colour (light grey-green) and enlarged kidneys in all treated animals.
600 mg/kg/day: change of colour (light grey-green or light brown) and enlargement of kidneys during necropsy.
1000 mg/kg/day: enlarged kidneys in all treated males and changed colour (lighter) of the kidneys in 4 males; enlarged spleen and liver in one male.

Females - all dose levels: no serious macroscopical changes at necropsy

MAIN STUDY
Males
Control: no macroscopical findings were recorded in all 6 males.
50 mg/kg/day: no macroscopical findings were recorded in 3 of 6 males. Light brown colour of kidneys in 3 males (and enlarged kidneys in one of them) and light brown colour of liver in one male were recorded.
200 mg/kg/day: enlarged kidneys and/or light colour of kidneys were recorded in all 6 males. Enlarged liver in 3 males and lighter colour of liver in one male were noted.
400 mg/kg/day: enlarged kidneys with light colour with irregular ochre foci were recorded in all 6 males. Enlarged liver in all 6 males was also noted. In one male marked dilatation of urether and in other males reduced epididymides, testes and prostate gland were recorded.

Satellite males
Control satellite: no macroscopical findings were recorded.
400 mg/kg/day satellite: no macroscopical findings were recorded in 2 of 6 males. In 4 males slightly enlarged kidneys were recorded.

Females
Control: no macroscopical findings were recorded in all 6 females.
50 mg/kg/day: no macroscopical findings were recorded in all 6 females.
200 mg/kg/day: no macroscopical findings were recorded in all 6 females.
400 mg/kg/day: no macroscopical findings were recorded in all 6 females.

Satellite females
Control satellite: no macroscopical findings were recorded.
400 mg/kg/day satellite: no macroscopical findings were recorded (except dilatation of uterus in 2 females).

Neuropathological findings:

not examined

Description (incidence and severity):

All results are reported in the paragraph below.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Full histopathology of the preserved organs and tissues was performed for high dose and control animals and satellite animals. Because of probably treatment related changes at the highest dose level and some macroscopical findings at the lowest and the middle dose level, the histopathological examination of macroscopically changed organs and then of kidneys and liver was performed at the middle and the lowest dose level.

The incidence of affected males is expressed in numeric form and ranged in sequence of dose levels 0-50-200-400 mg/kg/day and 0S-400S in satellite groups.

Males
In liver and kidneys histological changes related to test substance treatment were recorded. Hepatocellular hypertrophy (hypertrophic hepatocytes present in centrilobular area) of various intensity was detected in liver of 0-1-5-6 males. Hyaline droplets occurred in tubular epithelium of kidneys in 0-6-6-6 males and this finding was accompanied by chronic progressive nephropathy (very early or early stage) in 0-3-5-5 males. Also susp. decreased volume of colloid and/or susp. condensation of colloid in thyroid gland of 0-1-2-6 males was probably caused by the test substance administration.
Then sporadic findings without connection to the test substance treatment were found out. Inflammation of stomach mucosa was diagnosed in 0-/-/-2 males. Sporadic atrophic acines in prostate gland of 2-0-0-0 males and chronic inflammation (of various intensity) in prostate gland of 1-1-2-2 males were detected. In testes the following microscopical changes were recorded: tubular dystrophy (affection of max. 10 % of tubules) in 0-0-3-0 males, tubular dystrophy (affection of 100 % of tubules) only in 0-0-0-1 male and tubular atrophy (affection of max. 10 % of tubules) in 0-2-0-1 males. Mild hemosiderosis was found out in spleen of 0-/-/-3 males.
Incidence of other microscopical findings was very sporadic.

Satellite males
Chronic progressive nephropathy (very early, early or advanced stage) detected in kidneys of 0-6 satellite males related to the test substance treatment.
Also susp. decreased volume of colloid and/or susp. condensation of colloid in thyroid gland of 0-2 satellite males was probably caused by the test substance administration.
Other findings did not relate to the test substance treatment. In prostate gland of 0-2 satellite males chronic inflammation was detected. Mild hemosiderosis was recorded in spleen of 1-3 satellite males. Erythrocytosis of lymph nodes was noted only in satellite control males: 2-0.
Incidence of other microscopical findings was very sporadic.

Because of changed weight of liver connected with macroscopical finding, examination for focal fatty changes in liver was performed.
No findings related to the test substance treatment were recorded in males at the dose level 400 mg/kg/day compared to control males (that is why this examination was not performed for lower dose levels).

Females
In liver histological changes related to the test substance treatment were recorded. Hepatocellular hypertrophy (hypertrophic hepatocytes present in centrilobular area) of various intensity was detected in liver of 0-0-3-6 females. In kidneys and thyroid gland of females treatment related changes were not found out.
The changes related to previous pregnancy were found in both control and treated females: accumulation of siderophages in mesometrium of uterus in 4-/-/-5 females and hemosiderin in mucosa in 4-/-/-4 females.
Then sporadic findings without connection to the test substance treatment were observed: cysts in thymus of 1-/-/-2 females, mild hemosiderosis in spleen of 0-/-/-2 females, erythrocytosis of sinuses in lymph nodes of 0-/-/-2 females and cysts in thyroid gland of 0-0-1-1 females.
Incidence of other microscopical findings was very sporadic.

Satellite females
Only findings without relation to the test substance treatment were observed: hydrometra of uterus in 0-2 satellite females, cysts in thymus of 2-1 satellite females and mild hemosiderosis of spleen in 1-4 females.
Incidence of other microscopical findings was very sporadic.
Because of changed weight of liver connected with macroscopical finding, examination for focal fatty changes in liver was performed.
No findings related to the test substance treatment were recorded in females at the dose level 400 mg/kg/day compared to control females (that is why this examination was not performed for lower dose levels).

Description (incidence and severity):

HEALTH CONDITIONS CONTROL
The males and females were not distributed into the groups during the check-in, acclimatisation and the pre-exposure period with monitoring of oestrous cycle in females. No signs of diseases were recorded during that time.

Males
In control males and treated males of all dose levels no signs of diseases were recorded during the application period.

Satellite males
In satellite control males and satellite treated males of all dose levels no signs of diseases were recorded during application period. During the observation (recovery) period no changes of health status were noted in satellite treated males.

Females
In control females and treated females of all dose levels no signs of diseases were recorded during the application period.

Satellite females
In satellite control females and satellite treated females of all dose levels no signs of diseases were recorded during the application period.
During the observation (recovery) period no changes of health status were noted in satellite treated females.

Details on results:

Repeated oral administration of the test substance by gavage at the dose levels of 50, 200 and 400 mg/kg/day did not cause any mortality.
The test substance treatment did not produce changes detected in health condition control, daily clinical observation after application and functional observation of animals.
The test substance did not interfere with normal growth of treated parental animals. Body weight and body weight increments of treated animals were not significantly affected by the test substance administration.
 
Haematological examination of males revealed some parameters affected by the test substance treatment. Irreversible statistically significant changes of some parameters - decrease of total erythrocyte count (RBC) associated with decreased haemoglobin concentration and value of haematocrite (HCT) were recorded. The value of reticulocytes (RT) in treated group of males was statistically significantly increased. The mentioned haematological parameters were changed in a dose-dependent manner. Haematological examination of females demonstrated only delayed statistically significantly decreased haemoglobin concentration in satellite treated females. Non-significantly decreased value of haemoglobin and haematocrite was recorded in treated females. The value of reticulocytes was increased in females at the dose level 400 mg/kg (reversible - this increase was due to an extremely high finding in one female).
 
Some biochemical parameters were affected by treatment at 400 mg/kg/day. For males, statistically significantly increased level of total protein (T-Pro), total cholesterol (T-Chol), albumin (ALB), calcium (Ca), phosphorus (IP) and cholinesterase (CHE) were observed. Statistically significantly decreased value of total bilirubin (T-Bil) and glucose (GLU) in males at 400 mg/kg was recorded.
At 200 mg/kg/day statistically significantly increased level of total cholesterol (T-Chol), phosphorus (IP) and cholinesterase (CHE) were observed. Statistically significantly decreased value of total bilirubin (T-Bil) and glucose (GLU) was recorded.
At 50 mg/kg/day statistically significantly decreased value of total bilirubin (T-Bil) was recorded.
The increase of values of T-Chol, Ca and IP were dose-dependent.
The decrease of values of T-Bil and GLU were dose-dependent.
The values of alanine aminotranferase (ALT) were dose-dependently increased in treated males (without statistical significance).
In females some parameters were affected by treatment at 400 mg/kg/day. Statistically significantly increased concentration of total protein (T-Pro) and total cholesterol (T-Chol) were recorded and value of creatinine (Crea) was decreased. At 200 and 50 mg/kg/day, the value of alanine aminotransferase (ALT) was statistically significantly increased.
The value of creatinine (Crea) was statistically significantly decreased in a dose-dependent manner, irreversibly.
 
Biometry of organs showed significant changes in weight of liver and kidneys. These changed weights of liver and kidneys can be related to the test substance treatment, which was confirmed by the pathological and histopathological examinations.
Absolute and relative weights of liver and kidneys were statistically significantly increased in males in dose-dependent manner and irreversible. Also in females increased weight of kidneys and liver was recorded. Absolute weight of liver was increased statistically insignificantly but relative weight of liver was increased statistically significantly and in dose dependent manner. In kidneys, absolute and relative weight was increased statistically significantly and in dose dependent manner.
 
Macroscopical findings of liver and kidneys were diagnosed in treated males during the pathological examination. Enlarged liver and kidneys (with light colour and irregular ochre foci) were recorded in all males at the dose level 400 mg/kg/day (in other dose levels the same findings were noted but not in all animals). In females no macrscopical findings were recorded.
 
Histological examination recorded changes related to the test substance treatment in males. Hepatocellular hypertrophy (hypertrophic hepatocytes present in centrilobular area) of various intensity was detected in liver of males at all dose levels. Hyaline droplets occurred in tubular epithelium of kidneys in males of all dose levels and this finding was accompanied by chronic progressive nephropathy. Also susp. decreased volume of colloid and/or susp. condensation of colloidin thyroid gland of treated males was probably caused by the test substance administration.Chronic progressive nephropathy (very early, early or advanced stage) detected in kidneys of satellite males related to the test substance treatment.
In liver of treated females, histological changes related to the test substance treatment were recorded. Hepatocellular hypertrophy (hypertrophic hepatocytes present in centrilobular area) of various intensity was detected.In kidneys and thyroid gland of females treatment related changes were not found out.

Effect levels

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

haematology

histopathology: neoplastic

organ weights and organ / body weight ratios

Target system / organ toxicity

1

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

2

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

haematopoietic

Organ:

blood

erythrocyte development

Treatment related:

yes

Dose response relationship:

yes

3

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Applicant's summary and conclusion

Conclusions:

NOAEL < 50 mg/kg/day
LOAEL = 50 mg/kg/day, in males and females, based on changes of absolute and relative weight and microscopical structure of kidneys and liver, irreversible changes in haematological and biochemical values.

Executive summary:

Method

The substance was tested for reproduction and subacute toxicity using OECD guideline 422 (2016).

Wistar rats of SPF quality were used for testing. The substance was administered in the form of solution in olive oil. Oral application by stomach tube was performed daily. The study includes 4 main groups and 2 satellite groups of animals. Each main group consisted of 12 males and 12 females; each satellite group consisted of 6 males and 6 females. Main groups contained 3 treated groups (doses 50, 200, 400 mg/kg/day) and one control group (vehicle only). The satellite groups contained one control group (vehicle only) and one treated group (400 mg/kg/day). The dose levels for study were determined on the basis of results of a dose-range finding experiment.

The treated groups were dosed daily for the following periods:

males and females – 2 weeks prior to the mating period and during the mating period,

pregnant females  – during pregnancy and till the 12th day of lactation,

males  –  after mating period – totally for 49 days,

nonpregnant females (mated females without parturition) – for 25 days after the confirmed mating,

non-mated females – for 25 days after the end of mating period.

At the end of the administration period, animals of main groups were sacrificed and satellite animals were observed for the next 14 days without treatment.    

During the study, clinical observation and health status controls were performed daily. Body weight and food consumption were measured weekly or in the specified time intervals. Detailed clinical observation was carried out weekly. Functional observation was performed at the end of application and observation period. Vaginal smears were prepared daily, 2 weeks before start of administration period (oestrous cycle monitoring), during the mating period (until the presence of spermatozoa) and at necropsy day. 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) were also recorded.

The study was finished by urinalysis, haematological and biochemical analysis and gross necropsy of animals. In all males of main groups the sperm parameters, sperm motility and sperm morphology were examined. The selected organs from adult animals and pups were removed for weighing and histopathological examination.

 

Results

Repeated doses of test substance by oral route did not cause any mortality.

No changes were recorded in health condition control, daily clinical observation after application and functional observation of animals.

Test substance did not interfere with normal growth of treated parental animals; body weight and body weight increments of treated animals were not significantly affected.

Haematology

Haematological examination of males revealed irreversible statistically significant decrease of total erythrocyte count associated with decreased haemoglobin concentration and value of haematocrite. The value of reticulocytes in treated group of males was statistically significantly increased. The above mentioned haematological parameters were changed in a dose-dependent manner. These changes in males were probably connected with the test substance treatment.

Haematological examination of females demonstrated only delayed statistically significantly decreased haemoglobin concentration in satellite treated females. Non-significantly (but dose dependently) decreased value of haemoglobin and haematocrite was recorded in treated females. Decreased value of haemoglobin was recorded also in treated satellite females at the end of recovery period and the decrease was statistically significant in comparison with the control animals.  

Biochemistry

During biochemical examination of males, statistically significantly changed values were recorded. Statistically significantly increased concentration of total protein (T-Pro) in males at the dose level 400 mg/kg/day, total cholesterol (T-Chol) in males at the dose level 200 and 400 mg/kg/day (dose dependent), albumin (ALB) in males at the dose level 400 mg/kg, concentration of calcium (Ca) in males at the dose level 400 mg/kg/day (dose dependent), phosphorus (IP) in males at the dose level 200 and 400 mg/kg/day (dose dependent) and cholinesterase (CHE) in males at the dose level 200 and 400 mg/kg/day were recorded.

Statistically significantly decreased concentration of total bilirubin (T-Bil) in males at the dose levels 50, 200 and 400 mg/kg/day (dose dependent), glucose (GLU) in males at the dose level 200 and 400 mg/kg/day (dose dependent) were recorded.  

The values of alanine aminotranferase (ALT) were dose-dependently increased in treated males (without statistical significance); statistically significant increase of urea (BUN) concentration and glucose (GLU) were recorded in satellite treated males.

In females were recorded these statistically significantly changed values: increased concentration of total protein (T-Pro) in females at the dose level 400 mg/kg/day; total cholesterol (T-Chol) in females at the dose level 400 mg/kg/day and alanine aminotranferase (ALT) in females at the dose levels 50 and 200 mg/kg; statistically significantly decreased value of creatinine (Crea) at all dose levels (dose dependent, irreversible) was recorded.

The following statistically significant differences were found out in treated satellite females: increased value of alkaline phosphatase (ALP), triglycerides (TG), glucose (GLU); and decreased value of creatinine (Crea).

Biometry

Biometry of organs showed significant changes in weight of liver and kidneys.

Absolute and relative weights of liver and kidneys were statistically significantly increased in males in a dose-dependent manner and irreversible. Also in females increased weight of kidneys and liver was recorded. Absolute weight of liver was increased statistically insignificantly but relative weight of liver was increased statistically significantly and in dose dependent manner. In kidneys, absolute and relative weight was increased statistically significantly and in dose dependent manner.

These changes of liver and kidneys weights were related to the test substance treatment, as confirmed by pathological and histopathological examinations.

Pathology

Macroscopical findings of liver and kidneys were diagnosed in treated males during the pathological examination. Enlarged liver and kidneys (with light colour and irregular ochre foci) were recorded in all males at the dose level 400 mg/kg/day (in other dose levels the same findings were noted but not in all animals). In females no macroscopical findings were recorded.

Histological examination showed changes related to test substance treatment in males. Hepatocellular hypertrophy (hypertrophic hepatocytes present in centrilobular area) of various intensity was detected in liver of males at all dose levels. Hyaline droplets occurred in tubular epithelium of kidneys in males of all dose levels and this finding was accompanied by chronic progressive nephropathy. Also suspected decreased volume of colloid and/or suspected condensation of colloid in thyroid gland of treated males was probably caused by the test substance administration. Chronic progressive nephropathy (very early, early or advanced stage) detected in kidneys of satellite males related to the test substance treatment.

In liver of treated females, histological changes related to the test substance treatment were recorded. Hepatocellular hypertrophy (hypertrophic hepatocytes present in centrilobular area) of various intensity was detected. In kidneys and thyroid gland of females treatment related changes were not found out.

 

Conclusions

A NOAEL in males and females was established as lower than 50 mg/kg/day, based predominantly on changes of absolute and relative weight and microscopical structure of kidneys and liver, irreversible changes in haematological and biochemical values. The dose of 50 mg/kg/day was identified as LOAEL.