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Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

A Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test (OECD TG 422) in rats by the oral route (dietary) with ZMB2 resulted in a LOAEL value for fertility of 60 mg/kg/day.

Based on preliminary data from the ongoing extended one-generation reproductive toxicity study (OECD TG 443) for ZMB2, fertility effects have been observed. As this study is ongoing not all data are available at this point in time. Study data has not been populated in IUCLID as the study is ongoing, and it is not possible to partially populate a study summary and still pass the technical completeness check.

The dose levels administered to F0 and F1 generation rats were 5, 15, and 40 mg/kg/day. Duration and timing of treatment as well as all aspects of the study followed the OECD TG 443.

Among the F0 generation rats treated with a dose level of 40 mg/kg/day, three cases of dystocia were observed and considered treatment-related. For F1 litters, live litter size on Day 1 of age was lower than control at 40 mg/kg/day. In addition, post-implantation survival index was lower than

control in all groups of treated females, although in the absence of a dose response relationship. Despite these changes, the mean number of implantations were essentially similar in all groups. It is not known whether these findings in the F1 litters were related to changes seen in the F0 generation rats, which include changes in kidney weights (40 mg/kg/day) and thymus weights (15 and 40 mg/kg/day). No other findings were found to be significant so far.

The main, leading toxicological effect at this point in time is dystocia. This effect is observed at doses ≥ 40 mg/kg/day; not at 15 or 5 mg/kg/day.

Since the F0 animals breeding phase is completed the dystocia data will not change for the first breeding and, consequently, the NOAEL for dystocia is 15 mg/kg/day at this point in time.

Once all data on this OECD 443 study will be available, a definitive NOAEL will be derived.

Link to relevant study records

Referenceopen allclose all

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
Combined Repeated Dose Toxicity Study with Reproduction/Developmental Toxicity Screening Test (OECD TG 422)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
December 2002 - February 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Conducted to GLP and OECD Guidelines.
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
A sufficient number of male and female Sprague-Dawley Crl:CD (SD) IGS BR strain rats were obtained from Charles River UK. On receipt the animals were examined for signs of ill health or injury. The animals were acclimatised for 16 days during which time their health status was assessed. A total of eighty animals (forty males and forty females) were accepted into the study. At the start of the treatment the males weighed 298 to 375g and the females weighed 200 to 248g.
Upon arrival, the animals were housed in groups of four in polypropylene cages with stainless steel grid floors and tops, suspended over paper-lined polypropylene trays. During the mating period animals were transferred to a similar type cage on a one male to one female per cage basis.
Following evidence of successful mating, the males were returned to their original cages and transferred to a separate animal room of comparable conditions. The females were housed, individually, in polypropylene cages with solid floors and stainless steel tops. Mated females were given softwood chips, as bedding, throughout gestation and lactation.
The animals were allowed free access to food and water. A ground diet PMI 5002 was offered ad libitum. Main water was supplied from polycarbonate bottles attached to the cage. The diet and drinking water were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.
The animals were housed in air conditioned rooms. 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 by a computerised system. Both animal rooms used for this study were maintained to operate within a target temperature range of 21 +/-2 degC and a relative humidity range of 55 +/- 15%. On isolated occasions the room temperature and/or humidity fell outside the protocol limites but this was not considered to have affected the purpose or integrity of the study.
The animals were allocated to dose groups using a randomisation procedure based on stratified bodyweights and the group mean bodyweights were then determined to ensure similarity between the dose groups.
The animals were uniquely identified within the study, by an ear punching system routinely used at the laboratory. Color coded cage labels were used to assist recognition of dose groups.
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
Experimental Preparation:
The test material was prepared as a direct dietary admixture. For which dose level, an appropriate aliquot of test material was weighed and then added to approximately one third of the required amount of untreated diet in a mixing bowl. For week 1 test diet preparation the test material and diet were mixed for 20 minutes, for each dose level using a Hobart QE 200 mixer. For subsequent test diet preparations an initial mix of the test material and diet was performed using a Hobart QE 200 mixer for ten minutes. Further untreated diet was then added to the initial mix to give the required concentration and the entire amount was then mixed for ten minutes using the Hobart QE 200 mixer.
Prior to the start of the study, samples of test diet preparation were analyzed for achieved concentration, homogeneity and stability of test material in diet. The results of analysis of the original preparations showed the test material to be stable for at least 14 days. Subsequent preparations of the dietary admixtures were performed weekly. Samples of these preparations were taken on three occasions during the study to determine the achieved concentration of test material in the dietary admixture.
Details on mating procedure:
After the maturation period, the parental generation adults were paired on a one male to one female basis for a period of up to fourteen days. Following pairing, the polypropylene trays beneath each cage were checked for the presence of ejected copulation plugs. Additionally each female was checked for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of the estrous cycle 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. Mated females were then separated from the male and housed individually during the period of gestation and lactation. The males were returned to their original holding cages.

Each female was observed at 08:30, 12:30 and 16:30 hours at or around the period of expected parturition. At weekends, observations were carried out at 08:30 and 12:30 only. The following was recorded for each female:
i) Date of mating
ii) Date and time of observed start of parturition
iii) Date and time of observed completion of parturition.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of test substance in the dietary formulations was determined by high performance liquid chromatography (HPLC) using an external standard technique. Prior to the start of the study, samples of the dietary formulations were analysed for homogeneity, stability, and validation of the dietary dose group concentrations. The results of analysis of the original preparations showed the test material to be stable for at least 14 days. Subsequent preparations of the dietary admixtures were performed weekly. Samples of these preparations were taken on three occasions during the study to determine the achieved concentration of test material in the dietary admixture.
Duration of treatment / exposure:
47 days.
Frequency of treatment:
Daily.
Details on study schedule:
-Groups of ten male and ten female rats were dosed according to dose groups for fourteen days prior to pairing.
-All animals were given a detailed clinical examination once every week for four weeks to observe functional/behavioral changes in an open arena.
- One day prior to pairing (Day 13) five males and five females, randomly selected, were sampled for clinical chemistry and hematology.
- On Day 14 all animals were paired on a one male: one female basis within each dose group for a maximum of fourteen days.
- At the observation of mating or at the end of the fourteen day mating period males were returned to their original cages and females were transferred to individual cages.
- At the end of the mating phase five males per dose group were evaluated for functional/sensory responses to various stimuli. The males used were those selected for hematology/clinical chemistry.
- Pregnant females were allowed to give birth and maintain their offspring until Day 4 post partum. Evaluation of each litter size and weight were performed during this period.
- At Day 4 post partum, five females per dose group were evaluated for functional/sensory responses to various stimuli. The females used were those selected for hematology/clinical chemistry.
- At Day 5 post partum following completion of functional assessments all females and offspring were killed and examined macroscopically.
- Following completion of the female gestation and lactation phases, the males were killed and examined macroscopically.
Remarks:
Doses / Concentrations:
Initial: 1000ppm; Adjusted: 900ppm after 29 days
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
Initial: 2750ppm; Adjusted: 2500ppm after 29 days
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
Initial: 7500ppm; Adjusted: 6750ppm after 29 days; Adjusted: 5500ppm after 33 days
Basis:

No. of animals per sex per dose:
Control: 10 male, 10 female
1000ppm: 10 male, 10 female
2750ppm: 10 male, 10 female
7500ppm: 10 male, 10 female
Control animals:
yes, plain diet
Parental animals: Observations and examinations:
Morbidity/Mortality:
All animals were checked twice daily during the normal working week and once daily at weekends.

Clinical Observations:
All animals were observed daily, immediately before and one hour after dosing, for clinical signs of toxicity.

Functional Observations:
On the day of initiation of treatment and on Days 8, 15 and 22, all animals were observed for signs of functional/behavioral toxicity. Functional performance tests were also performed on five selected females per group on Day 22 for males and Day 4 of lactation for females, together with an assessment of sensory reactivity to difference stimuli.

Behavioral Assessments:
Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed:
Gait
Tremors
Twitches
Convulsions
Bizarre/Abnormal/Stereotypic behavior
Salivation
Pilo-erection
Exophthalmia
Lachrymation
Hyper Hypothermia
Skin colour
Respiration
Palpebral closure
Urination
Defecation
Transfer arousal
Tail elecation

Functional Performance Tests:
- Motor Activity
Twenty purpose built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals of one sex were tested at each occasion and were randomly allocated to the activity monitors. The tests were performed at approximately the same time each day, under similar laboratory conditions. The evaluation period was five hours for each animal. The percentage of time each animal was active and mobile was recorded for the overall five hour period and also during the final 20% of the period (considered to be the asymptotic period). The motor activity for females at 2750 ppm was not recorded as females were killed in extremis prior to evaluation.
- Forelimb/Hindlimb Grips Strength
An automated grip strength meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was drawn along the trough of the meter by the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. The animal was pulled by the base of the tail until its grip was broken. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal. Fore and hinf limb grip strength was not recorded for females at 2750 ppm as females were killed in extremes prior to evaluation.
-
Sensory Reactivity:
Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. The following parameters were observed:
Grasp response
Vocalisation
Toe pinch
Tail pinch
Finger approach
Touch escape
Pupil reflex
Startle reflex
Blink reflex
The sensory reactivity for intermediate level females was not recorded as the females were killed in extremis prior to evaluation.

Bodyweight:
During the maturation and mating period the parental generation animals were weighed weekly. Following mating the parental males were weighed weekly until termination. Parental generation females showing evidence of mating were weighed on Days 0, 7, 14 and 20 post coitum. Parental generation females with a live litter were weighted on Days 1 and 4 post partum.

Food Consumption:
During the maturation period, food consumption was recorded for each cage of adults weekly. For females showing evidence of mating, food consumption was recorded for the periods covering Days 1 to 7, 7 to 14 and 14 to 21 post coitum. For females with live litters, fod consumption was recorded for the period covering Days 1 to 4 post partum.

Food conversion efficiency was determined as:
Food conversion ratio = group mean body weight gain (g bw/day) during week / group mean food consumption (g food/rat/day)

Laboratory Investigations:
Hematological and blood chemical investigations were performed on five males and five females selected from each test and control group on Study Day 13. Blood samples were obtained from the lateral tail vein. Animals were not fasted prior to 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 leucocyte count (WBC)
Differential leucocyte count - neutrophils (Neut), lymphocytes (Lymph), monocytes (Mono), eosinophil’s (Eos), basophils (Bas)
Platelet count (PLT)
Prothrombin time (CT) was assessed by 'Hepato Quick' and Activated partial thromboplastic time (APTT) was assess by 'Preci Clot' using samples collected into sodium citrate solution (0.11 mol/l).

Blood Chemistry:
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:
Urea
Glucose
Total protein (Tot.Prot)
Albumin
Albumin/Globulin (A/G) ratio (by calculation)
Sodium (Na+)
Potassium (K+)
Chloride (Cl-)
Calcium (CA++)
Inorganic phosphorus (P)
Aspartate aminotransfease (ASAT)
Alanine aminotransfease (ALAT)
Alkaline phosphatase (AP)
Creatinine (Creat)
Total cholestrol (Chol)
Total bilirubin (Bili)
Oestrous cyclicity (parental animals):
A vaginal smear was prepared for each female and stage of the estrous cycle or the presence of sperm was recorded.
Sperm parameters (parental animals):
A vaginal smear was prepared for each female and stage of the estrous cycle or the presence of sperm was recorded.
Litter observations:
At the observation of completion of parturition, the number of live and dead offspring was recorded. The subsequent date and time of Day 1 post partum litter observations were standardized.
The following observations were recorded for all individual offspring alive on the particular day of observation:

- Individual offspring weights were recorded on Days 1 and 4 post partum
- The number of offspring was recorded daily up to weaning, and reporting for Days 1 and 4 post partum. Offspring sex was recorded for days 1 and 4 post partum.
The clinical condition of individual offspring was observed daily and any findings recorded.
Postmortem examinations (parental animals):
At Day 5 of lactation all the surviving adults, including non-fertile animals, were killed by carbon dioxide asphyxiation; followed by exsanuination by cardiac puncture for selected animals and cervical dislocation for unselected animals. All animals were examined macroscopically for both internal and external abnormalities. Selected organs and tissues were retained in fixative.
The following list of organs were weighed for all animals at necropsy where applicable:
Adrenals
Brain
Epididymides
Heart
Kidneys
Liver
Ovaries
Spleen
Testes
Thymus
Paired organs were weighed together.

Tissue preservation:
Samples of the following tissues were preserved from all animals in buffered 10% formalin except where stated:
Adrenals
Aorta (thoracic)
Bone & bone marrow (femur including stifle joint)
Bone & bone marrow (sternum)
Brain (including cerebrum, cerebellum and pons)
Caecum
Colon
Duodenum
Epididymides (preserved in bouins fluid)
Eyes
Gross lesions
Heart
Ileium
Jejunum
Kidneys
Liver
Lungs (with bronchi) (inflated to normal inspiratory volume with buffered 10% formalin before immersion in fixture)
Lymph nodes (cervical and mesenteric)
Mammary gland
Muscle (skeletal(
Esophagus
Ovaries
Pancreas
Pituitary
Prostate
Salivary glands (submaxillary)
Sciatic nerve
Seminal vesicles
Thyroid/parathyroid
Trachea
Urinary bladder
Uterus + Cervix
Vagina
Coagulating gland
Skin (hind limb)
Spinal cord (cervical)
Spleen
Stomach
Testes (preserved in bouins fluid)
Thymus
Urinary bladder
Uterus

Histopathology:
The tissues listed above were examined from five males and five females selected from the control and high dose groups. Histopathology was extended to include examination of the liver, spleen, pituitary, salivary glands and sternum for five males and five females selected from both the low and intermediate dose groups.
The following list of tissues were examined from the remaining unselected males and females from the control and high dose groups:
Epididymides
Ovaries
Pituitary
Prostate
Seminal vesicles with coagulating gland
Uterus and cervix
Vagina
Postmortem examinations (offspring):
All offspring alive at Day 5 were killed by barbiturate overdose. All these offspring were examined macroscopically for internal and external abnormalities.
Statistics:
- Endpoint group 1: Adult male and female bodyweight during the maturation, gestation and lactation periods, adult male food consumption, female food consumption during maturation, gestation and lactation, litter size, litter weight, individual offspring bodyweight offspring landmarks of physical development, haematology, blood chemistry and organ weights: Values were analysed to establish homogeneity of group variances using Levene's test followed by one-way analysis of variance. If the variances were unequal subsequent comparisons between control and treated groups were performed using a pairwise 'T' test Comparison Method. If variances were equal, subsequent comparisons between control and treated groups were performed using Dunnett's Multiple Comparison Method. For haematology and blood chemistry values, an additional regression analysis of all values was also performed.
- Endpoint group 2: Adult pre-coital intervals, female gestation lengths, offspring reflexological responses and litter sex ratios, relative organ weights: Individual values were compared using the Kruskal-Wallis non-parametric rank sum test. Where significant differences were seen, pairwise comparison of control values against treated group values was performed using Mann-Whitney "U" test.
- Histopathology: For those tissues from selected animals only. Chi-squared analysis for differences in the incidence of lesions occurring with an overall frequency of 1 or greater. For the comparison of severity grades for the more frequently observed conditions, Kruskal-Wallis one-way non-parametric analysis of variance.
Reproductive indices:
- Mating index (%) = [number of animals mated / number of animals paired] x 100
- Pregnancy index(%) = [number of pregnant females / number of animals paired] x 100
- Parturition index(%) = [number of females delivering live pups / number of pregnant females] x 100
Offspring viability indices:
- Live birth index(%) = [number of pups alive on Day 1 / number of pups born] x 100
- Viability index(%) = [number of pups alive on Day 4 / number of pups alive Day 1] x 100
Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
effects observed, treatment-related
Reproductive function: oestrous cycle:
effects observed, treatment-related
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Mortality:
One high dose female (number 71) was killed in extremis during the late gestation phase of the study due to possible dystocia. At the intermediate dose level a total of eight females were killed in extremis during the gestation phase of the study. Six of the eight female mortalities were found to have offspring in utero at post mortem examination indicating possible impairment of parturition.
There were no mortalities at the low dose level or the control level.

Clinical Observations:
The treatment related clinical signs at the high dose were associated with the majority of females and one male. The most prevalent of findings were hunched posture and pilo-erection. Clinical signs of toxicity were seen early in the treatment period with hunched posture observed from week 2 and pilo erection from week 5 along with sporadic instants of tip-toe gait. These findings did not persist throughout the course of the study as the remaining nine high dose female animals showed no clinical signs of toxicity at the end of the treatment. One male also showed clinical signs of hunched posture during the treatment period.
Two intermediate dose level females showed hunched posture and three showed pilo erection, but the onset of these findings was later than those seen in high dose females.
No clinical effects were observed for males at this dose level.
At the low dose level there were no clinical signs of reaction to treatment.

Behavioral Evaluations:
The behavioral evaluation showed no evidence to suggest any significant treatment related observations associated with behavioral change. There was no indication of a treatment related effect on sensory reactivity to various stimuli or an effect on motor activity.

Bodyweight:
- Maturation/post mating
At the high dose level there was a reduction in group mean bodyweight for males during the first week of treatment which resulted in a statistically significant difference (p<0.001) compared to control values at Week 2 and 3 of the study. Subsequent bodyweight gain for males and females prior to mating was lower than control value and the difference in group mean bodyweight remained statistically significant (p<0.001). Post mating bodyweight gain was inconsistent and differences in group mean values remained statistically significant (p<0.001) compared to control values.
At the intermediate dose level there was a reduction in male bodyweight gain, which resulted in lower group mean bodyweight compared to control values. The difference was statistically significant (p<0.001) from Week 4 of the study until termination. There was no significant effect on female bodyweight gain prior to mating.
At the low dose level there was a reduction in male bodyweight gain which resulted in lower group mean bodyweight compared to control values. The difference was statistically significant (p<0.05) from Week 4 until termination. There were no significant effects upon female bodyweight gain prior to mating.

- Gestation
The two high dose females that were pregnant showed a significantly lower bodyweight gain during pregnancy than controls.
The females of the intermediate level showed a statistically lower bodyweight gain during gestation when compared to the controls. This resulted in statistically significant differences (p<0.001) in group mean bodyweight for Days 7, 14 and 20 of gestation.
At the low dose level there was a slight reduction in female bodyweight gain during gestation compared to controls. This resulted in a statistically significant difference in group mean bodyweight (p<0.01) for Day 20 of gestation.

- Lactation
At the high (one animal) and intermediate (two animals) dose level there were insufficient females with live litters to allow for meaningful evaluation of bodyweight change during lactation.
At the low dose level, while female bodyweights during lactation were lower than control values, there was no significant difference in bodyweight gain between Days 1 and 4 of lactation.

Food Consumption:
- Maturation/Post Mating
At the high dose level there was a marked reduction in group mean food consumption for males and females prior to mating. This reduction in food consumption was also evident for males during the post mating phase of the study.
At the intermediate dose level there was a reduction in group mean food consumption for males and females prior to mating. The reduction in food consumption was also evident for males during the post mating phase of the study.
At the low dose level there was a reduction in male and female food consumption prior to mating. Post mating male food consumption was reduced compared to control values.

- Gestation
At the high dose level from the small number of pregnant females, there was notably lower food consumption throughout gestation when compared to control values.
At the intermediate dose level there was a lower group mean food consumption throughout gestation when compared with control values. The difference was statistically significant (p<0.001) of each measurement point.
At the low dose level there was a lower group mean food consumption throughout gestation when compared with control values. The differences were statistically significant (p<0.01 - Days 1 to 7 of gestation, p<0.001 Days 7 to 14 and 14 to 21 of gestation).

- Lactation
At the high and intermediate dose levels there were too few females (two females at the intermediate dose level and one female at the high dose level) with live litters to allow for a meaningful evaluation of food consumption. Of the females evaluated, the food consumption values were notably lower than controls.
At the low dose level there was no significant difference in female food consumption between Days 1 and 4 of lactation when compared it control values.

- Food conversion ratios
Due to the low number of females with live litters, food conversion ratio evaluation is restricted to pre-mating only.
At the high dose level the foo conversion ratio for females was notably lower than control values during the first week of treatment whereas the male value was higher than control. The post mating food conversion ratios for males were inconsistent compared with controls.
At the intermediate dose level both male and, more obviously, female values were lower than controls prior to mating. Post mating male values were more comparable with controls.
At the low dose level male or female food conversion ratios prior to mating or for males post mating were comparable with control values.

- Chemical intake
As the high and intermediate dose level, there was an increase in test material intake for males and females in the second week of treatment prior to mating which reflects the increased food consumption seen. This was also seen for females at the low dose level, Post mating male test material intake was lower during the sixth week of the study which reflects the reduction in dose levels for all the treated groups.

Haematology:
At the high dose level there was a slight decrease in platelet count for males compared to control values. The differences was statistically significant (p<0.01). There was a slight increase in clotting time for females compared to ctrol values. The difference was statistically signicant (p<0.05). There was a reduction in mean cell volume and an increase in activated partial thromboplastin time for males only, which was statistically significant (p<0.01) compared to control values.
At the intermediate dose level there was a slight reduction in mean cell volume for males only. The difference was statistically significant (p<0.05) compared to control values.
At the low dose level there were no significant treatment related effects seen in the hematological parameters examined.

Clinical Chemistry:
At the high dose level notable statistically differences, compared to control values were associated with decreased plasma phosphorus values for both sexes (p<0.01), decreased plasma chloride for males and females (p<0.01 and p<0.001 respectively), increased plasma cholesterol for both sexes (p<0.001) and an increased plasma creatinine (p<0.001) for males only. The statistically significant difference for plasma bilirubin for females (p<0.05) was considered to be incidental.
At the intermediate dose level notable statistically significant differences comparted to control values were associated with a decreased plasma phosphorus for males and females (p<0.01 and p<0.001 respectively), an increased plasma cholesterol for males and females (p<0.01 and p<0.05 respectively) a decreased plasma chloride for females only (p<0.01) and an increased plasma creatinine for males only (p<0.05). A reduction in plasma asparate aminotransferase for females (p<0.01) was also observed which is considered to be incidental.
At the low dose level statistically significant differences when compared to control values were associated with a decreased plasma phosphorus for both sexes (p<0.05) and an increased plasma cholesterol for both sexes (p<0.01) and an increased plasma creatinine for males only (p<0.05).

Reproductive Performances:
- Fertility
At the high dose level there was a marked reduction in the number of mating pairs with positive evidence of mating (four females mated). In addition, of those females with positive evidence of mating, only 50% achieved pregnancy (two females achieved pregnancy). The females with no evidence of mating generally showed a lack of estrous cyclist. Two of the mating pairs with positive evidence of mating also showed an increased pre coital interval. These findings were considered to be of toxicological importance and treatment-related.
At the intermediate and low dose levels there were no treatment related effects upon fertility. All mating pairs showed positive evidence mating and pregnancy.

- Gestation and Parturition
At the high dose level one of the two pregnant females was killed due to possible dystocia.
At the intermediate dose level eight females were killed in extremis during late gestation. The appearance of offspring, in utero in six of these females at post mortem examination was indicative of difficulties at parturation. Of the females that started delivery, there was an apparent increase in the gestation length.
At the low dose level all females produced a live litter but there was a slight increase in the gestation lengths compared to controls.

Organ Weights:
At the high dose group the low number of pregnant females resulted in too few animals of the same physiological status as the control group females to allow direct comparison of organ weights. Statistically significant reductions in male absolute organ weight values compared to control values were observed for kidneys, epididymides, heart, thymus, spleen (p<0.001) and adrenals (p<0.01). Only the spleen and thymus weight relative to bodyweight was significantly lower than controls (p<0.01). The weight of testis and brain relative to bodyweight were significantly higher (p<0.001) than controls but this is likely to be fortuitous and a result of reduced bodyweight among males. The increase in liver weight, relative to bodyweight (p<0.001), may well represent a true effect of treatment.
At the intermediate dose group the low number of females at termination resulted in too few animals to allow for a meaningful evaluation. Statistically significant reductions in male absolute organ weight values were observed for kidneys, epididymides, spleen and thymus (p<0.01) plus adrenals and heart (p<0.01). Only the difference for the thymus weight, relative to bodyweight, remained statistically significant (p<0.001). Liver weight, relative to bodyweight was significantly higher (p<0.05) than controls. An increased brain and testis weight, relative to bodyweight, was considered to be fortuitous and directly related to lower bodyweights among males.
At the low dose group statistically significant reductions in organ weights were observed for thymus and spleen (p<0.001) kidneys (p<0.01) and heart (p<0.05) for males and adrenals (p<0.001) and heart (p<0.05) for females. Concomitant statistically significant reductions in organ weight, relative to bodyweight, were seen for spleen (p<0.01) and thymum (p<0.001) in males and females (p<0.01).

Uterine Exams:
At the high dose level there were limited numbers of pregnancies to allow for meaningful data evaluation of the uterine parameters.
At the intermediate dose level there was a statistically significant lower group mean corpora lutea count (p<0.05) and total implantation count (p<0.01) compared to control values.
At the low dose group the mean corpora lutea count and total implantation count were lower than control values. None of these differences were statistically significant but evidence of inherent variability of results among females of this dose group may be of toxicological significance

Histopathology:
LIVER:
Centrilobular hepatocyte enlargement was observed in relation to treatment for rats of with sex of all treatment levels. Hepatocyte enlargement is commonly observed in the rodent liver following the administration of xenobiotics and, in the absence of associated inflammatory or degenerative changes, is generally considered to be adaptive in nature.

SPLEEN:
Generally lower grades of severity of extramedullary haemopoiesis and higher grades of severity of pigment accumulation were observed in relation to treatment in high dose females. An effect at the intermediate dose level was difficult to determine but at the low dose level the effect on haemopoiesis was in the opposite direction with generally higher grades of severity of the condition prevailing compared with the control group. The toxicological significance, if any, of this finding is uncertain. There was probably no effect on pigment deposition at the intermediate dose level and no effect at the low dose level. Male rats were not similarly affected.

PITUITARY:
Higher grades of severity of cellular vacuolation in the pars anterior were seen for rats of either sec treated with the high and intermediate dose levels and for male rats only of the low dose group.

THYROIDS:
Generally higher grades of severity of follicular cell hypertrophy were seen in relation to treatment for rats of either sex of all treatment groups.

SALIVARY GLANDS:
Lower severity grades of serous secretion in the submandibular glands were more prevalent among rats of either sex of all treatment groups compared with the controls.

BONE MARROW:
Higher grades of severity of adipose infiltration, representing myeloid hypoplasia, were seen in relation to treatment for rats of either sex treated at the high and intermediate dose levels, and for male rats only of the low dose level.

HEART:
Focal myocarditis was observed in several control and treated rats and is a common background entity in laboratory maintained rats. The severity of the condition was never greater than minimal, or one or two foci, and should not be interpreted as being indicative of any ongoing myocardial disease.

LIVER:
Scattered mononuclear cell foci were observed in the majority of animals examined in the study. Such are commonly observed in the rodent liver and are not indicative of any adverse condition at the severities encounted.

KIDNEYS:
Isolated groups of basophilic tubules are frequently encountered in the renal cortex of laboratory maintained rats and have no pathological significance at the severities or frequencies reported in this study. Similarly focal mineralization is a commonly observed background condition amongst female rats.

LUNGS:
A minimal severity of bronchus associated lymphoid tissue was reported for most animals examined in the study and is not indicative of respiratory disease. Minor severities and low incidences of focal pneumonitis and accumulations of alveolar macrophages are commonly observed pulmonary changes in laboratory maintained rats of this age and are not suggestive of significant respiratory disease.

MAMMARY GLAND:
Glandular hyperplasia was observed in the mammary tissue of all femaled control rats examined and in two female high dose rats. The appearance of the mammary tissue is consistent with pregnancy and lactation and the group distribution of the condition in this study is not a primary effect of treatment but related to the incidence of pregnancy.

UTERUS:
Areas of haemorrhage and fibrosis were seen in the myometrium of the uterus in all control terminal death animals and in one high dose terminal death animal. These conditions are consistent with normal post partum uterine changes in the rat and the group distribution is not a primary effect of treatment. Areas of haemorrhage and fibrosis were also seen for most female premature death animals from Group 3 and in single premature death female rat from Group 4, together with uterine dilatition in all cases. Pyometra was reported for two Group 3 female rats.

All remaining morphological changes were those commonly observed in laboratory maintained rats of the age and strain employed and, since there were no differences in incidence or severity between control and treatment groups, all were considered to be without toxicological significance.
Dose descriptor:
LOAEL
Effect level:
60 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: LOAEL, P, female, 900/1000 ppm diet (60 mg/kg bw/day). Basis - increased gestation length.
Clinical signs:
no effects observed
Description (incidence and severity):
No significant clinical findings
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
not specified
Description (incidence and severity):
Too few numbers of live litters to allow meaningful evaluation.
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Histopathological findings:
not examined
Live Litter Size and Viability:
At high and intermediate dose levels there were a small number of litters for evaluation; of those available there was a notable decrease in litter size at birth and the live litter birth index was reduced for both groups. At the intermediate dose level there was one total litter loss during lactation.
At the low dose level there was a slight reduction in group mean live litter size at birth. This did not achieve statistical significance due to the high intragroup variability but is considered to be of toxicological importance.

Offspring Clinical Condition:
There were no significant clinical findings associated with live offspring during the study.

Offspring Bodyweight Gain:
At the high and intermediate dose level, there were limited numbers of live litters to allow for meaningful evaluation of offspring weight gain.
At the low dose level there were slightly lower live litter weights compared to control values but this did not attain statistical significance and was due to lower group mean litter sizes. Group mean individual offspring bodyweights were comparable with control values.

Offspring Sex Ratio:
At the high and intermediate dose group the limited number of live litters did not allow meaningful evaluation.
At the low dose group there were no significant treatment related differences in offspring sex ratios.

Macroscopic post mortem findings:
At high and intermediate dose level there were limited numbers of offspring available for evaluation, but it is of note that the offspring of the intermediate dose level showed an increase in the number that had no milk in the stomach at examination.
At the low dose group there were no significant findings.
Dose descriptor:
LOAEL
Generation:
F1
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
viability
mortality
food consumption and compound intake
other: At the intermediate dose level, there were limited numbers of live litters to allow for meaningful evaluation of offspring weight gain, sex ratio and for macroscopic examination
Reproductive effects observed:
not specified
Conclusions:
The administration of ZMB2 to male and female Sprague-Daley rats at dose levels up to 7500 ppm diet (adjusted to 6750 ppm and then to 5500 ppm; study average of 371 mg/kg bw/day), for a period of up to 47 days, resulted in treatment-related toxic effects upon adults, both in terms of systemic and fertility endpoints. At the lowest dose (60 mg/kg bw/day), effects on fertility included decreased mean corpora lutea and total implantation counts, and increased gestation length. While these low dose effects were not statistically significant, toxicological significance cannot be ruled out based on the impacts on fertility at the higher doses and due to the high intragroup variability for the low dose group. The No Observed Adverse Effect Levels (NOAELs) for systemic and fertility endpoints were not established.

Endpoint:
extended one-generation reproductive toxicity - with F2 generation (Cohorts 1A, and 1B with extension)
Study period:
Upon approval of the ECHA.
Data waiving:
other justification
Justification for data waiving:
other:
Justification for type of information:
A study for this endpoint is in progress. This information will be submitted later based on ECHA communication/decision number TPE-D-2114355514-50-01/F.
Qualifier:
according to
Guideline:
other: OECD 443
Deviations:
no
Principles of method if other than guideline:
An OECD Test Guideline 443 Extended One Generation Reproductive Toxicity Study (EOGRTS) is proposed for the substance 1,3-dihydro-4(or 5) -methyl-2H-benzimidazole-2-thione, zinc salt (ZMB2) (CAS Number 61617-00-3 and EC Number 262-872-0).
This updated testing proposal takes into account the European Chemicals Agency’s (ECHA) request to update the testing proposal to EOGRTS from the OECD 416 two-generation reproduction study. Following review of the global regulatory requirements of both the Lead Registrant (LR) (European Union (EU) entity) and a non-EU entity represented in the EU by an Only Representative (OR) who is involved with this registration, it has been deemed appropriate to propose an EOGRTS.
In a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test (OECD TG 422) in Sprague-Dawley rats by the oral route (diet) with ZMB2 increased liver weight and histopathology, thyroid hypertrophy and increased cholesterol were reported (Safepharm, 2006). Due to these effects, the LR proposes that EOGRTS include the examination of potential endocrine related toxicity with cohort 1B extended to include the F2 generation to clarify toxicity to thyroid.
In the OECD TG 422, statistically significant reductions in male absolute organ weight values compared to control values were observed for the thymus in all dose groups. Due to these effects, the LR proposes the EOGRTS to include the examination of potential immunotoxicological effects (cohort 3).
There are no triggers to include specific neurotoxicity examinations.
In addition, the LR proposes that this study be used as read-across to 1,3-dihydro-4(or 5)-methyl-2H-benzimidazole-2-thione (MB2) (CAS Number 53988-10-6 and EC Number 258-904-8), which is reflected within the testing proposal of MB2.
Exposure via diet:
According to OECD TG 443 and EU.56 “Dietary exposure is the preferred method of administration”. Based on the complexity of this study, with extension of Cohort 1B to include the F2 generation and taking into account that the available OECD TG 422 dosed rats also via diet, diet is considered to be the appropriate dosing route.
Reproductive effects observed:
not specified
Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
20 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
2
Additional information

Considerations on a potential impact on DNELs originating from preliminary data from the ongoing extended one-generation reproductive toxicity study (OECD TG 443) for ZMB2:

The resulting DNELs based on the main, leading toxicological effect dystocia observed would be above the current ones (based on the OECD 422 LOAEL). Thus for the time being, the current DNELs are still considered protective.

Once the OECD 443 study will be completed, a definitive NOAEL as well as definitive DNELs will be derived taking into account all available data.

Effects on developmental toxicity

Description of key information
A Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test (OECD TG 422) in rats by the oral route (dietary) with ZMB2 resulted in a LOAEL value for developmental toxicity of 60 mg/kg/day.
Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
December 2002 - February 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Conducted to GLP and OECD Guidelines.
Qualifier:
according to
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
A sufficient number of male and female Sprague-Dawley Crl:CD (SD) IGS BR strain rats were obtained from Charles River UK. On receipt the animals were examined for signs of ill health or injury. The animals were acclimatised for 16 days during which time their health status was assessed. A total of eighty animals (forty males and forty females) were accepted into the study. At the start of the treatment the males weighed 298 to 375g and the females weighed 200 to 248g.
Upon arrival, the animals were housed in groups of four in polypropylene cages with stainless steel grid floors and tops, suspended over paper-lined polypropylene trays. During the mating period animals were transferred to a similar type cage on a one male to one female per cage basis.
Following evidence of successful mating, the males were returned to their original cages and transferred to a separate animal room of comparable conditions. The females were housed, individually, in polypropylene cages with solid floors and stainless steel tops. Mated females were given softwood chips, as bedding, throughout gestation and lactation.
The animals were allowed free access to food and water. A ground diet PMI 5002 was offered ad libitum. Main water was supplied from polycarbonate bottles attached to the cage. The diet and drinking water were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.
The animals were housed in air conditioned rooms. 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 by a computerised system. Both animal rooms used for this study were maintained to operate within a target temperature range of 21 +/-2 degC and a relative humidity range of 55 +/- 15%. On isolated occasions the room temperature and/or humidity fell outside the protocol limites but this was not considered to have affected the purpose or integrity of the study.
The animals were allocated to dose groups using a randomisation procedure based on stratified bodyweights and the group mean bodyweights were then determined to ensure similarity between the dose groups.
The animals were uniquely identified within the study, by an ear punching system routinely used at the laboratory. Color coded cage labels were used to assist recognition of dose groups.
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
Experimental Preparation:
The test material was prepared as a direct dietary admixture. For which dose level, an appropriate aliquot of test material was weighed and then added to approximately one third of the required amount of untreated diet in a mixing bowl. For week 1 test diet preparation the test material and diet were mixed for 20 minutes, for each dose level using a Hobart QE 200 mixer. For subsequent test diet preparations an initial mix of the test material and diet was performed using a Hobart QE 200 mixer for ten minutes. Further untreated diet was then added to the initial mix to give the required concentration and the entire amount was then mixed for ten minutes using the Hobart QE 200 mixer.
Prior to the start of the study, samples of test diet preparation were analyzed for achieved concentration, homogeneity and stability of test material in diet. The results of analysis of the original preparations showed the test material to be stable for at least 14 days. Subsequent preparations of the dietary admixtures were performed weekly. Samples of these preparations were taken on three occasions during the study to determine the achieved concentration of test material in the dietary admixture.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations”: Add “The concentration of test substance in the dietary formulations was determined by high performance liquid chromatography (HPLC) using an external standard technique. Prior to the start of the study, samples of the dietary formulations were analysed for homogeneity, stability, and validation of the dietary dose group concentrations. The results of analysis of the original preparations showed the test material to be stable for at least 14 days. Subsequent preparations of the dietary admixtures were performed weekly. Samples of these preparations were taken on three occasions during the study to determine the achieved concentration of test material in the dietary admixture.
Details on mating procedure:
After the maturation period, the parental generation adults were paired on a one male to one female basis for a period of up to fourteen days. Following pairing, the polypropylene trays beneath each cage were checked for the presence of ejected copulation plugs. Additionally each female was checked for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of the estrous cycle 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. Mated females were then separated from the male and housed individually during the period of gestation and lactation. The males were returned to their original holding cages.

Each female was observed at 08:30, 12:30 and 16:30 hours at or around the period of expected parturition. At weekends, observations were carried out at 08:30 and 12:30 only. The following was recorded for each female:
i) Date of mating
ii) Date and time of observed start of parturition
iii) Date and time of observed completion of parturition.
Duration of treatment / exposure:
47 days
Frequency of treatment:
Daily.
Duration of test:
47 days
No. of animals per sex per dose:
Control: 10 male, 10 female
1000ppm: 10 male, 10 female
2750ppm: 10 male, 10 female
7500ppm: 10 male, 10 female
Control animals:
yes, plain diet
Details on study design:
-Groups of ten male and ten female rats were dosed according to dose groups for fourteen days prior to pairing.
-All animals were given a detailed clinical examination once every week for four weeks to observe functional/behavioral changes in an open arena.
- One day prior to pairing (Day 13) five males and five females, randomly selected, were sampled for clinical chemistry and hematology.
- On Day 14 all animals were paired on a one male: one female basis within each dose group for a maximum of fourteen days.
- At the observation of mating or at the end of the fourteen day mating period males were returned to their original cages and females were transferred to individual cages.
- At the end of the mating phase five males per dose group were evaluated for functional/sensory responses to various stimuli. The males used were those selected for hematology/clinical chemistry.
- Pregnant females were allowed to give birth and maintain their offspring until Day 4 post partum. Evaluation of each litter size and weight were performed during this period.
- At Day 4 post partum, five females per dose group were evaluated for functional/sensory responses to various stimuli. The females used were those selected for hematology/clinical chemistry.
- At Day 5 post partum following completion of functional assessments all females and offspring were killed and examined macroscopically.
- Following completion of the female gestation and lactation phases, the males were killed and examined macroscopically.
Maternal examinations:
Morbidity/Mortality:
All animals were checked twice daily during the normal working week and once daily at weekends.

Clinical Observations:
All animals were observed daily, immediately before and one hour after dosing, for clinical signs of toxicity.

Functional Observations:
On the day of initiation of treatment and on Days 8, 15 and 22, all animals were observed for signs of functional/behavioral toxicity. Functional performance tests were also performed on five selected females per group on Day 22 for males and Day 4 of lactation for females, together with an assessment of sensory reactivity to difference stimuli.

Behavioral Assessments:
Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed:
Gait
Tremors
Twitches
Convulsions
Bizarre/Abnormal/Stereotypic behavior
Salivation
Pilo-erection
Exophthalmia
Lachrymation
Hyper Hypothermia
Skin colour
Respiration
Palpebral closure
Urination
Defecation
Transfer arousal
Tail elecation

Functional Performance Tests:
- Motor Activity
Twenty purpose built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals of one sex were tested at each occasion and were randomly allocated to the activity monitors. The tests were performed at approximately the same time each day, under similar laboratory conditions. The evaluation period was five hours for each animal. The percentage of time each animal was active and mobile was recorded for the overall five hour period and also during the final 20% of the period (considered to be the asymptotic period). The motor activity for females at 2750 ppm was not recorded as females were killed in extremis prior to evaluation.
- Forelimb/Hindlimb Grips Strength
An automated grip strength meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was drawn along the trough of the meter by the tail until its grip was broken. The animal was 2012 drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. The animal was pulled by the base of the tail until its grip was broken. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal. Fore and hind limb grip strength was not recorded for females at 2750 ppm as females were killed in extremes prior to evaluation.
- Sensory Reactivity:
Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. The following parameters were observed:
Grasp response
Vocalisation
Toe pinch
Tail pinch
Finger approach
Touch escape
Pupil reflex
Startle reflex
Blink reflex
The sensory reactivity for intermediate level females was not recorded as the females were killed in extremis prior to evaluation.

Bodyweight:
During the maturation and mating period the parental generation animals were weighed weekly. Following mating the parental males were weighed weekly until termination. Parental generation females showing evidence of mating were weighed on Days 0, 7, 14 and 20 post coitum. Parental generation females with a live litter were weighted on Days 1 and 4 post partum.

Food consumption and compound intake:
During the maturation period, food consumption was recorded for each cage of adults weekly. For females showing evidence of mating, food consumption was recorded for the periods covering Days 1 to 7, 7 to 14 and 14 to 21 post coitum. For females with live litters, food consumption was recorded for the period covering Days 1 to 4 post partum.

- Chemical Intake:
Chemical intake was determined based on study food consumption and body weight results as follows:
Chemical intake (mg chemical/kg bw) = dose level in feed (ppm) x group mean food consumption (g food/rat/day) / Group mean bodyweight for week (g)

Food efficiency:
Food efficiency (conversion) was determined as:
Food conversion ratio = group mean body weight gain (g bw/day) during week / group mean food consumption (g food/rat/day)

Laboratory Investigations:
Hematological and blood chemical investigations were performed on five males and five females selected from each test and control group on Study Day 13. Blood samples were obtained from the lateral tail vein. Animals were not fasted prior to 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 leucocyte count (WBC)
Differential leucocyte count - neutrophils (Neut), lymphocytes (Lymph), monocytes (Mono), eosinophil’s (Eos), basophils (Bas)
Platelet count (PLT)
Prothrombin time (CT) was assessed by 'Hepato Quick' and Activated partial thromboplastic time (APTT) was assess by 'Preci Clot' using samples collected into sodium citrate solution (0.11 mol/l).

Blood Chemistry:
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anticoagulant:
Urea
Glucose
Total protein (Tot.Prot)
Albumin
Albumin/Globulin (A/G) ratio (by calculation)
Sodium (Na+)
Potassium (K+)
Chloride (Cl-)
Calcium (CA++)
Inorganic phosphorus (P)
Aspartate aminotransferase (ASAT)
Alanine aminotransferase (ALAT)
Alkaline phosphatase (AP)
Creatinine (Creat)
Total cholesterol (Chol)
Total bilirubin (Bili)

Ovaries and uterine content:
All adult animals killed in extremis, found dead during the course of the study, or which were alive at termination (lactation Day 5) were examined macroscopically for internal and external abnormalities. All significant abnormalities in animals found dead or killed were retained in fixative for possible further study. Adult animals were killed by carbon dioxide asphyxiation; followed by exsanguination by cardiac puncture for selected animals andcervical dislocation for unselected animals.
- Organ weights: Ovaries were weighed (in pairs) at necropsy
- Endpoints evaluated for pregnant females: number of corpora lutea of all ovaries, number of uterine implantation sites; number of early resorptions; number of late resorptions
- Other examinations: Additionally, the uteri of apparently non-pregnant females were examined
- Tissue preservation: Samples of the following tissues were preserved from all animals in buffered 10% formalin: ovaries, uterus + cervix, vagina
- Histopathology: The following tissues were examined from five females selected from the control and high dose groups: ovaries, uterus + cervix, vagina.
Fetal examinations:
All offspring that died, were killed in extremis during lactation or were alive at termination (lactation Day 5) were examined macroscopically for internal and external abnormalities. Offspring were killed via barbiturate overdose.
Statistics:
- Endpoint group 1: Adult male and female bodyweight during the maturation, gestation and lactation periods, adult male food consumption, female food consumption during maturation, gestation and lactation, litter size, litter weight, individual offspring bodyweight offspring landmarks of physical development, haematology, blood chemistry and organ weights: Values were analysed to establish homogeneity of group variances using Levene's test followed by one-way analysis of variance. If the variances were unequal subsequent comparisons between control and treated groups were performed using a pairwise 'T' test Comparison Method. If variances were equal, subsequent comparisons between control and treated groups were performed using Dunnett's Multiple Comparison Method. For haematology and blood chemistry values, an additional regression analysis of all values was also performed.
- Endpoint group 2: Adult pre-coital intervals, female gestation lengths, offspring reflexological responses and litter sex ratios, relative organ weights: Individual values were compared using the Kruskal-Wallis non-parametric rank sum test. Where significant differences were seen, pairwise comparison of control values against treated group values was performed using Mann-Whitney "U" test.
- Histopathology: For those tissues from selected animals only. Chi-squared analysis for differences in the incidence of lesions occurring with an overall frequency of 1 or greater. For the comparison of severity grades for the more frequently observed conditions, Kruskal-Wallis one-way non-parametric analysis of variance.
Indices:
- Offspring live birth index(%) = [number of pups alive on Day 1 / number of pups born] x 100
- Offspring viability index(%) = [number of pups alive on Day 4 / number of pups alive Day 1] x 100
- Offspring sex ratio (%) = [number male pups / number pups of determined sex] x 100; estimated for each litter on Day 1 and on Day 4.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Results specific to parental males may be found in Section 7.5.1
Mortality:
One high dose female (number 71) was killed in extremis during the late gestation phase of the study due to possible dystocia. At the intermediate dose level a total of eight females were killed in extremis during the gestation phase of the study. Six of the eight female mortalities were found to have offspring in utero at post mortem examination indicating possible impairment of parturition. There were no mortalities at the low dose level or the control level.

Clinical Observations:
The treatment related clinical signs at the high dose were associated with the majority of females. The most prevalent of findings were hunched posture and pilo-erection. Clinical signs of toxicity were seen early in the treatment period with hunched posture observed from week 2 and pilo erection from week 5 along with sporadic instants of tip-toe gait. These findings did not persist throughout the course of the study as the remaining nine high dose female animals showed no clinical signs of toxicity at the end of the treatment. Two intermediate dose level females showed hunched posture and three showed pilo erection, but the onset of these findings was later than those seen in high dose females. At the low dose level there were no clinical signs of reaction to treatment.

Behavioural Evaluations:
The behavioural evaluation showed no evidence to suggest any significant treatment related observations associated with behavioural change. There was no indication of a treatment related effect on sensory reactivity to various stimuli or an effect on motor activity.

Bodyweight (pre-mating, gestation, lactation):
- Pre-mating
At the high dose level, bodyweight gain for females prior to mating was lower than control value and the difference in group mean bodyweight remained statistically significant (p<0.001). At the intermediate and low dose levels, there was no significant effect on female bodyweight gain prior to mating.

- Gestation
The two high dose females that were pregnant showed a significantly lower bodyweight gain during pregnancy than controls. The females of the intermediate level showed a statistically lower bodyweight gain during gestation when compared to the controls. This resulted in statistically significant differences (p<0.001) in group mean bodyweight for Days 7, 14 and 20 of gestation. At the low dose level there was a slight reduction in female bodyweight gain during gestation compared to controls. This resulted in a statistically significant difference in group mean bodyweight (p<0.01) for Day 20 of gestation.

- Lactation
At the high (one animal) and intermediate (two animals) dose level there were insufficient females with live litters to allow for meaningful evaluation of bodyweight change during lactation. At the low dose level, while female bodyweights during lactation were lower than control values, there was no significant difference in bodyweight gain between Days 1 and 4 of lactation.

Food Consumption (pre-mating, gestation, lactation):
- Pre-Mating
At the high dose level, there was a marked reduction in group mean food consumption for females prior to mating. At the intermediate dose level, there was a reduction in group mean food consumption for females prior to mating. At the low dose level, there was a reduction in female food consumption prior to mating.

- Gestation
At the high dose level from the small number of pregnant females, there was notably lower food consumption throughout gestation when compared to control values. At the intermediate dose level there was a lower group mean food consumption throughout gestation when compared with control values. The difference was statistically significant (p<0.001) of each measurement point. At the low dose level there was a lower group mean food consumption throughout gestation when compared with control values. The differences were statistically significant (p<0.01 - Days 1 to 7 of gestation, p<0.001 Days 7 to 14 and 14 to 21 of gestation).

- Lactation
At the high and intermediate dose levels there were too few females (two females at the intermediate dose level and one female at the high dose level) with live litters to allow for a meaningful evaluation of food consumption. Of the females evaluated, the food consumption values were notably lower than controls. At the low dose level there was no significant difference in female food consumption between Days 1 and 4 of lactation when compared it control values.

- Chemical intake:
As the high and intermediate dose level, there was an increase in test material intake for females in the second week of treatment prior to mating which reflects the increased food consumption seen. This was also seen for females at the low dose level.

Food Conversion:
Due to the low number of females with live litters, food conversion ratio evaluation is restricted to pre-mating only. At the high dose level, the food conversion ratio for females was notably lower than control values during the first week of treatment. At the intermediate dose level, female values were obviously lower than controls prior to mating. At the low dose level, female food conversion ratios prior to mating were comparable with control values.

Hematology:
At the high dose level, there was a slight increase in clotting time for females compared to control values. The difference was statistically significant (p<0.05). At the intermediate and low dose levels, there were no significant treatment related effects seen in the female haematological parameters examined.

Clinical Chemistry:
At the high dose level, notable statistically differences for females, compared to control values were associated with decreased plasma phosphorus values (p<0.01), decreased plasma chloride (p<0.001), and increased plasma cholesterol (p<0.001). The statistically significant difference for plasma bilirubin (p<0.05) was considered to be incidental. At the intermediate dose level, notable statistically significant differences for females compared to control values were associated with a decreased plasma phosphorus females (p<0.001), an increased plasma cholesterol (p<0.05), and a decreased plasma chloride (p<0.01). A reduction in plasma aspartate aminotransferase (p<0.01) was also observed which is considered to be incidental. At the low dose level, statistically significant differences for females when compared to control values were associated with a decreased plasma phosphorus (p<0.05) and an increased plasma cholesterol (p<0.01).

Macroscopic Post Mortem Findings:
There were no significant treatment-related macroscopic abnormalities seen at post mortem macroscopic examination. A significant number (six) of intermediate level females were found to have offspring present in utero as a consequence of dystocia.
Organ Weights:
Findings for non-reproductive female organs are provided in Section 7.5.1.

At the high dose group the low number of pregnant females resulted in too few animals of the same physiological status as the control group females to allow direct comparison of organ weights. At the intermediate dose group, the low number of females at termination resulted in too few animals to allow for a meaningful evaluation. At the low dose group, no effects on ovary weights were observed.
Uterine examination:
Findings for reproductive-related uterine content (corpora lutea and total implantation counts) are provided in Section 7.8.1.

At the high dose level, there were limited numbers of pregnancies to allow for meaningful data evaluation of the uterine parameters. At the intermediate dose level, the high post implantation loss can be attributed to the number of offspring found dead in utero, as a consequence of dystocia seen among females of this dose group. At the low dose group, the post implantation loss count was higher than control values. This low dose difference was not statistically significant but evidence of inherent variability of results among females of this dose group may be of toxicological significance.

Histopathology:
Findings for non-reproductive female organs are provided in Section 7.5.1.

OVARIES: No effects observed.

UTERUS: Areas of haemorrhage and fibrosis were seen in the myometrium of the uterus in all control terminal death animals and in one high dose terminal death animal. These conditions are consistent with normal postpartum uterine changes in the rat and the group distribution is not a primary effect of treatment. Areas of haemorrhage and fibrosis were also seen for most female premature death animals from Group 3 and in the single premature death female rat from Group 4, together with uterine dilatation in all cases. Pyometra was reported for two Group 3 female rats.

VAGINA: No effects observed.
MAMMARY GLAND:
Glandular hyperplasia was observed in the mammary tissue of all female control rats examined and in two female high dose rats. The appearance of the mammary tissue is consistent with pregnancy and lactation and the group distribution of the condition in this study is not a primary effect of treatment but related to the incidence of pregnancy.

All remaining morphological changes were those commonly observed in laboratory maintained rats of the age and strain employed and, since there were no differences in incidence or severity between control and treatment groups, all were considered to be without toxicological significance.
Key result
Dose descriptor:
LOAEL
Effect level:
60 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Litter size and viability:
At high and intermediate dose levels there were a small number of litters for evaluation; of those available there was a notable decrease in litter size at birth and the live birth index was reduced for both groups. At the intermediate dose level there was one total litter loss during lactation. At the low dose level there was a slight reduction in group mean live litter size at birth. This did not achieve statistical significance due to the high intragroup variability but is considered to be of toxicological importance.

Clinical signs:
There were no significant clinical findings associated with live offspring during the study

Bodyweight gain:
At the high and intermediate dose level, there were limited numbers of live litters to allow for meaningful evaluation of offspring weight gain. At the low dose level there were slightly lower live litter weights compared to control values but this did not attain statistical significance and was due to lower group mean litter sizes. Group mean individual offspring bodyweights were comparable with control values

Sex ratio:
At the high and intermediate dose group the limited number of live litters did not allow meaningful evaluation.
At the low dose group there were no significant treatment-related differences in offspring sex ratios.

Macroscopic exam:
At high and intermediate dose level there were limited numbers of offspring available for evaluation, but it is of note that the offspring of the intermediate dose level showed an increase in the number that had no milk in the stomach at examination. At the low dose level there were no significant findings.
Dose descriptor:
LOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
reduction in number of live offspring
changes in litter size and weights
changes in postnatal survival
other: At the intermediate dose level, there were limited numbers of live litters to allow for meaningful evaluation of offspring weight gain, sex ratio and for macroscopic examination
Dose descriptor:
LOEL
Effect level:
60 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
changes in litter size and weights
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
The administration of ZMB2 to male and female Sprague-Daley rats at dose levels up to 7500 ppm diet (adjusted to 6750 ppm and then to 5500 ppm; study average of 371 mg/kg bw/day), for a period of up to 47 days, resulted in treatment-related toxic effects upon the dams and offspring. At the lowest dose (60 mg/kg bw/day), developmental effects included increased post-implantation loss and decreased mean litter size. While these low dose effects were not statistically significant, toxicological significance cannot be ruled out based on the impacts on these endpoints at the higher doses and due to the high intragroup variability for the low dose group. No internal or external abnormalities were identified during the offspring macroscopic exam. The No Observed Adverse Effect Levels (NOAELs) for systemic and developmental endpoints were not established.
Endpoint:
developmental toxicity
Study period:
Upon approval of the ECHA
Data waiving:
other justification
Justification for data waiving:
other:
Justification for type of information:
A study for this endpoint is in progress. This information will be submitted later based on ECHA communication/decision number TPE-D-2114355514-50-01/F.

An OECD Test Guideline 414 Prenatal Development Toxicity Study is proposed for the substance 1,3-dihydro-4(or 5) -methyl-2H-benzimidazole-2-thione, zinc salt (ZMB2) (CAS Number 61617-00-3 and EC Number 262-872-0). In a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test (OECD TG 422) in Sprague-Dawley rats by the oral route (diet) with ZMB2 there were limited numbers of pregnancies at the 371 mg/kg bw/day dose group and high post implantation loss in the 150 mg/kg bw/day dose group (attributed to the number of offspring found dead in utero, as a consequence of dystocia). Further investigation of developmental toxicity is required, therefore, the prenatal Development Toxicity Study (OECD Test Guideline 414) in rats, oral route is proposed. The Lead Registrant proposes that this study be used as read-across to 1,3-dihydro-4(or 5)-methyl-2H-benzimidazole-2-thione (MB2) (CAS Number 53988-10-6 and EC Number 258-904-8).
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Abnormalities:
not specified
Developmental effects observed:
not specified
Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
20 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
2
Additional information

A Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test (OECD TG 422) in Sprague-Dawley rats by the oral route (dietary) with ZMB2 resulted in increased post-implantation loss and decreased mean litter size at the lowest dose 1000/900 ppm diet (ca. 60 mg/kg/day, based on study-specific chemical intake data).

Following ECHA (2010) guidance on DNEL calculation, a NOAEL value for developmental endpoints for ZMB2 is estimated as 20 mg/kg bw/day, and equals one-third the LOAEL value.

An OECD Guidelines for the Testing of Chemicals Test No. 443 Extended One Generation Reproductive Toxicity Study (EOGRTS) and OECD Guidelines for the Testing of Chemicals Test No. 414: Prenatal Development Toxicity Study are proposed to further assess the reproductive and developmental properties of ZMB2.


Justification for selection of Effect on developmental toxicity: via oral route:
Only one study available.

Justification for classification or non-classification

Following ECHA (2010) guidance on DNEL calculation, a NOAEL value for developmental endpoints for ZMB2 is estimated as 20 mg/kg bw/day, and equals one-third the LOAEL value.

Based on the limited animal data available (OECD 422 diet study) and concurrent systemic toxicity observed at the lowest dose the substance exhibits potential adverse effects to reproduction/development.

In accordance with Regulation No 1272/2008 Table 3.7.1(a) Hazard categories for reproductive toxicants, ZMB2 should be classified as CATEGORY 2: Suspected human reproductive toxicant based on the OECD 422 study.

However, based on preliminary data from the ongoing extended one-generation reproductive toxicity study (OECD 443), ZMB2 is being classified as a category 1B reproductive toxicant. A robust summary has not been created as the study is still ongoing and the IUCLID system will not accept the creation of a summary from a partially complete study.

Classification is justified as in the main OECD 443 study dystocia was observed amongst dams at the top dose.

Since dystocia is a discrete event, no additional information created in the study will refute or confirm the finding, and thus immediate classification is deemed necessary.