Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April - June 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April - June 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
1996
Deviations:
yes
Remarks:
Deviations are considered not to have affected the integrity or purpose of the study.
Principles of method if other than guideline:
Deviations were noted but were not considered to have affected the integrity or purpose of the study.
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
Source:
The animals were acclimatized for eight days. A total of one hundred and twenty animals (sixty males and sixty females) were accepted into the study. At the start of treatment the males weighed 302 to 351g, the females weighed 184 to 231g, and were approximately twelve weeks old.

Initially, all animals were housed in groups of four by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding. During the pairing phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation, parturition and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.

The animals were allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad Global Certified Pelleted Diet) was used. Tap drinking water was supplied from polycarbonate bottles attached to the cage. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels except for paired animals and mated females during gestation and lactation. Mated females were also given softwood flakes, as bedding, throughout gestation and lactation.

The animals were housed in a single air-conditioned room. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored. The temperature remained within 22 ± 3 °C during the study. Transient deviations from the target range for relative humidity of 50 ± 20% were considered not to have affected the purpose or integrity of the study.

The animals were uniquely identified within the study by an ear punching system and a tail mark.
Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on oral exposure:
The test item was prepared at the appropriate concentrations in Arachis oil BP. The homogeneity of the test item formulations were confirmed by analysis. Formulations were prepared on a daily basis; all animals were dosed within 2.5 hours after dose preparation. The formulations were considered to be stable for the period between preparation and dosing.

Test item formulation were analyzed for concentration of 5,5’-Dithiodi-1,3,4-thiadiazole-2(3H)-thione. The results indicate that the prepared formulations were within 80-107% of the nominal concentration.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test item concentration in the test samples was determined spectrophotometrically.
Spectrophotometer: Camspec M550
Wavelength: ¿max at ~334 nm
Cell path length: 1 cm
Reference medium: tetrahydrofuran

The detection system was found to have acceptable linearity. The analytical procedure had acceptable recoveries of test item in the vehicle. The method of analysis was validated and proven suitable for use.

Duration of treatment / exposure:
41-47 days. Males were dosed daily throughout the study until the day prior to scheduled necropsy. Females were dosed for a two week pre-pairing period, throughout pairing and gestation and early lactation (Days 1-4). Females in parturition were not dosed on that day.
Frequency of treatment:
Once daily by gavage.

Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
12 males and 12 females per dose.
Control animals:
yes, concurrent vehicle
Details on study design:
Five dose groups (0, 30, 100, 300, and 1000) each comprising 24 animals (12 males and 12 females) were used. Dose levels were chosen based on available toxicity data including a fourteen day range-finding toxicity study.
Observations and examinations performed and frequency:
All animals were inspected twice daily for morbidity/mortality.

Clinical Observations
All animals were examined for overt signs of toxicity, ill-health and behavioral change immediately before dosing, within 30 minutes after dosing, and one hour after dosing (except for females during parturition where applicable).

Functional Observations
Prior to the start of treatment and at weekly intervals thereafter, all animals were observed at least two hours after dosing for signs of functional/behavioral toxicity. Functional performance tests were also performed on five selected males and five selected females from each dose level, on Day 4 post partum (or Day 24 post coitum for the two contingency females in Group 5), together with an assessment of sensory reactivity to various stimuli, grip strength and motor activity.

Behavioral Assessments
Detailed individual observations were performed for each animal using a purpose built arena. The following parameters were observed:

Gait
Hyper/Hypothermia
Tremors
Skin colour
Twitches
Respiration
Convulsions
Palpebral closure
Bizarre/Abnormal/Stereotypic behaviour
Urination
Salivation
Defecation
Pilo-erection
Transfer arousal
Exophthalmia
Tail elevation
Lachrymation

Functional Performance Tests
* Motor Activity;
* Forelimb/Hindlimb Grip Strength;
* Sensory Reactivity.

The following parameters were observed:

Grasp response
Touch escape
Vocalization
Pupil reflex
Toe pinch
Blink reflex
Tail pinch
Startle reflex
Finger approach

Body Weight
Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until pairing. During the pairing phase females were weighed daily until mating was confirmed. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum. Body weights were also recorded at terminal kill.

Food Consumption
During the pre-pairing period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded on Days 1 through 4 post partum.
Food efficiency (the ratio of body weight change/dietary intake) was calculated retrospectively for males throughout the study period (with the exception of the mating phase) and for females during the pre-pairing phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated for females during gestation and lactation.

Water Consumption
As increased water consumption was observed during the fourteen day range finding study , formal daily gravimetric measurement of daily water intake was performed during the pre-pairing period (beginning on Day 5) for both sexes.

Laboratory Investigations
Hematological and blood chemical investigations were performed on five males and five littering females selected from each test and control group prior to termination (Day 42 for males and Day 4 post partum or Day 24 post coitum for females). Blood samples were obtained from the lateral tail vein. Repeat blood samples were taken by cardiac puncture at termination, if required. Animals were not fasted prior to blood sampling.

Hematology
The following parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant:

Hemoglobin (Hb)
Erythrocyte count (RBC)
Hematocrit (Hct)
Erythrocyte indices
- mean corpuscular hemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular hemoglobin concentration (MCHC)
Total leukocyte count (WBC)
Differential leukocyte count
- neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count (PLT)

Blood Chemistry
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:

Urea
Calcium (Ca++)
Glucose
Inorganic phosphorus (P)
Total protein (Tot.Prot.)
Aspartate aminotransferase (ASAT)
Albumin
Alanine aminotransferase (ALAT)
Albumin/Globulin (A/G) ratio (by calculation)
Alkaline phosphatase (AP)
Sodium (Na+)
Creatinine (Creat)
Potassium (K+)
Total cholesterol (Chol)
Chloride (Cl-)
Total bilirubin (Bili)
Bile acids
Sacrifice and pathology:
Adult animals were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination. Males were killed on Day 43 or Day 44, females that littered were killed on Day 5 post partum, females that failed to achieve pregnancy or produce a litter were killed on Day 25 post coitum, and one female was killed in extremis during parturition. Surviving offspring were terminated via intracardiac overdose of suitable barbiturate agent.

All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Organ Weights
The following organs were dissected free from fat and weighed before fixation from the five selected males and five selected females from each dose group. Tissues shown with* were weighed from all remaining animals:

Adrenals
Prostate*
Brain
Seminal vesicles*
Epididymides*
Spleen
Heart
Testes*
Kidneys
Thymus
Liver
Thyroid (weighed post-fixation with Parathyroid)
Ovaries*
Uterus (weighed with Cervix)*
Pituitary (post fixation)*

Histopathology
Samples of the following tissues were removed from the five selected males and five selected females from each dose group and preserved in buffered 10% formalin, except where stated. Tissues shown with* were preserved from all remaining animals:

Adrenals
Muscle (skeletal)
Aorta (thoracic)
Ovaries*
Bone & bone marrow (femur including stifle joint)
Pancreas
Bone & bone marrow (sternum)
Pituitary*
Brain (including cerebrum, cerebellum and pons)
Prostate*
Caecum
Rectum
Coagulating gland*
Salivary glands (submaxillary)
Colon
Sciatic nerve
Duodenum
Seminal vesicles
Epididymides*•
Skin (hind limb)
Esophagus
Eyes+
Spinal cord (cervical, mid-thoracic and lumbar)
Gross lesions
Spleen
Heart
Stomach
Ileum (including peyer’s patches)
Thyroid/parathyroid
Jejunum
Trachea
Kidneys
Testes*•
Liver
Thymus
Lungs (with bronchi) #
Urinary bladder
Lymph nodes (mandibular and mesenteric)
Uterus/Cervix*
Mammary gland*
Vagina*

+ = eyes fixed in Davidson’s fluid
• = preserved in Modified Davidsons fluid
# = lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative

The tissues from five selected control and 1000 mg/kg bw/day dose group animals, any animals dying during the study, and any animals which failed to mate or did not achieve a pregnancy were prepared as paraffin blocks, sectioned at a nominal thickness of 5 µm and stained with hematoxylin and eosin for subsequent microscopic examination. The tissues shown with* from the remaining control and 1000 mg/kg bw/day animals and animals which did not achieve a pregnancy were also processed. In addition, sections of testes from all control and 1000 mg/kg bw/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined.

Detailed qualitative examination of the testes was undertaken, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell-or stage-specificity of testicular findings was noted.
Following the initial results of histopathology for high dosage animals, histopathological examination was extended to five animals of either sex (except where stated) for the low and both intermediate dosage groups for the following tissues: adrenal glands (males only), lymph nodes, pituitary (all animals), salivary gland, spleen (females only), thyroid glands, kidney, liver and thymus.
Microscopic examination was conducted by the Study Pathologist and followed by a peer review.
Statistics:
Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters:

Grip Strength, Motor Activity, Body Weight, Body Weight Change, Food Consumption during gestation and lactation.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Effects were not toxicologically significant
Mortality:
mortality observed, treatment-related
Description (incidence):
Effects were not toxicologically significance
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day, reduced body weight gain was apparent in males; by Day 43, overall body weight gain was less than 50% of their control counterparts.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Reduced food consumption (1000 and 300 mg/kg bw/day) in males; no adverse affect in females.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Males post pairing only - Food conversion efficiency was slightly lower than the control at 1000 mg/kg bw/day.
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
A number of statistically significant differences from control at 300 and 1000 mg/kg bw/day may have been associated with treatment.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Please see details on results.
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Microscopic changes in the kidney of both sexes at 1000 mg/kg bw/day, including diffuse nephropathy for one female. Histological changes were also noted for the liver at 1000 mg/kg bw/day and the thyroid gland.
Histopathological findings: neoplastic:
no effects observed
Details on results:
Mortality
One female was killed in extremis on day 40 of the study. Necropsy revealed enlarged adrenals, eleven dead foetuses in the uterus and one dead foetus in the vagina. While this death appears to be related to difficulties associated with parturition, as a number of females were observed with similar, but less severe, clinical signs at the same stage of the study, a direct association with treatment cannot be discounted.

Clinical Observations
At 1000 mg/kg bw/day, treatment of males was associated with a low incidence of increased post-dosing salivation. Although all males were affected to some extent, the incidence for individual animals was infrequent given the length of dosing for the study; eight males showed this sign on five or less occasions, with a further four males showing increased salivation on seven or eight occasions during the study (one of these males also showed noisy respiration on one single occasion). Similar post-dosing salivation was also observed for females at 1000 mg/kg bw/day but at a lower incidence; only eight females were affected and for all these females the sign was only observed for three occasions or less.
At 300 mg/kg bw/day, eight males showed increased post dosing salivation; for five males it was only observed on one single occasion during the study and for the remaining two males it was only observed on two separate occasions. One of these males also showed noisy respiration on a single occasion. At 100 mg/kg bw/day, five males showed increased post dosing salivation; for three males it was only observed on one single occasion during the study and for the remaining two males it was only observed on two separate occasions.
Increased post-dosing salivation was not observed for either sex at 30 mg/kg bw/day or for females at 100 or 300 mg/kg bw/day.
The low incidence of salivation on the study did not indicate any obvious systemic toxicity. Similar post-dosing salivation is frequently observed when the test item formulations are slightly irritant or unpalatable but the low incidence for this finding did not indicate a significant problem relating to irritancy or palatability. The increased post-doing salivation incidence observed in this study most probably represents occasional difficulties in dosing animals, differences in individual dosing techniques and slight differences in subjective calling levels for this observation and was considered to be of no toxicological significance.
At 1000 mg/kg bw/day, one male (number 105) showed piloerection and hunched posture on the morning of Day 44 (day of scheduled termination). No similar findings were apparent for other males on the study.
Also at 1000 mg/kg bw/day, female number 118, as previously discussed, showed adverse clinical signs and was killed around the time of expected parturition (Day 40). Female number 116 showed piloerection and hunched posture on Days 41 and 42, gave birth but cannibalized her single offspring on Day 43 and showed further piloerection and hunched posture on Days 44 to 46. Female number 117 showed piloerection during Days 41 and 42, which for this animal represented Days 22 and 23 post coitum (the time of expected partition), but was not observed to give birth to a litter. Female number 119 showed piloerection during Days 39 to 42, but this animal was not pregnant.
Control male number 2 was observed to have a small eye during Days 8 and 9 but, as this animal was not treated, it was considered to be of no toxicological significance.

Behavioural Assessments
There were no treatment-related changes in the behavioural parameters for either sex at any of the dosages investigated.

Functional Performance Tests
There were no changes in functional performance for either sex that were considered to be related to treatment.
For females at 1000 mg/kg bw/day, higher values for hind limb grip strength during trial two attained statistical significance when compared with control. However, in the absence of any statistically significant differences from control in the other assessments of grip strength for these animals, this finding was considered incidental and of no toxicological significance.

Sensory Reactivity Assessments
Results from sensory reactivity assessments of females with litters at Day 4 post partum did not indicate any effect of treatment at any of the dosages investigated.
For one non-pregnant female (number 119) at 1000 mg/kg bw/day assessed on Day 41 no pupil reflex was apparent; this animal had also been observed to have piloerection during routine assessment of clinical signs.

Body Weight
At 1000 mg/kg bw/day, body weight gain of males was lower than control from Day 8, with differences from control attaining statistical significance. By Day 43, overall body weight gain was less than 50% of their control counterparts.
At 30, 100 and 300 mg/kg bw/day overall body weight gain of males on Day 43 was lower than control, although not to the same degree as observed at 1000 mg/kg bw/day. Although differences in bodyweight gain for males at these lower dosages attained statistical significance on occasions when compared to control, intergroup differences did not reveal any consistent pattern that indicated a clear effect of treatment and it is considered that much of these differences in body weight performance may reflect normal biological variation of rats of this age.
For females at all dosages there was no adverse effect of treatment on mean body weights or body weight gain during the two week pre-pairing period. At 1000 mg/kg bw/day, body weight gain of females was superior to control; such gain is unlikely to represent an adverse effect and probably reflects normal biological variation for female rats of this age.

Food Consumption
At 1000 mg/kg bw/day, food consumption of males was lower than control throughout the two week pre-pairing phase of the study and this lower intake continued to a slightly greater extent during the post-pairing phase of the study.
At 300 mg/kg bw/day food consumption of males was slightly lower than control during the second week of the pre-pairing phase of the study and during the post-pairing phase.
Food consumption of males at 30 and 100 mg/kg bw/day appeared to be unaffected by treatment.
There was no effect on food consumption of females during the two week pre-pairing phase of the study at any of the dosages investigated.


Food Conversion Efficiency
Food conversion efficiency for both sexes during the two week pre-pairing phase of the study was considered to be unaffected by treatment at 30, 100, 300 and 1000 mg/kg bw/day.
For males at 1000 mg/kg bw/day, food conversion efficiency was slightly lower than control during the post-pairing phase of the study. Food conversion efficiency of males during the post pairing at 30, 100 and 300 mg/kg bw/day was considered to be unaffected by treatment.

Water Consumption
Intergroup differences in water intake did not indicate any effect of treatment for either sex at 30, 100, 300 or 1000 mg/kg bw/day.

Laboratory Investigations
Haematology
For males at 300 and 1000 mg/kg bw/day, mean cell haemoglobin concentration was lower than control with differences attaining statistical significance. Individual values for the treated animals were within the historical control range while one control value exceeded this historical range, however. In the absence of any histological correlates this isolated finding was considered incidental and unrelated to treatment.
There were no other statistically significant differences from control observed for haematology parameters in either sex.

Blood Chemistry
At all dosages, mean alanine aminotransferase levels were statistically significantly lower than control for both sexes. While the lowest mean value for females was at 1000 mg/kg bw/day, intergroup values for each sex did not show any consistent dosage relationship; however, with the exception of one female value at 300 mg/kg bw/day, all values for treated animals were below the historical control range. For males at 1000 mg/kg bw/day, this decrease was accompanied by lower alkaline phosphatase levels which also attained statistical significance compared with control, although all individual values for these treated animals were within the historical control range.
For males at 100 and 300 mg/kg bw/day and both sexes at 1000 mg/kg bw/day, mean total cholesterol was statistically significantly lower than control. Individual values for males at 100 and 300 mg/kg bw/day were within the historical control range, but values for one male and three females at 1000 mg/kg bw/day were lower than this historical range.
For males at 1000 mg/kg bw/day, mean total protein and albumin levels were statistically significantly lower than control and were accompanied by a statistically significant increase in albumin/globulin ratio compared with control. All individual values for these treated animals were within the historical control range. Higher mean glucose levels for males at 1000 mg/kg bw/day also attained statistical significance when compared to control but all individual values for these treated animals were again within the historical control range.
For females at 1000 mg/kg bw/day, higher levels of inorganic phosphorus attained statistical significance when compared with control and the majority of individual values for these treated animals exceeded the historical control range.
For females at 1000 mg/kg bw/day, mean levels of potassium and bilirubin were lower than control while the mean level of creatinine was higher than control; differences from control for these parameters attained statistical significance but all individual values for these treated animals were within the historical control range.

Pathology
Necropsy
The type, incidence and distribution of macroscopic findings observed at terminal necropsy did not indicate any obvious effect of treatment at 30, 100, 300 or 1000 mg/kg bw/day.

Organ Weights
For males at all dosages, lower absolute and body weight-relative pituitary weights attained statistical significance when compared to control; however mean absolute values showed no dosage relationship and, with the exception of one higher body weight-relative value at 1000 mg/kg bw/day, all values for treated males were within the historical control range.
Additionally for males at 1000 mg/kg bw/day, lower absolute and body weight-relative thymus weights attained statistical significance compared to control but, with the exception of one absolute value at 1000 mg/kg bw/day, all values for these treated animals were within the historical control range.
For females at 300 and 1000 mg/kg bw/day, absolute and body weight-relative kidney weights were higher than control, with differences attaining statistical significance and showing a dosage relationship. The majority of individual body weight-relative values at 300 mg/kg bw/day and absolute and body weight relative values at 1000 mg/kg bw/day exceeded the historical control range.
For females at 1000 mg/kg bw/day, there was a statistically significant increase in absolute and body weight-relative thyroid weights, with the majority of body weight-relative values exceeding the historical control range.
For females at 300 and 1000 mg/kg bw/day, absolute and body weight-relative liver weights were higher than control, however there was no dosage relationship and the majority of individual values for these treated animals were within the historical control range.
For females at 300 mg/kg bw/day, absolute and body weight-relative brain weights were statistically significantly higher than control, and for two treated animals absolute and body weight-relative values exceeded the historical control range. However, in the absence of any similar increase in weights or evidence of microscopic change for the brain at 1000 mg/kg bw/day, this finding was considered fortuitous and unrelated to treatment.
At 30 mg/kg bw/day, absolute and body weight-relative pituitary weights for females were statistically significantly higher than control, but only one body weight-relative value for these treated females exceeded the historical control range. In the absence of any similar increase in weights or evidence of microscopic change for the pituitary at 100 and 300 mg/kg bw/day, this finding was considered fortuitous and unrelated to treatment.

Histopathology
Increased cortical vacuolation in the adrenal gland was present in 4/5 males at 1000 mg/kg bw/day along with 1 animal at 30 mg/kg bw/day (moderate vacuolation).
Decreased cellularity was present in the mesenteric lymph node of 1 female and the mandibular lymph node of 1 male and one female at 1,000 mg/kg bw/day.
Increased vacuolation of the pars distalis of the pituitary gland was present in males and females at 1000 mg/kg bw/day and males at 300 mg/kg bw/day.
Acinar cell atrophy in the submaxillary salivary gland was present in 2 males and females at 1000 mg/kg bw/day only.
Increased hematopoiesis in the spleen was present in 3/6 females at 1000 mg/kg bw/day. Decreased white pulp cellularity was present in 1/6 1000 mg/kg bw/day females.
In the thyroid gland enlarged follicles, describing follicles with slight dilation and hypertrophy of the epithelial cells, was present in males and females at 300 and 1000 mg/kg bw/day.
Centrilobular hypertrophy was noted in the liver of 1/6 females and 4/5 males at 1000 mg/kg bw/day only.
Thymic atrophy was increased in incidence and/or severity at 1000 mg/kg bw/day males and females only.
In the kidney, multifocal tubular basophilia, mild or moderate, was present in all males and 5/6 females at 1000 mg/kg bw/day. In the other 1000 mg/kg bw/day female, the changes were even more notable and diffuse nephropathy was recorded. Within the basophilic tubules there was apoptosis and mitosis and in all 1000 mg/kg bw/day animals karyomegaly (very large and sometimes atypical nuclei) was noted. Tubular cell vacuolation was noted in 2/5 300 mg/kg bw/day females – large balloon-like vacuoles scattered in the tubular cells. Pigment was noted in the tubular cells (usually superficial near the lumen and yellow/brown in colour) in all Groups treated with the test item.
Hyaline droplets were present in males, but they did not appear to be increased in animals treated with the test item when compared with controls.

Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Histopathological changes observed at 300 and 1000 mg/kg bw/day.
Critical effects observed:
not specified
Conclusions:
Within the confines of this study, the No Observed Adverse Effect Level (NOAEL) for toxicity was considered to be 100 mg/kg bw/day. A No Observed Effect Level (NOEL) for toxicity was not established due to lower levels of alanine aminotransferase levels and deposition of yellow/ brown pigment in the tubular cells of kidneys in all animals treated with 5,5’-Dithiodi-1,3,4 -thiadiazole-2(3H)-thione, despite the absence of histopathological lesions at 30 and 100 mg/kg bw/day.
Executive summary:

The test item was administered by gavage to four groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for up to eight weeks, including a two week prepairing phase, pairing, gestation and early lactation (Days 1 through 4 post partum) for females,

at dose levels of 30, 100, 300 and 1000 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (Arachis oil BP) over the same treatment period.

One female at 1000 mg/kg bw/day was killed prior to dosing on Day 40 due to adverse clinical signs including piloerection, lethargy, hunched posture, ptosis, decreased respiration rate and ataxia; piloerection had been observed on the previous day. Necropsy revealed enlarged adrenals, eleven dead foetuses in the uterus and one dead foetus in the vagina. While this death appears to be related to difficulties associated with parturition, an association with treatment cannot be discounted.

Treatment was associated with an increased post-dosing salivation at 100, 300 and 1000 mg/kg bw/day. At 1000 mg/kg bw/day, one male showed piloerection and hunched posture on Day 44 and similar clinical signs were occasionally observed for four females (including the decedent) from Day 39. There were no treatment-related changes in the behavioural parameters, no changes in functional performance for either sex and no changes from sensory reactivity assessments of females with litters at Day 4 post partum.

At 1000 mg/kg bw/day, lower body weight gain and low food consumption were apparent for males. For females at this dosage lower body weight gain and lower food consumption were apparent during gestation and lactation but this was based on a small group size (6 and 3 females respectively). There was no clear effect of treatment on body weight gain and food consumption for either sex at 30, 100 or 300 mg/kg bw/day.

There was no effect of treatment on water consumption for either sex at 30, 100, 300 or 1000 mg/kg bw/day.

There was no effect of treatment on hematology parameters for either sex at 30, 100, 300 or 1000 mg/kg bw/day.

At all dosages, mean alanine aminotransferase levels were statistically significantly lower than control for both sexes. For males at 1000 mg/kg bw/day, this decrease was accompanied by a statistically significantly lower level of alkaline phosphatase. For males at 100 and 300 mg/kg bw/day and both sexes at 1000 mg/kg bw/day, lower mean total cholesterol was statistically significantly lower than control. For males at 1000 mg/kg bw/day, mean total protein and albumin levels were statistically significantly lower than control and were accompanied by a statistically significant increase in albumin/globulin ratio. For females at 1000 mg/kg bw/day, higher levels of inorganic phosphorus and creatinine and lower levels of potassium and billirubin attained statistical significance when compared with control.

Macroscopic necropsy examination did not indicate any obvious effect of treatment for either sex at 30, 100, 300 or 1000 mg/kg bw/day.

For males at all dosages, absolute and body weight relative pituitary weights were statistically significantly lower than control. For males at 1000 mg/kg bw/day, absolute and body weight relative thymus weights were statistically significantly lower than control. For females at 300 and 1000 mg/kg bw/day, absolute and body weight relative liver and kidney weights were statistically significantly higher than control. Additionally for females at 1000 mg/kg bw/day, there was a statistically significant increase in absolute and body weight relative thyroid weights.

Changes were noted in the adrenal glands of males, kidneys, pituitary, thyroid glands and livers of both sexes when treated with 5,5’-Dithiodi-1,3,4-thiadiazole-2(3H)-thione at 1000 mg/kg bw/day. Changes were also apparent in the spleens of females, lymph nodes, thymus and

salivary gland of both sexes at this dose, which are considered likely to be secondary. When 5,5’-Dithiodi-1,3,4-thiadiazole-2(3H)-thione was administered at 300 mg/kg bw/day changes were noted in the pituitary glands of males, thyroid glands and kidneys of both sexes.

Deposition of yellow/brown pigment in the tubular cells of the kidneys of animals treated with 30 and 100 mg/kg bw/day of 5,5’-Dithiodi-1,3,4-thiadiazole-2(3H)-thione was considered unlikely to be of toxicological importance, within the confines of this study, as no associated

pathology was present.

Within the confines of this study, the No Observed Adverse Effect Level (NOAEL) for toxicity was considered to be 100 mg/kg bw/day. A No Observed Effect Level (NOEL) for toxicity was not established due to lower levels of alanine aminotransferase levels and deposition of yellow/ brown pigment in the tubular cells of kidneys in all animals treated with 5,5’-Dithiodi-1,3,4 -thiadiazole-2(3H)-thione, despite the absence of histopathological lesions at 30 and 100 mg/kg bw/day.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016

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:
1996
Deviations:
yes
Remarks:
Deviations are considered not to have affected the integrity or purpose of the study.
Principles of method if other than guideline:
Deviations are noted but do not affect the integrity or purpose of the study.
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
5,5'-dithiodi-1,3,4-thiadiazole-2(3H)-thione
EC Number:
276-763-0
EC Name:
5,5'-dithiodi-1,3,4-thiadiazole-2(3H)-thione
Cas Number:
72676-55-2
Molecular formula:
C4-H2-N4-S6
IUPAC Name:
5-[(5-sulfanylidene-4,5-dihydro-1,3,4-thiadiazol-2-yl)disulfanyl]-2,3-dihydro-1,3,4-thiadiazole-2-thione
Test material form:
solid: particulate/powder

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
Source:
The animals were acclimatized for eight days. A total of one hundred and twenty animals (sixty males and sixty females) were accepted into the study. At the start of treatment the males weighed 302 to 351g, the females weighed 184 to 231g, and were approximately twelve weeks old.

Initially, all animals were housed in groups of four by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding. During the pairing phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation, parturition and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.

The animals were allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad Global Certified Pelleted Diet) was used. Tap drinking water was supplied from polycarbonate bottles attached to the cage. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels except for paired animals and mated females during gestation and lactation. Mated females were also given softwood flakes, as bedding, throughout gestation and lactation.

The animals were housed in a single air-conditioned room. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored. The temperature remained within 22 ± 3 °C during the study. Transient deviations from the target range for relative humidity of 50 ± 20% were considered not to have affected the purpose or integrity of the study.

The animals were uniquely identified within the study by an ear punching system and a tail mark.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
The test item was prepared at the appropriate concentrations in Arachis oil BP. The homogeneity of the test item formulations were confirmed by analysis. Formulations were prepared on a daily basis; all animals were dosed within 2.5 hours after dose preparation. The formulations were considered to be stable for the period between preparation and dosing.

Test item formulation were analyzed for concentration of 5,5’-Dithiodi-1,3,4-thiadiazole-2(3H)-thione . The results indicate that the prepared formulations were within 80-107% of the nominal concentration.
Details on mating procedure:
Animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of estrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages. Mated females were housed individually during the period of gestation and lactation.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test item concentration in the test samples was determined spectrophtometrically.
The detection system was found to have acceptable linearity. The analytical procedure had acceptable recoveries of test item in the vehicle. The method of analysis was validated and proven suitable for use.
Duration of treatment / exposure:
41-47 days. Males were dosed daily throughout the study until the day prior to scheduled necropsy. Females were dosed for a two week pre-pairing period, throughout pairing and gestation and early lactation (Days 1-4). Females in parturition were not dosed on that day.
Frequency of treatment:
Once daily by gavage.
Details on study schedule:
Each pregnant female was observed at least three times a day (nominally early morning, mid-day and as late as possible during the normal working day) around the period of expected parturition. Observations were carried out at early morning (approximately 0830) and as late as possible at weekends and public holidays. The following was recorded for each female:

i. Date of pairing
ii. Date of mating
iii. Date and time of observed start of parturition (see Deviations from Study Plan)
iv. Date and time of observed completion of parturition (see Deviations from Study Plan)

On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded. Offspring were individually identified within each litter by tattoo on Day 1 post partum.

For each litter the following was recorded:

i. Number of offspring born
ii. Number of offspring alive recorded daily and reported on Days 1 and 4 post partum (see deviations from Study Plan)
iii. Sex of offspring on Days 1 and 4 post partum
iv. Clinical condition of offspring from birth to Day 5 post partum
v. Individual offspring weights on Days 1 and 4 post partum (litter weights were calculated retrospectively from these data)
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
12 males + 12 females
Control animals:
yes, concurrent vehicle
Details on study design:
Five dose groups (control, low, intermediate I, intermediate II and high) each comprising 24 animals (12 male and 12 female) were used. Dose levels were chosen based on available toxicity data including a fourteen day range-finding toxicity study. In this range-finding study, a dosage of 1000 mg/kg bw/day was well tolerated but increases in liver and kidneys weights were apparent for both sexes and a low level of brown/yellow pigment disposition was observed in the kidneys for females. Dosages of 0 (Control), 30, 100, 300 and 1000 mg/kg bw/day were chosen in collaboration with the Study Monitor for the Sponsors.

i. Groups of twelve male and twelve female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). The first day of dosing was designated as Day 1 of the study.
ii. Prior to the start of treatment and once weekly thereafter, all animals in an open arena were observed for signs of functional/behavioral changes.
iii. On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
iv. Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages with suitable nesting material. Dosing continued through pairing and subsequent female gestation and lactation phase (females in parturition were not dosed).
v. During Week 6, five selected males per dose group were evaluated for functional/sensory responses to various stimuli, grip strength and motor activity assessments.
vi. Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Litter size, offspring weight and sex, surface righting and clinical signs were recorded during this period.
vii. At Day 4 post partum or Day 25 post coitum, five selected females per dose group were evaluated for functional/sensory responses to various stimuli, grip strength and motor activity assessments.
viii. Blood samples were taken from the same five males from each dose group for hematological and blood chemical assessments on Day 42.
ix. On Day 43, six males from each dose group were killed and examined macroscopically . The remaining six males from each dose group were weighed.
x. On Day 44, the remaining six males from each dose group were killed and examined macroscopically.
xi. Blood samples were taken from five randomly selected females from each dose group for hematological and blood chemical assessment on Day 4 post partum or Day 25 post coitum for non-pregnant females. At Day 5 post partum, all females and their surviving offspring were killed and examined macroscopically. Any female which did not produce a pregnancy or litter was also killed and examined macroscopically on or after Day 25 post coitum.

Positive control:
not required

Examinations

Parental animals: Observations and examinations:
See Repeated Dose toxicity, 7.5.1., Key Study for general evaluation of parental animals related to the repeat dose portion of the study.
Oestrous cyclicity (parental animals):
not observed
Sperm parameters (parental animals):
Detailed qualitative examination of the testes was undertaken, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell-or stage-specificity of testicular findings was noted.
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities.

GROSS EXAMINATION OF DEAD PUPS: no

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no
Postmortem examinations (parental animals):
Necropsy
Adult animals were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination. Males were killed on Day 43 or Day 44, females that littered were killed on Day 5 post partum, females that failed to achieve pregnancy or produce a litter were killed on Day 25 post coitum, and one female was killed in extremis during parturition. Surviving offspring were terminated via intracardiac overdose of suitable barbiturate agent.
For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced, as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution (Salewski 1964). The corpora lutea were also counted.
All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Organ Weights
The following organs were dissected free from fat and weighed before fixation from the five selected males and five selected females from each dose group. Tissues shown with* were weighed from all remaining animals:

Adrenals
Prostate*
Brain
Seminal vesicles*
Epididymides*
Spleen
Heart
Testes*
Kidneys
Thymus
Liver
Thyroid (weighed post-fixation with Parathyroid)
Ovaries*
Uterus (weighed with Cervix)*
Pituitary (post fixation)*

Histopathology
Samples of the following tissues were removed from the five selected males and five selected females from each dose group and preserved in buffered 10% formalin, except where stated. Tissues shown with* were preserved from all remaining animals:

Adrenals
Muscle (skeletal)
Aorta (thoracic)
Ovaries*
Bone & bone marrow (femur including stifle joint)
Pancreas
Bone & bone marrow (sternum)
Pituitary*
Brain (including cerebrum, cerebellum and pons)
Prostate*
Caecum
Rectum
Coagulating gland*
Salivary glands (submaxillary)
Colon
Sciatic nerve
Duodenum
Seminal vesicles
Epididymides*•
Skin (hind limb)
Esophagus
Eyes+
Spinal cord (cervical, mid-thoracic and lumbar)
Gross lesions
Spleen
Heart
Stomach
Ileum (including peyer’s patches)
Thyroid/parathyroid
Jejunum
Trachea
Kidneys
Testes*•
Liver
Thymus
Lungs (with bronchi) #
Urinary bladder
Lymph nodes (mandibular and mesenteric)
Uterus/Cervix*
Mammary gland*
Vagina*

+ = eyes fixed in Davidson’s fluid
• = preserved in Modified Davidsons fluid
# = lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative

Tissues were dispatched to the Test Site (Propath UK Ltd) for processing. The tissues from five selected control and 1000 mg/kg bw/day dose group animals, any animals dying during the study, and any animals which failed to mate or did not achieve a pregnancy were prepared as paraffin blocks, sectioned at a nominal thickness of 5 µm and stained with hematoxylin and eosin for subsequent microscopic examination. The tissues shown with* from the remaining control and 1000 mg/kg bw/day animals and animals which did not achieve a pregnancy were also processed. In addition, sections of testes from all control and 1000 mg/kg bw/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined.
Detailed qualitative examination of the testes was undertaken, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell-or stage-specificity of testicular findings was noted.
Following the initial results of histopathology for high dosage animals, histopathological examination was extended to five animals of either sex (except where stated) for the low and both intermediate dosage groups for the following tissues: adrenal glands (males only), lymph nodes, pituitary (all animals), salivary gland, spleen (females only), thyroid glands, kidney, liver and thymus.
Microscopic examination was conducted by the Study Pathologist and a peer review being conducted by Peter Millar Associates Ltd.
Statistics:
Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05.

Additional data are available upon request.

Reproductive indices:
Mating Performance and Fertility

The following parameters were calculated from the individual data during the mating period of the parental generation:

i. Pre-coital Interval

Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.

ii. Fertility Indices

For each group the following were calculated:

Mating Index (%) = number of animals mated/number of animals paired x 100

Pregnancy Index (%) = number of pregnant females/number of animals mated x 100

The following parameters were calculated from individual data during the gestation and parturition period of the parental generation:

i. Gestation Length

Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.

ii. Parturition Index

The following was calculated for each group:

Parturition Index (%) = number of females delivering live offspring/number of pregnant females x 100

Offspring viability indices:
The standard unit of assessment was considered to be the litter, therefore values were first calculated for each litter and the group mean was calculated using their individual litter values. Group mean values included all litters reared to termination (Day 5 of age).

i. Implantation Losses (%)

Group mean percentile pre-implantation and post-implantation loss were calculated for each female/litter as follows:

Pre–implantation loss (%) = Number of corpora lutea - number of implantation sites/number of corpora lutea x100

Post–implantation loss (%) = Number of implantation sites - total number of offspring born/ number of implantation sites x100

ii. Live Birth and Viability Indices

The following indices were calculated for each litter as follows:

Live Birth Index (%) = Number of offspring alive on Day 1/number of offspring born x 100

Viability Index (%) = Number of offspring alive on Day 4/Number of offspring alive on Day 1 x 100

iii. Sex Ratio (% males)

Sex ratio was calculated for each litter value on Days 1 and 4 post partum, using the following formula:

Number of male offspring/Total number of offspring x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
See Repeated Dose toxicity, 7.5.1. Key Study(2016) for general evaluation of parental animals.
Mortality:
mortality observed, treatment-related
Description (incidence):
See Repeated Dose toxicity, 7.5.1. Key Study(2016) for general evaluation of parental animals.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
See Repeated Dose toxicity, 7.5.1. Key Study(2016) for general evaluation of parental animals.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
See Repeated Dose toxicity, 7.5.1. Key Study(2016) for general evaluation of parental animals.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
See Repeated Dose toxicity, 7.5.1. Key Study(2016) for general evaluation of parental animals.
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
See Repeated Dose toxicity, 7.5.1. Key Study(2016) for general evaluation of parental animals.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
See Repeated Dose toxicity, 7.5.1. Key Study(2016) for general evaluation of parental animals.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
See Repeated Dose toxicity, 7.5.1. Key Study(2016) for general evaluation of parental animals.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
See Repeated Dose toxicity, 7.5.1. Key Study(2016) for general evaluation of parental animals.
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, treatment-related

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day there was not effect on mating but the pregnancy rate was poor.
At 1000 mg/kg bw/day, there was no effect on mating (all 12 females mated), but the subsequent pregnancy rate was poor with three females failing to achieve pregnancy. Three of the nine pregnant females were not observed to give birth to a litter and one pregnant female was killed around the time of parturition. Two of the remaining females showed total litter loss post partum and only three females successfully reared their young to Day 5 of lactation.

There was no effect of treatment on mating and fertility at 30, 100 or 300 mg/kg bw/day.

At 1000 mg/kg bw/day, there was a clear increase in the length of gestation for the five females observed to give birth to a litter, with a mean gestation length of 24.9 days for the high dose compared to 22.5 days for the control group.

There was no effect of treatment on gestation length at 30, 100 or 300 mg/kg bw/day.

At 1000 mg/kg bw/day, assessment of any effect of treatment on corpora lutea count, pre-implantation loss, numbers of implantations, post-implantation loss, litter size and sex ratio at birth/Day 1 and subsequent offspring survival to Day 4 of age was frustrated by the small group size, although a clear increase in post implantation loss was apparent and supported by the incidence of post partum total litter loss observed at this dosage.

There was considered to be no effect of treatment on the corpora lutea count, pre-implantation loss, numbers of implantations, post-implantation loss, litter size and sex ratio at birth/Day 1 and subsequent offspring survival to Day 4 of age at 30, 100 or 300 mg/kg bw/day.

In total 10 females from control, 11 females each from 30 and 100 mg/kg bw/day, 12 females from 300 mg/kg bw/day and 3 females from 1000 mg/kg bw/day dose groups gave birth to live litters and successfully reared young to Day 5 of age. At 1000 mg/kg bw/day data from pregnant females that failed to litter and females that showed total litter loss post partum were also taken into consideration.

Details on results (P0)

There were no test item related microscopic findings in the testes, including following the qualitative examination of the stages of spermatogenesis in the testes (no test item related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle) or following the evaluation of the uterus or evaluation of follicles and corpora lutea in the ovaries. Although fewer Group 5 animals showed implantation sites reflecting the in-life data there was no evidence that the non-pregnant or non-lactating animals were not cycling normally.

Effect levels (P0)

open allclose all
Dose descriptor:
NOEL
Remarks:
(reproductive performance)
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: absence of effect at 300 mg/kg
Dose descriptor:
NOAEL
Remarks:
(parental toxicity)
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic

Target system / organ toxicity (P0)

Critical effects observed:
not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
See below results for additional details. Mean litter weights were lower 1000 mg/kg bw/day reflecting the lower litter size.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
not examined
Nipple retention in male pups:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

Offspring Litter Size, Sex Ratio and Viability
At 1000 mg/kg bw/day the mean number of corpora lutea was lower than control, however considering the small group size, this probably reflect normal biological variation and, at this stage, an association with treatment is not considered proven. The mean number of implantation sites was also lower than control but this mainly reflected the previous low corpora lutea count and, while higher mean pre-implantation loss was observed this was mainly due to one litter. However there was a clear increase in mean post-implantation loss with a corresponding reduction in live litter size on Day 1. This finding has to be viewed in the context of a further three females that either lost their litter in utero or very shortly after delivery. Overall offspring viability of the litters reared to weaning was lower than control and this value excludes two litters that both showed total litter loss post partum. It should be noted that many dams at this dosage showed excessively long gestation length and this prolonged gestation length would have been expected to compromise the offspring at parturition and lead to lower post partum survival, particularly during the early lactation period assessed as part of this study. Sex ratio of the offspring appeared unaffected by maternal treatment indicating that there was no selective effect on survival for either sex.

There was considered to be no effect of treatment on the corpora lutea count, pre-implantation loss, numbers of implantations, post-implantation loss, litter size at birth/Day 1 and subsequent offspring survival to Day 4 of age at 30, 100 or 300 mg/kg bw/day. Sex ratio for the offspring was similar in all groups and did not indicate any selective effect of maternal treatment on survival for either sex.

Offspring Growth and Development
At 1000 mg/kg bw/day there was no adverse effect of treatment on the initial body weight of the offspring on Day 1 or subsequent body weight gain to Day 4. It could be argued that given the longer gestation period and smaller size for these litters, a higher offspring body weight at Day 1 may have been expected. It should also be noted that two litters showed total litter loss and these “inferior” litters have not been included in the mean calculation which is based on a very small group size. Mean litter weights were lower at this dosage reflecting the lower litter size.

There was considered to be no adverse effect of treatment on offspring body weight on Day 1 and subsequent body weight gain to Day 4 at 30, 100 or 300 mg/kg bw/day.

Clinical signs apparent for offspring were typical of the age and neither the incidence or distribution indicated any adverse effect of treatment at 30, 100 or 1000 mg/kg bw/day although the number of offspring assessed at the high dosage was much lower than the other groups on the study.

For all dosages the success rate at assessment of surface righting for the offspring at Day 1 of age was lower than control, however there was no dosage relationship and these differences probably reflect normal biological variation rather than any treatment related effect.

Necropsy - Offspring
Necropsy findings apparent for offspring were typical for the age observed and the low incidence and distribution of these observations did not indicate any effect of maternal treatment at 30, 100, 300 or 1000 mg/kg bw/day.

Effect levels (F1)

Dose descriptor:
NOEL
Generation:
F1
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Including the survival, growth and development of the offspring

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
The No Observed Effect Level (NOEL) for reproduction, including the survival, growth and development of the offspring, was considered to be 300 mg/kg bw/day.
Executive summary:

The test item was administered by gavage to four groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for up to eight weeks, including a two week prepairing phase, pairing, gestation and early lactation (Days 1 through 4 post partum) for females, at dose levels of 30, 100, 300 and 1000 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (Arachis oil BP) over the same treatment period.

At 1000 mg/kg bw/day, there was no effect on mating (all 12 females mated), but the subsequent pregnancy rate was poor with three females failing to achieve pregnancy. Three of the nine pregnant females were not observed to give birth to a litter and one pregnant female was killed

around the time of parturition. Two of the remaining females showed total litter loss post partum and only three females successfully reared their young to Day 5 of lactation. There was no effect of treatment on mating and fertility at 30, 100 or 300 mg/kg bw/day.

At 1000 mg/kg bw/day, there was a clear increase in the length of gestation for the five females observed to give birth to a litter, with a mean gestation length of 24.9 days for the high dose compared to 22.5 days for the control group. There was no effect of treatment on gestation length at 30, 100 or 300 mg/kg bw/day.

At 1000 mg/kg bw/day, assessment of any effect of treatment on corpora lutea count, preimplantation loss, numbers of implantations, post-implantation loss, litter size and sex ratio at birth/Day 1 and subsequent offspring survival to Day 4 of age was frustrated by the small group

size, although a clear increase in post implantation loss was apparent and supported by the incidence of post partum total litter loss observed at this dosage.

There was considered to be no effect of treatment on the corpora lutea count, pre-implantation loss, numbers of implantations, post-implantation loss, litter size and sex ratio at birth/Day 1 and subsequent offspring survival to Day 4 of age at 30, 100 or 300 mg/kg bw/day.

At 1000 mg/kg bw/day, assessment of any effect of treatment on offspring growth and development was frustrated by the small group size. There were, however, no adverse effects of treatment observed on pup clinical signs or pup body weights on post partum (lactation) Days 1

and 4.

Offspring body weight and body weight gain, surface righting ability on Day 1, clinical signs and necropsy findings did not indicate any effect of treatment at 30, 100 or 300 mg/kg bw/day.

Within the confines of this study, the No Observed Adverse Effect Level (NOAEL) for toxicity was considered to be 100 mg/kg bw/day.

The No Observed Effect Level (NOEL) for reproduction, including the survival, growth and development of the offspring, was considered to be 300 mg/kg bw/day.