Zinc bis(benzenesulphinate)

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

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

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

Vehicle:

olive oil

Details on exposure:

Test substance was weighed 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.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: from day 29 to 42
- Proof of pregnancy: presence of spermatozoa in vaginal smears, referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Determination of stability and homogeneity of the application form
1- Analytical method
Stability and homogeneity were determined by means of measuring of a content of the 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 the 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 the 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 the 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

Details on study schedule:

- Age at mating of the mated animals in the study: sexually adult

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

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.

Positive control:

No

Examinations

Parental animals: Observations and examinations:

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: 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;

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

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

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

- 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 administration/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

- 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

- biochemical examination (main study only):
males and nonpregnant females: after the end of application period
parental females: on day 13 of lactation
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 parental males: serum levels of thyroid hormone thyroxine (T4); further assessment of T4 in blood samples from the dams, if relevant.

- observation of sperms: sperm motility and sperm morphology in parental males – during and after necropsy

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

Oestrous cyclicity (parental animals):

- vaginal smears from day 1 to 14 (pre-exposure period) for monitoring oestrus cycle; only females with regular cyclicity were selected for the study.
- daily in mating period (max. 14 days).
- vaginal smears on necropsy day to determine the stage of oestrus cycle.

Sperm parameters (parental animals):

Observations in all males (except the satellite group): sperm motility and sperm morphology.

Sperm motility
Sperm samples were taken from one epididymis and sperm motility was assessed from these samples. The motility of sperm was determined by microscopic examination of the prepared sperm suspension. The result of observation was evaluated subjectively according to following grades: 1 - fast progressive motility, 2 - slow progressive motility, 3 - no progressive motility, 4 - non-motile sperm.

Sperm morphology
Sperm samples were taken from one epididymis and sperm morphology was assessed from these samples. A smear from the sperm suspension was prepared and stained (Giemsa staining). The morphology of sperm was determined by microscopic examination.
All deviations, e.g. broken tail, abnormal form of tail, double head, amorphous head, abnormal form of neck, were recorded.

Litter observations:

- mortality/viability: twice daily

- body weight:
pups (litters): day 1, 4, 12 and 13
pups (individually): day 4 of lactation

- anogenital distance measurement: pups, day 4 of lactation

- 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.

- biochemical examination (main study only):
2 pups per litter: on day 4 of lactation
pups: on day 13 of lactation
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: serum levels of thyroid hormone thyroxine (T4); further assessment of T4 in blood samples from the day 4 pups, if relevant.

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

Postmortem examinations (parental animals):

BIOMETRY OF ORGANS:
At the end of study, the experimental animals were narcotised and sacrificed by cutting the neck spine and medulla. After the gross necropsy of the cranial, thoracic and abdominal cavities, the organs for weighing and further histological examination were collected.
The absolute weights of testes or ovaries, epididymis or uterus, prostate gland + seminal vesicles, pituitary gland were determined in the reproduction part of study for 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.

PATHOLOGICAL EXAMINATION
DRFE: necropsy of all animals of each group; observation of all macroscopic abnormalities

Main study: necropsy of all animals of each group
Tissues and organs collected from all males and females at necropsy and fixed for further histopathology evaluation: all gross lesions, pituitary gland, prostate gland, seminal vesicles, testes, epididymis/epididymides, cervix of uterus, ovaries, uterus and vagina.

HISTOPATHOLOGICAL EXAMINATION (ORGANS COLLECTED DURING NECROPSY)
In reproductive part of the study, full histopathology of 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).

Postmortem examinations (offspring):

HISTOPATHOLOGY / ORGAN WEIGTHS
Weight of thyroid glands of one male and female day 13 pup from each litter.

Reproductive indices:

All males and females basic groups were used

Fertility - group parameters
Male mating index = number of males with confirmed mating × 100 / number of males cohabited
Female mating index = number of sperm-positive females × 100 / number of females cohabited
Male fertility index = number of males impregnating a females × 100 / number of males cohabited
Female fertility index = number of pregnant females × 100 / number of sperm-positive females
Gestation index = number of females with live born pups × 100 / number of pregnant females

Offspring viability indices:

Loss of offspring - individual parameters
Pre-implantation loss = number of corpora lutea - number of implantations
Post-implantation loss = number of implantations - number of live births
Post-natal loss = number of live births - number of alive at postnatal day 4/13

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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.

Females
No clinical changes were recorded in control and treated females during the application period.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

MEAN BODY WEIGHT INCREMENT
MALES
Body weight of treated males of the dose levels 200 and 400 mg/kg/day at the end of the study was quite analogous to control group. Body weight of males of the dose level 50 mg/kg/day was slightly higher compared to control males.
Body weight increments were similar in control and treated males for the whole study.
Statistically significant differences in necropsy body weight were not found in treated males.

FEMALES
Statistically significant differences of body weights were not detected in treated females.

Pre-mating period
Different body weight on the 1st day of administration was caused by sequential including of animal groups to the study. Body weights of treated females were well-balanced among the treated groups but decreased in comparison with the control group of females.
Weight increments were variable.

Pregnancy
Mean body weight of all treated groups was slightly decreased in comparison with control (without dose dependence). Weight increments of treated females and control females were quite balanced during pregnancy.

Lactation
Only mothers (females with live pups born) were included in the evaluation of body weight increments during lactation period. Mean body weight of all treated groups was slightly decreased in comparison with control (without dose dependence). Weight increments of treated mothers were slightly higher at the end of lactation period in comparison with the control females.

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).

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.

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.

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).

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Males - hormone T4
Blood samples from the all adult parental males were assessed for serum levels for thyroid hormone thyroxine (T4 total).
Mean concentrations of T4 hormone at the dose levels 200 and 400 mg/kg/day were statistically significantly decreased in comparison with control (dose-dependently).

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.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

All results are reported in the paragraph below

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Males
Full histopathology of the preserved organs and tissues was performed for high dose and control 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 (all males) and then of kidneys was performed at the middle and the lowest dose levels.

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

Microscopical examination of reproductive organs and pituitary gland did not reveal presence of treatment related changes, only the spontaneous changes were found out in males. In testes the following microscopical changes were recorded: tubular dystrophy (affection of max. 10 % of tubules) in 0-0-4-1 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 0-3-0-1 males.
Germ cells or/and cell detritus were noted in epididymal tubules of 0-0-0-2 males. In prostate gland of 1-2-2-4 males chronic inflammation (of various intensity) was observed.
Sporadic atrophic acines occurred in prostate gland of 2-1-0-0 males. Susp. decreased volume of colloid and/or susp. condensation of colloid in thyroid gland of 0-3-3-11 males was probably caused by the test substance administration.
Microscopical findings detected in other organs (esp. in liver, kidneys and thyroid gland – related to the test substance treatment) are described in repeated dose toxicity part of study.

Females
Full histopathology of the preserved organs and tissues was performed for high dose and control animals.
Because of probably treatment related changes at the highest dose level (in kidneys) and some macroscopical findings at the lowest and the middle dose level the histopathological examination of macroscopically changed organs (all females) and then of kidneys was performed at the middle and the lowest dose level.
The incidence of affected females is expressed in numeric form and ranged in sequence of dose levels 0-50-200-400 mg/kg/day further in the text.

Microscopic examination of reproductive organs, thyroid gland and pituitary gland did not reveal presence of treatment related changes. The changes related to previous pregnancy were found in both control and treated females: accumulation of siderophages in mesometrium in 8-/-/-9 females and hemosiderin in mucosa of uterus in 9-/-/-9 females. Hydrometra of uterus (non-pathological finding related to the oestrous cycle) occurred in 0-/-1-1 females.
Microscopical findings detected in other organs (esp. in liver – related to the test substance treatment) are described in repeated dose toxicity part of study.

Other effects:

no effects observed

Description (incidence and severity):

HEALTH CONDITION CONTROL
In control and treated animals of all dose levels no signs of diseases were recorded during the application period.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Oestrous cycles were monitored before treatment starts to select for the study females with regular cyclicity. Vaginal smears of all females were monitored daily for two weeks.
Three females were placed into the satellite group for irregular oestrus cycle.

Reproductive function: sperm measures:

effects observed, treatment-related

Description (incidence and severity):

Motility of sperms was worse in groups of treated males in comparison with the control group of males (dose dependently).
Increased percentage of affected sperms was recorded during sperm morphology examination in males at treated dose levels. The highest percentage of affected sperms was recorded in males at the dose level 400 mg/kg/day.
Male ability to produce sperm that can fertilise eggs was not affected by the test substance administration.

Reproductive performance:

no effects observed

Description (incidence and severity):

Evidence of copulation was found out in all females.
Decreased number of females achieving pregnancy was recorded in females at the dose level 200 mg/kg/day. All pregnant females gave birth live pups. No abortion was recorded.
Mean duration of pregnancy was similar in treated and control groups.
Mean number of implantations and number of live born pups were not significantly changed in treated females.

Details on results (P0)

Fertility parameters
The values of mating indexes showed that mating was not negatively affected by the test substance treatment.
Fertility indexes were lower at the dose level 200 mg/kg/day.
Gestation index was comparable among the control and treated groups.
Post-implantation losses were not significantly changed in treated groups in comparison with the control group.
Post-natal losses were decreased at the dose levels 200 and 400 mg/kg.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive function (oestrous cycle)

reproductive function (sperm measures)

reproductive performance

2

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

haematology

clinical biochemistry

organ weights and organ / body weight ratios

histopathology: neoplastic

Target system / organ toxicity (P0)

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

Results: F1 generation

General toxicity (F1)

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

The total number of live pups at first check of litter after parturition, on the 4th day and 13th day of lactation was insignificantly decreased at the dose levels 200 mg/kg/day compared to control group.
The four stillborn pups were recorded in females at the dose level 50 mg/kg/day only.
Mean number of live pups at first check of litter after parturition was quite well balanced among the treated groups and the control group.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant differences were recorded. The mean body weight of pup at the dose levels 200 and 400 mg/kg/day at the first check of litter after parturition was statistically significantly decreased compared to control group. Insignificantly decreased body weight of pups of these dose levels was also recorded at the 4th day of lactation, but at the end of lactation period the mean body weight of pups among the groups was similar.
Mean weight of litter within all weighing intervals was insignificantly decreased in treated groups 200 and 400 mg/kg/day compared to control.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Hormone T4
Blood samples from the 13 day old pups were assessed for serum levels of thyroid hormone (T4). Pup blood was pooled by litter.
No statistically significant differences were recorded in mean concentration of hormone T4 in pups from treated groups against control pups.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Weight of thyroid gland was not statistically significantly changed in male and female pups of treated mothers in comparison with the control group of mothers.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The macroscopic examination was performed in all pups (except cannibalism).

Control: 167 pups were examined. One female pup died - empty stomach was recorded and one female pup was not examined due to cannibalism in one litter. In another litter one male and one female pups were not examined due to cannibalism. No macroscopical findings were recorded in other pups.
50 mg/kg/day: 171 pups were examined – in one litter one male pup and one female pup died – malnutrition, empty stomach, flatulence of stomach and intestines were recorded and two pups (male and female) were not examined due to cannibalism. In another litter four stillborn pups were found and one male pup was not examined due to cannibalism. No macroscopical findings were recorded in other pups.
200 mg/kg/day: 123 pups were examined – in one litter two male pups died - one had empty stomach, in second the cause of death was not detected. In another litter one female pup was not examined due to cannibalism. No macroscopical findings were recorded in other pups.
400 mg/kg: 148 pups were examined – litter from female No.165: one male pup was not examined due to cannibalism; female No. 166: in one female pup – malnutrition, flatulency were recorded; female No. 167: three pups (one male and two females) were not examined due to cannibalism; female No.168: in three male and four female pups swollen liver with whitish edges was recorded and eight pups (two males and six females) were not examined due to cannibalism, one dead female was not examined due to autolysis of organs; female No. 169: one male pup was not examined due to cannibalism; female No. 171: one male pup died – malnutrition and marked flatulency of gastrointestinal tract were recorded, three male and two female pups had to be euthanized due to moribund condition – pink blood was recorded and two female pups were not examined due to cannibalism; female No. 172: four pups (two males and two females) were not examined due to cannibalism. No macroscopical findings were recorded in other pups.

Developmental immunotoxicity (F1)

Description (incidence and severity):

 

Details on results (F1)

Development of pups
The presence and number of nipples in male pups were counted on day 13 of lactation - the presence of nipples in male pups was not recorded.
The anogenital distance (AGD) of each pup was measured on 4th of lactation. Statistically significantly decreased mean AGD was recorded in male pups in all treated groups, but corrected AGD values were insignificantly lower only in male pups at the dose level 50 mg/kg/day.
No differences in postnatal development of pups were observed at the control and treated groups.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

food efficiency

haematology

other: malnutrition, marked flatulency o0f gast

Overall reproductive toxicity

Reproductive effects observed:

yes

Lowest effective dose / conc.:

400 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

not specified

Dose response relationship:

yes

Relevant for humans:

not specified

Any other information on results incl. tables

The test substance treatment did not affect physical growth of parental animals (body weight, body weight increment, food consumption) in any phase of the study (before mating, during mating period, pregnancy and lactation period). 

 

Microscopical examination of reproductive organs and pituitary gland in males did not reveal presence of treatment related changes, only the spontaneous changes were found out.

In thyroid gland in males, absolute and relative weight of thyroid gland was increased – at the dose levels 200 and 400 mg/kg/day statistically significantly. Suspected decrease in volume of colloid and/or suspected condensation of colloid in thyroid gland was probably caused by the test substance administration. Serum level for thyroid hormone thyroxine in treated males was decreased in dose dependent manner, at the dose levels 200 and 400 statistically significantly. However, all findings mentioned above did not have influence to ability of reproduction of males.

Microscopic examination of reproductive organs, thyroid gland and pituitary gland in females did not reveal presence of treatment related changes.

 

Examination of sperm motility and morphology in parental males showed worsened sperm quality in treated males in comparison with the control males, but the ability of males to fertilize the females was not affected.

 

Numbers of implantations and number of pups were not influenced by the test substance treatment at any dose level. No adverse effect of the test substance treatment was observed during examination of blood concentration of thyroxine hormone in pups. Corrected anogenital distance in treated pups was comparable to the control pups. 

Macroscopical examination of pups showed increased number of cannibalism of pups and number of pups with macroscopical findings at the dose level of 400 mg/kg. Malnutrition, empty stomach, marked flatulency of gastrointestinal tract, pink blood and moribund condition were recorded in pups at the dose level 400 mg/kg/day. Cannibalism and unhealthy conditions were not associated to dams inability, as dams care well for healthy pups.

Applicant's summary and conclusion

Conclusions:

NOAEL for reproduction = 400 mg/kg/day, as all changes in parental males and females at all dose levels were considered to be of non toxicological significance.
NOAEL for development = 200 mg/kg/day, based predominantly on increased number of cannibalism and macroscopical findings as malnutrition, marked flatulency of gastrointestinal tract, pink blood and moribund condition of some pups at the dose level 400 mg/kg.

Executive summary:

Method

The substance was tested for reproduction and subacute toxicity according to OECD guideline 422.

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.

In the reproduction toxicity part of the study, 4 main groups of animals were used; each main group consisted of 12 males and 12 females. Main groups contained 3 treated groups (doses 50, 200, 400 mg/kg/day) and one control group (vehicle only). The dose levels for study were determined on the basis of results of a dose-range finding experiment at doses of 150, 300, 600 and 1000 mg/kg/day.

The treated groups were administered 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,

After the end of administration period the animals of main groups were sacrificed.  

During the study clinical observation and health status controls were performed daily. The body weight and food consumption were measured weekly or in the specified time intervals. Detailed clinical observation was carried out weekly. The 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 oral administration of the test substance to rats by gavage at the dose levels of 50, 200 and 400 mg/kg/day did not cause any mortality.

As for the reproduction part of study, the treatment with the substance did not affect physical growth of parental animals (body weight, body weight increment, food consumption) in any phase of the study (before mating, during mating period, pregnancy and lactation period).  

Slight decrease of absolute and relative weight of epididymides, testes and prostate gland with seminal vesicles at treated males was recorded. Microscopical examination of reproductive organs and pituitary gland did not reveal presence of treatment related changes, only the spontaneous changes were found out.

In thyroid gland in males, absolute and relative weight of thyroid gland was increased – at the dose levels 200 and 400 mg/kg/day statistically significantly. Suspected decreased in volume of colloid and/or suspected condensation of colloid in thyroid gland was probably caused by the test substance administration. Serum level for thyroid hormone thyroxine was decreased in dose dependent manner, at the dose levels of 200 and 400 mg/kg/day statistically significantly. However, all findings mentioned above did not influence the reproduction ability of males.

Microscopic examination of reproductive organs, thyroid gland and pituitary gland in females did not reveal presence of treatment related changes.

Examination of sperm motility and morphology in parental males showed worsened sperm quality in treated males in comparison with the control males, but the ability of males to fertilize the females was not affected.

Numbers of implantations and pups were not influenced by the test substance treatment at any dose level. No adverse effect of the test substance treatment was observed during examination of thyroxine hormone blood concentration in pups. Corrected anogenital distance in treated pups was comparable to the control pups.  

Macroscopical examination of pups showed increased number of cannibalised pups and pups with macroscopical findings upon treatment at 400 mg/kg/day. Malnutrition, empty stomach, marked flatulency of gastrointestinal tract, pink blood and moribund condition were recorded in pups at the dose level 400 mg/kg/day. It can be related to the test item treatment.

Therefore, a NOAEL of 400 mg/kg/day was established for reproduction, as all changes in parental males and females were of no toxicological significance.

A NOAEL of 200 mg/kg/day was established for development, based on dose-dependent increased number of cannibalism and macroscopical findings as malnutrition, marked flatulency of gastrointestinal tract, pink blood and moribund condition of some pups at dose level of 400 mg/kg/day.