3-methyl-1-vinyl-1H-imidazolium methyl sulphate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline Study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

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)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

3-methyl-1-vinyl-1H-imidazolium methyl sulphate

EC Number:

247-832-2

EC Name:

3-methyl-1-vinyl-1H-imidazolium methyl sulphate

Cas Number:

26591-72-0

Molecular formula:

C6H9N2.CH3O4S

IUPAC Name:

1-ethenyl-3-methyl-1H-imidazol-3-ium methyl sulfate

Details on test material:

- Name of test material (as cited in study report): 3-Methyl-1-vinyl-1 H-imidazolium methyl sulfate
- Physical state: solid
- Analytical purity: 99.97 area-% determined by HPLC-Fingerprint
- Lot/batch No.: 77761968E0
- Stability under test conditions: The stability of the test item under storage conditions over the study period was guaranteed by the sponsor,
and the sponsor holds this responsibility.
- Storage condition of test material: Room temperature

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 12 - 13 weeks
- Weight at study initiation: males: 339.0 g - 372.4g; females: 191.1 g - 225.1 g
- Housing: During the study period, the rats were housed individually in Makrolon type M III cages supplied by Becker & Co., Castrop-Rauxel, Germany (floor area of about 800 cm²), with the following exceptions:
- During overnight matings, male and female mating partners were housed together in Makrolon type M III cages.
- Pregnant animals and their litters were housed together until PND 4.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance solutions in drinking water were prepared at the beginning of the administration period and thereafter in intervals, which took into account the analytical results of the stability verification.
For the preparation of the administration solutions the test substance was weighed in a calibrated beaker depending on the dose group, topped up with drinking water and subsequently intensely mixed with a magnetic stirrer until it was completely dissolved.

VEHICLE
- Justification for use and choice of vehicle (if other than water): good sulubility in water
- Amount of vehicle (if gavage): 10 mL/kg bw (including test substance)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analyses were carried out at the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology of BASF SE, Ludwigshafen, Germany.
Analytical verifications of the stability of the test substance in drinking water for a period of 7 days at room temperature were carried out prior to the start of the study.
Given that test substance was completely miscible with drinking water, solutions were considered to be homogenous without further analysis.
Samples of the test substance solutions were sent to the analytical laboratory once at the beginning of the study for verification of the concentrations.
Of each sample, one additional reserve sample was retained. Details of the sampling schedule were recorded with the raw data.

Duration of treatment / exposure:

The duration of treatment covered a 2-week pre-mating and a mating period in both sexes, approximately 3 weeks post-mating in males and in one not pregnant female, and the entire gestation period as well as approximately 2 weeks of the lactation period.

Frequency of treatment:

once daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 100, 300 and 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on range-finder
- Rationale for animal assignment (if not random): random

Positive control:

N/A

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS/DETAILED CLINICAL OBSERVATIONS: Yes
A check for moribund or dead animals was made twice daily on working days or once daily (Saturday, Sunday or on public holidays). If animals were in a moribund state, they were sacrificed and necropsied. The examinations of these animals were carried out according to the methods established at the BASF SE laboratory for pathology, Experimental Toxicology and Ecology, Ludwigshafen, Germany.
A cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. Abnormalities and changes were documented daily for each animal.
The parturition and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams. Only particular findings (e.g. inability to deliver) were documented on an individual dam basis.
On weekdays (except Saturday, Sunday and public holidays) the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings. The day of parturition was considered the 24-hour period from about 15.00 h of one day until about 15.00 h of the following day.

Detailed clinical observations were performed in all animals once prior to the first administration and at weekly intervals during the administration period. The examinations started in the morning. The findings were ranked according to the degree of severity, if applicable.
For observation, the animals were removed from their cages by the investigator and placed in a standard arena (50 x 37.5 cm wide, with side borders which are 25 cm high). The following parameters listed were assessed:
1. Abnormal behavior in “handling”
2. Fur
3. Skin
4. Posture
5. Salivation
6. Respiration
7. Activity/arousal level
8. Tremors
9. Convulsions
10. Abnormal movements
11. Gait abnormalities
12. Lacrimation
13. Palpebral closure
14. Exophthalmos
15. Assessment of the feces discharged during the examination (appearance/consistency)
16. Assessment of the urine discharged during the examination
17. Pupil size

BODY WEIGHT: Yes
In general, the body weight of the male and female parental animals was determined once a week at the same time of the day (in the morning) until sacrifice.
The body weight change of the animals was calculated from these results.
The following exceptions are notable for the female animals:
- During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
- Females with litter were weighed on the day of parturition (PND 0) and on PND 4.
Females without positive evidence of sperm, without litter or waiting for necropsy, were weighed weekly. These body weight data were solely used for the calculations of the dose volume; therefore these values are not reported in the Summary but in the Individual Tables.

FOOD CONSUMPTION:
Generally, food consumption was determined once a week for male and female parental animals, with the following exceptions:
- Food consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (male and female F0 animals).
- Food consumption of the F0 females with evidence of sperm was determined on gestation days (GD) 0, 7, 14 and 20.
- Food consumption of F0 females, which gave birth to a litter was determined on PND 1 and 4.
Food consumption was not determined in females without positive evidence of sperm during gestation periods and in females without litter during lactation period.

HAEMATOLOGY: Yes
The following parameters were determined in blood with EDTA K3 as anticoagulant using a particle counter (Advia 120 model; Bayer, Fernwald, Germany): Leukocyte count (WBC), Erythrocyte count (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (PLT), Differential blood count, Reticulocytes.
Clotting tests were carried out using a ball coagulometer (AMAX destiny plus model; Trinity biotech, Lemgo, Germany).
Prothrombin time (Hepato Quick’s test) (HQT)
Furthermore, blood smears were prepared and stained according to WRIGHT without being evaluated, because of non-ambiguous results of the differential blood cell counts measured by the automated instrument. (reference: Hematology: Principles and Procedures, 6th Edition, Brown AB, Lea & Febiger, Philadelphia, 1993, page 101).

CLINICAL CHEMISTRY: Yes
An automatic analyzer (Hitachi 917; Roche, Mannheim, Germany) was used to examine the clinicochemical parameters :
Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), gamma-Glutamyltransferase (GGT), Sodium (NA), Potassium (K), Chloride (CL), Inorganic phosphate (INP), Calcium (CA), Urea (UREA), Creatinine (CREA), Glucose (GLUC), Total bilirubin (TBIL), Total protein (TPROT), Albumin (ALB), Globulins (GLOB), Triglycerides (TRIG), Cholesterol (CHOL), Bile acids (TBA)

URINALYSIS: Yes
The dry chemical reactions on test strips (Combur 10 test M, Roche, Mannheim, Germany) used to determine urine constituents semiquantitatively were evaluated with a reflection photometer (Miditron M; Roche, Mannheim, Germany).
Urine-Parameters examined: pH, Protein, Glucose, Ketones, Urobilinogen, Bilirubin, Blood, Specific gravity, Sediment, Color, turbidity, Volume

NEUROBEHAVIOURAL EXAMINATION: Yes
Functional observation battery (FOB)
A functional observational battery was performed in five male and 5 female animals (with litter) per group at the end of the administration period starting at about 10.00 h. The FOB started with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable. The observations were performed at random.
Home cage observations:
The animals were observed in their closed home cages; any disturbing activities (touching the cage or rack, noise) were avoided during these examinations in order not to influence the behavior of the animals. Attention was paid to:
1. Posture
2. Tremors
3. Convulsions
4. Abnormal movements
5. Impairment of gait
6. Other findings
Open field observations:
The animals were transferred to a standard arena (50 x 50 cm wide, with side borders which are 25 cm high) and observed for at least 2 minutes. The following parameters were examined:
1. Behavior when removed from cage
2. Fur
3. Skin
4. Salivation
5. Nose discharge
6. Lacrimation
7. Eyes/pupil size
8. Posture
9. Palpebral closure
10. Respiration
11. Tremors
12. Convulsions
13. Abnormal movements/stereotypy
14. Impairment of gait
15. Activity/arousal level
16. Feces excreted within 2 minutes (number of scybala discharged/appearance/consistency)
17. Urine excreted within 2 minutes (amount/color)
18. Number of rearings within 2 minutes

Sensory motor tests/Reflexes:
The animals were removed from the open field and subjected to following sensory motor or reflex tests:
1. Approach response
2. Touch response
3. Vision (“visual placing response”)
4. Pupillary reflex
5. Pinna reflex
6. Audition (“startle response”)
7. Coordination of movements (“righting response”)
8. Behavior during “handling”
9. Vocalization
10. Pain perception (“tail pinch”)
11. Other findings
12. Grip strength of forelimbs and hindlimbs
13. Landing foot-splay test

Motor activity measurement (MA)
The MA was measured on the same day as FOB was performed in 5 parental males and females (with litter) per group. The examinations were performed using the Multi-Varimex system supplied by Columbus Instruments Int. Corp., Ohio, U.S.A. For this purpose, the animals were placed in clean polycarbonate cages for the time of measurement. The number of beam interrupts was counted over 12 intervals for 5 minutes in each case.
The sequence at which the animals were placed in the polycarbonate cages was selected at random. The measurement was started at about 14.00 h. On account of the measuring variant "staggered", the starting time was varied by the time needed to place the animals in the cages. For each animal, measurement was started individually when the 1st beam was interrupted and ended exactly 1 hour later. The animals received no food or water during the measurements. After the transfer of the last animal in each case, the room where the measurements were carried out was darkened.

Sacrifice and pathology:

GROSS PATHOLOGY / HISTOPATHOLOGY: Yes
Necropsy
With one exception, all parental animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology; special attention was given to the reproductive organs. Female animal 137 was the exception, which was found dead on study day 12 in phase pre-mating.

Weight parameters
Weight assessment was carried out on all animals. The following weights were determined:

1. Anesthetized animals
2. Epididymides
3. Testes

The following weights were determined in 5 animals/sex and test group (females with litters, same animals as used for clinical pathology examinations):

1. Adrenal glands
2. Brain
3. Heart
4. Kidneys
5. Liver
6. Spleen
7. Thymus

Organ / Tissue preservation list
The following organs / tissues were preserved in neutral-buffered 4% formaldehyde or in modified Davidson’s solution:

1. All gross lesions
2. Adrenal glands
3. Aorta
4. Bone marrow (femur)
5. Brain
6. Cecum
7. Cervix
8. Coagulating gland
9. Colon
10. Duodenum
11. Eyes with optic nerve
12. Esophagus
13. Extraorbital lacrimal glands
14. Epididymides (modified Davidson’s solution)
15. Femur with knee joint
16. Heart
17. Ileum
18. Jejunum (with Peyer’s patches)
19. Kidneys
20. Larynx
21. Liver
22. Lungs
23. Lymph nodes (axillary and mesenteric)
24. Mammary gland (male and female)
25. Nose (nasal cavity)
26. Ovaries (modified Davidson’s solution)
27. Oviducts
28. Pancreas
29. Parathyroid glands
30. Pharynx
31. Pituitary gland
32. Prostate gland
33. Rectum
34. Salivary glands (mandibular and sublingual)
35. Sciatic nerve
36. Seminal vesicles
37. Skeletal muscle
38. Spinal cord (cervical, thoracic and lumbar cord)
39. Spleen
40. Sternum with marrow
41. Stomach (forestomach and glandular stomach)
42. Testes (modified Davidson’s solution)
43. Thymus
44. Thyroid glands
45. Trachea
46. Urinary bladder
47. Uterus
48. Vagina

The ovaries of animal 137 that died were fixed in 4% buffered formaldehyde solution.
The uteri of all female F0 parental animals were examined for the presence and number of implantation sites. The uteri of apparently non-pregnant animals or empty corpora uteri were placed in 1% ammonium sulfide solutions for about 5 minutes in order to be able to identify
early resorptions or implantations (SALEWSKI's method). Then the uteri were rinsed carefully with 0.9% NaCl solution. When the examinations were completed, the uteri were transferred to the Pathology Laboratory for further processing.

Paraffin embedding, sectioning and staining
After the organs were fixed, processing, examination by light microscopy and evaluation of findings were performed, staining with Hematoxylin-eosin (H&E):
all gross lesions (all affected animals per group),
control and high dose animals only:
Adrenal glands (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Bone marrow (femur) (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Brain (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Cecum (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Cervix (all animals per group)
Coagulation glands (all animals per group)
Colon (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Duodenum (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Epididymides (all animals per group)
Heart (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Ileum (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Jejunum (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Kidneys (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Liver (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Lung (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Lymph nodes (mesenteric and axillary lymph nodes) (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Ovaries (all animals per group)
Oviducts (all animals per group)
Peyer’s patches (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Prostate (all animals per group)
Rectum (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Sciatic nerve (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Seminal vesicles (all animals per group)
Spinal cord (cervical, thoracic and lumbar cords) (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Spleen (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Stomach (forestomach and glandular stomach) (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Testes (all animals per group)
Thymus (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Thyroid glands (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Trachea (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Urinary bladder (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Uterus (all animals per group)
Vagina (all animals per group)

Special attention was given on stages of spermatogenesis in the male gonads.
Female animal 137 that died unscheduled was processed histotechnically and assessed like planned.
A correlation between gross lesions and histopathological findings was attempted.

Statistics:

Detailed statistical analyses were conducted

Results and discussion

Results of examinations

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no substance-related mortalities in any of the male and female parental animals in any of the groups.
On premating day 12, one high dose F0 female (No. 137 - 1000 mg/kg body weight/day) was found dead after a gavage error.
With the exception of salivation after treatment, no clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any male or female F0 generation parental animals during the whole study including gestation and lactation periods.
Several male and female animals of dose group 3 (1000 mg/kg bw/d) showed salivation after treatment. This transient salivation for a few minutes immediately after each treatment was likely to be induced by the unpleasant taste of the test substance or by local irritation of the upper digestive tract. It is not considered to be a sign of systemic toxicity.
One sperm positive high-dose female (1000 mg/kg bw/d - No. 136), one sperm positive lowdose female (100 mg/kg bw/d - No. 118) and one sperm negative low-dose female (No. 116) did not deliver F1 pups.
One high-dose female (No. 135) showed complete litter loss (all pups stillborn or found dead on PND 0)
Male and female animals of all dose groups (1000, 300 and 100 mg/kg bw/d) did not show any abnormalities during detailed clinical observations.

BODY WEIGHT AND WEIGHT GAIN
Mean body weights and mean body weight change of the male and female F0 generation parental animals in all test substance-treated groups (test groups 1 - 3) were comparable to the concurrent control group during the entire study period.

FOOD CONSUMPTION
Food consumption of the male and female F0 generation parental animals in all test substance-treated groups (test groups 1 - 3; 100, 300 and 1000 mg/kg bw/d) was comparable to the concurrent control group during the entire study period.

HAEMATOLOGY
No treatment-related changes among hematological parameters were observed.
In males of test groups 2 and 3 (300 and 1000 mg/kg bw/d) absolute and relative eosinophil counts and additionally in males of test group 1 (100 mg/kg bw/d) relative eosinophil counts were increased. The values were within historical control ranges whereas the means of the
control were at the low part of these ranges (absolute eosinophil counts: 0.07-0.18 Giga/L, relative eosinophil counts: 1.3-3.0 %). Therefore, these alterations were regarded as incidental and not treatment-related.

CLINICAL CHEMISTRY
No treatment-related changes among clinical chemistry parameters were observed.
In males of test group 2 (300 mg/kg bw/d) alanine aminotransferase (ALT) activities were lower, whereas in females of the same test group ALT activities were higher compared to controls. Additionally, in males of test group 2 (300 mg/kg bw/d) potassium values were lower compared to controls. All these values were changed not dose-dependently and therefore these alterations were regarded as incidental and not treatment-related.
In males of test group 3 (1000 mg/kg bw/d) calcium levels were increased, but the means were within the historical control range (calcium: 2.49-2.76 mmol/L). Therefore, this change was regarded as incidental and not treatment-related.

URINALYSIS
No treatment-related changes among urinalysis parameters were observed.

NEUROBEHAVIOUR
Functional observational battery (FOB)
Home cage observations:
No test substance-related or spontaneous findings were observed in male and female animals of all test groups during the home cage observation.
Open field observations:
The open field observations did not reveal any test substance-related findings in male and female animals of all test groups.
Sensorimotor tests/reflexes:
There were no test substance-related findings in male and female animals of all test groups. Any deviations from "zero values" were equally distributed between test substance-treated groups and controls or occurred in single animals only. Therefore, these observations were considered as being incidental.
Quantitative Parameters:
No test substance-related impaired parameters were observed in male and female animals of all test groups.
Motor activity measurement (MA)
No treatment-related changes on motor activity data (summation of all intervals) was observed in the male and female animals of all dose groups in comparison to the concurrent control group.
The statistically significantly decreased number of interrupted beams in high-dosed males (test group 3 - 1000 mg/kg bw/d) in interval 2, the statistically significantly increased number of interrupted beams in mid-dosed males (test group 2 - 300 mg/kg bw/d) in interval 6 and the statistically significantly increased number of interrupted beams in high-dosed females (test group 3) in interval 2 were considered as spontaneous in nature and not treatment related.

ORGAN WEIGHTS
Absolute weights
When compared to the control group 0 (set to 100%), the mean absolute liver weight was significantly increased in male animals of the high-dose group (111 %). All other mean absolute weight parameters in males and all weight parameters in females did not show significant differences when compared to the control group 0.

When compared to the control group 0 (set to 100%), the mean relative liver weights were significantly increased in male animals of the high-dose group (112 %).
All other mean relative weight parameters did not show significant differences when compared to the control group 0.
The increase in absolute and relative liver weights in test group 3 (1000 mg/kg bw/day) in male animals was considered to be treatment - related but not adverse due to lack of histopathological correlate.

GROSS PATHOLOGY
Female animal 137 that died showed a ruptured esophagus.
All other findings occurred individually. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

HISTOPATHOLOGY:
Animal 137 showed rupture of the esophagus confirming the macroscopic diagnosis.
All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Effect levels

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

In an OECD 422 study, the test compound 3-Methyl-1-vinyl-1 H-imidazolium methyl sulfate was administered daily by gavage to groups of 10 male and 10 female Wistar rats to screen for potential repeated dose, reproductive and developmental toxicity. The animals were dosed throughout the study. For females this included two weeks prior to mating, the variable time of conception, the duration of pregnancy and at least four days after delivery.

Males were dosed for a minimum of four weeks. Thereby, both sexes were exposed up to and including the day before scheduled kill with 100, 300, and 1000 mg/kg bw/d.

Analyses confirmed the overall accuracy of the prepared concentrations and demonstrated the stability of the test substance in drinking water over a period of 7 days at room temperature.

In the clinical examinations of the parental animals F0 no toxicologically-relevant adverse differences were caused by 3-Methyl-1-vinyl-1 H-imidazolium methyl sulfate up to the limit dose of 1000 mg/kg bw/d. In the continuative investigations including the detailed clinical observation (DCO), the functional observational battery (FOB), and measurement of motor

activity (MA) no significant differences to control were observed at any dose level. No treatment related findings were observed during mating, gestation, delivery and lactation.

Concerning clinical pathology including the analyses of red and white blood cells, coagulations parameters, enzymes, substrates, electrolytes, and minerals no treatmentrelated, adverse effects were observed up to limit dose (1000 mg/kg bw/d).

Regarding pathology, the liver of male animals of test group 3 (1000 mg/kg bw/day) showed an increase in absolute (+11%) and relative (+12%) weights which was regarded to be adaptive and non- adverse in the absence of histological findings. All other pathological findings recorded were considered to be incidental in nature and not related to treatment.

The test substance led to no treatment-related changes in the genital organs of males (testes and epididymides) and females (ovaries) in the histotechnical assessment.