1,3-dihydro-4(or 5)-methyl-2H-benzimidazole-2-thione, zinc salt

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

In an extended one generation reproductive toxicity study according to OECD TG 443, Vulkanox ZMB2 was administered orally, by gavage, to CD rats at dose levels of 5, 15 or 40 mg/kg bw/day.  The evaluation included assessment of the integrity and performance of the adult male and female reproductive tract, and systemic toxicity in pregnant and lactating females and in young and adult offspring.  In addition, developmental neurotoxicity and developmental immunotoxicity assessments were included, along with an evaluation of the maturing reproductive tract and its integrity and function.

Based on the results obtained in this study it was concluded that the No-Observed-Adverse-Effect-Level (NOAEL) for reproductive performance of the F0 and F1 Cohort 1B animals was 15 mg/kg/day due to the incidences of prolonged parturition/dystocia in females of both generations receiving 40 mg/kg/day.

Aside from the above mentioned instances of prolonged parturition/dystocia among females at 40 mg/kg/day, increased incidences of liver hypertrophy, thyroid gland hypertrophy and involution/atrophy of the thymus were observed at 40 mg/kg/day, therefore the NOAEL for systemic toxicity in the F0 and F1 adult animals was concluded to be 15 mg/kg/day.

The NOAEL for the F1 and F2 offspring up to weaning was concluded to be 15 mg/kg/day due to reduced early post-partum survival at 40 mg/kg/day in both generations.

There was no evidence of developmental neurotoxicity or developmental immunotoxicity on this study, therefore the NOAEL for these endpoints was concluded to be 40 mg/kg/day.

Link to relevant study records

Reference

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:

supporting study

Study period:

December 2002 - February 2003

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system 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

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.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

15 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP guideline study according TG 443.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional 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.

Effects on developmental toxicity

Description of key information

In a Prenatal Developmental Toxicity Study according to OECD guideline 414 four groups of 20 females received Vulkanox ZMB2 at doses of 8, 25 or 70 mg/kg/day by oral gavage administration, from Day 6 to 19 after mating, at a volume dose of 5mL/kg body weight. A similarly constituted Control group received the vehicle, dried corn oil at the same volume dose as treated groups. Animals were killed on Day 20 after mating for reproductive assessment and fetal examination.

Clinical observations, body weight and food consumption were recorded. Adult females were examined macroscopically at necropsy on Day 20 after mating and the gravid uterus weight recorded. All fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination or skeletal examination.

Results

Oral administration of Vulkanox ZMB2 to pregnant female rats from Day 6 to 19 after mating, inclusive, at 8, 25 or 70 mg/kg/day was generally well tolerated and elicited no deaths. On Day 6 of gestation (Day 1 of treatment), clinical signs comprised decreased activity and/or unsteady gait in several females shortly after the administration of Vulkanox ZMB2 at 70 mg/kg/day. In addition, piloerection was evident in one female and splayed hindlimbs was evident in a separate second female on Day 6 of gestation when administered with ZMB2 at a concentration of 70 mg/kg/day. From Days 7 to 20 of gestation, there was no toxicity-related changes in clinical condition of the adult females that could be clearly related to treatment.

For females receiving 25 or 70 mg/kg/day, overall mean bodyweight gain from Day 6 to 20 of gestation was slightly reduced when compared with Controls. These differences however, were not considered adverse and differences when compared with Controls were only evident from Days 6-8 of gestation for females receiving 70 mg/kg/day (greater mean weight loss followed by low weight gain) and Days 7 to 8 of gestation for females receiving 25 mg/kg/day (low weight gain), respectively. When compared with Controls, there was no effect of treatment on mean gravid uterine weights however, when the contribution of the gravid uterus was taken into consideration, a dose-dependent and statistically significant decrease in adjusted maternal body weight gain was evident in females administered with Vulkanox ZMB2 at 25 or 70 mg/kg/day.

Mean food consumption for females receiving 70 mg/kg/day was slightly but consistently lower than that of Controls and attained statistical significance at each scheduled recording. For those females receiving 25 mg/kg/day, mean food consumption was slightly lower than that of Controls from Days 10-14 of gestation only.

There were no test item-related macroscopic abnormalities detected among the parent females or fetuses at scheduled termination.

There was no effect of maternal treatment with Vulkanox ZMB2 on litter data, as assessed by the mean number of implantations, resorptions, live young, sex ratio and pre- and post‑implantation losses. Placental and litter weights were essentially similar in all groups but, fetal weights were slightly low at 70 mg/kg/day.

The incidence of major and minor fetal abnormalities and skeletal variants showed no dose response relationship to maternal treatment with Vulkanox ZMB2. Across all treated groups there was an increase in the incidence of incompletely ossified cranial bones when compared to concurrent control, the fetal and litter incidences of which exceeded the historical control data range. The assessment of ossification is, however, an evaluation at a snapshot in time, occurring at a transitory stage in fetal development, and would continue as the animals matured, and therefore this minor skeletal abnormality is considered to have no long term consequence and is not adverse.

Conclusion:

Based on the results of this study, the No-Observed-Adverse-Effect-Level (NOAEL) for maternal toxicity and embryo-fetal survival and development was concluded to be 70 mg/kg/day.

Additional 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

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting 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

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system 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.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Starting Date: 24th October, 2018 Experimental Completion Date: 22nd November, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

Please see the primciples of method if other than guideline section directly below, for further details regarding the deviation. This deviation was considered to have not affected the integrity or validity of the study.

Principles of method if other than guideline:

Deviation 1:
Section 6.1.4 of the study plan stated that only females showing at least two copulation plugs would be allocated. During the mating period, it was necessary to allocate one female which had only shown one copulation plug (plug retained in vagina), although it showed 3+ (many scattered) sperm in the vaginal smear. This female was allocated as Group 1F No. 20. Whilst the allocation of a single female with only one copulation plug does carry a slightly increased risk of non-pregnancy, the other 19 females assigned to Group 1 showed strong mating evidence to provide the required regulatory minimum of 16 litters for assessment, this deviation from the study plan was considered not to impact upon the scientific integrity of the study. This deviation was considered to have not affected the integrity or validity of the study.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Test item: Vulkanox ZMB2
Test item identity (including alternative names): 1,3-dihydro-4(or 5)-methyl-2H-benzimidazole-2-thione, zinc salt
CAS number: 61617-00-3
Intended use: Industrial chemical.
Appearance: Solid, white powder.
Storage conditions: At ambient temperature (15 to 25C) in the dark.
Supplier: Sponsor.
Batch number: RA605102T6-Dor
Purity: 95.9%
Correction factor 1.05 (for purity)
Expiry date: 10 May 2019
Supplier’s responsibilities: Characterization of the test item and the documentation of the methods of synthesis, fabrication or derivation and stability.
Archive sample: A 0.5 g representative sample was taken from each batch of test item. This sample was placed in a well closed glass container and stored in the archives under the same conditions as the bulk material.

Species:

rat

Strain:

Crj: CD(SD)

Details on test animals or test system and environmental conditions:

A total of ninety-two time-mated female Crl:CD(SD) strain rats were obtained from Charles River (UK) Limited, Margate, Kent. Spare animals were removed from the study room after treatment commenced. The day that positive evidence of mating was observed was designated Day 0 of gestation. The weight range of the animals at the start of the study (Day 0 of gestation) were 221g to 301g.

The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The Sprague Dawley [Crl:CD(SD)] strain was used because of the historical control data available at this laboratory.

TEST ANIMALS
- Source: Charles River (UK) Limited
- Age at study initiation: Approximately 69 days old
- Weight at study initiation: 221 to 301 g.
- Fasting period before study: Not stated
- Housing: Cages comprised of a polycarbonate body with a stainless steel mesh lid and a solid (polycarbonate) bottom were used during the acclimatization and gestation periods; changed at appropriate intervals.
Grid bottomed cages were used during pairing. Cages were suspended above absorbent paper which was changed daily during pairing.
- Number of animals per cgae:
Acclimatization: Up to four animals
During pairing: One (stock) male and one female
Gestation: One female
- Diet (e.g. ad libitum): Non restricted access to SDS VRF1 Certified pelleted diet. The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
- Water (e.g. ad libitum): Non restricted access to potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.
- Acclimation period: 5 days before commencement of pairing

MATING:
Male/female ratio: 1:1 with identified stock males.
Daily checks for evidence of mating: Ejected copulation plugs in cage tray and vaginal smears were checked for the presence of sperm.
Day 0 of gestation: When positive evidence of mating was detected.
A colony of stock males from the same source was maintained specifically for the purpose of mating. These animals are not part of the study and are maintained as stock animals.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Monitored and maintained within the range of 20-24ºC
- Humidity (%): Monitored and maintained within the range of 40 - 70%
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light): Artificial lighting, 12 hours light : 12 hours dark.

IN-LIFE DATES: From: Day 0 To: End of study

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The required amount of test item was ground in a mortar using a pestle to a fine powder and mixed with a small amount of the vehicle to form a paste. Any agglomerates were broken down. Further amounts of vehicle were gradually added and mixed to produce a smooth, pourable suspension. The suspension was transferred to a measuring cylinder which had been wetted with vehicle, the mortar was rinsed with vehicle and this was added to the measuring cylinder. Vehicle was added to achieve the final volume and the suspension was transferred to a beaker and mixed using a high shear homogenizer. The suspension was transferred to the final containers, via syringe whilst magnetically stirring. All jars were flushed with Nitrogen.

Group Treatment Dose (mg/kg/day) Nominal concentration (mg/mL) Formulated concentration (mg/mL)* Volume dose (mL/kg/day)
1 Vehicle 0 0 0 5
2 Vulkanox ZMB2 8 1.6 1.667 5
3 Vulkanox ZMB2 25 5 5.210 5
4 Vulkanox ZMB2 70 14 14.588 5

Frequency of preparation: Weekly.
Storage of formulation: Refrigerated temperature (2-8˚C).

VEHICLE
Dried corn oil

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability and homogeneity: Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations at 1, 10 and 200 mg/mL were analyzed to assess the stability and homogeneity of the test item in the liquid matrix. Stability was confirmed as follows:
- Formulations at 1 mg/mL were confirmed to be stable at ambient temperature (15 to 2°C) for up to six hours and refrigerated temperature (2-8°C) for 8 days.
- Formulations in the concentration range 10 mg/mL and 200 mg/mL were confirmed to be stable at ambient temperature (15 to 25°C) for one day and at refrigerated temperature (2 to 8°C) for 15 days.

Achieved concentration: Samples of each formulation prepared for administration on Day 6 and 19 after mating were analyzed for achieved concentration of the test item.

The method of analysis and results are presented within Annex 1 of the report. The mean concentrations for all occasions were within the acceptance limits of +10/-15% of the nominal concentrations, confirming the accuracy of formulation. The differences from mean remained within 2%, confirming precise analysis. Procedural recoveries were prepared at each occasion as a quality control measure and were within acceptable limits. Results are not corrected for procedural recoveries.

Details on mating procedure:

Male/female ratio 1:1 with identified stock males.
Daily checks for evidence of mating: Ejected copulation plugs in cage tray and vaginal smears were checked for the presence of sperm.
Day 0 of gestation When positive evidence of mating was detected.

A colony of stock males from the same source was maintained specifically for the purpose of mating. These animals are not part of the study and are maintained as stock animals.

Allocation and identification:
Allocation On the day of positive evidence of mating (Day 0). Only females showing at least two copulation plugs were allocated with the exception of one female that was allocated after showing one copulation plug. (See section 4)
Method To group and cage position in the sequence of mating. Females mating on any one day were evenly distributed amongst the groups.
Allocation was controlled to prevent any stock male from providing more than one mated female in each treatment group.

Animal accommadation - Number of animals per cage:
Acclimatization: Up to four animals
During pairing: One (stock) male and one female
Gestation: One female

Duration of treatment / exposure:

The test item was administered from Day 6 to Day 19 of gestation, by gavage. Control animals were treated in an identical manner with the vehicle alone.

Frequency of treatment:

Daily

Duration of test:

15 days from day 1 of treatment to day 19.

Dose / conc.:

8 mg/kg bw/day (nominal)

Remarks:

Low

Dose / conc.:

25 mg/kg bw/day (nominal)

Remarks:

Intermediate

Dose / conc.:

70 mg/kg bw/day (nominal)

Remarks:

High

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control group

No. of animals per sex per dose:

20 mated females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels chosen for investigation in this study (0, 8, 25 and 70 mg/kg/day) were based on the results from a preliminary study for effects on embryo fetal development in the female CD rat (Envigo Study Number: HT08GM).

In that study (HT08GM), the dose levels investigated were initially 0, 15, 50 and 150 mg/kg/day. The dose level of 150 mg/kg/day exceeded the maximum tolerated dose with marked signs of toxicity apparent within approximately one hour of dose administration which required premature euthanasia of two females after the first or second dose (signs of unsteady gait, decreased activity, splayed hindlimbs, flattened posture, partially closed eyes, whole body pallor and/or reduced body temperature). Consequently, the high dose level was reduced to 100 mg/kg/day, however this dose level was also considered to exceed the maximum tolerated dose due to signs of decreased activity, unsteady gait, splayed hindlimbs and reduced body temperature which persisted for at least six hours after dosing. The high dose level was therefore further reduced to 75 mg/kg/day. One female showed transient swaying gait, decreased activity and irregular breathing after the first dose administration at 75 mg/kg/day, but no further significant post-dosing signs were apparent at 75 mg/kg/day during the remainder of the study, and no post-dosing signs were apparent in animals given 15 or 50 mg/kg/day throughout the study.
Females given 50 or 100/75 mg/kg/day showed mean body weight loss after the first dose administration, after which body weight performance was similar to control. Mean food consumption was slightly lower than Control from Day 6 to Day 10 of gestation in all groups of treated females, and remained slightly lower than Control until Day 14 of gestation in the 50 mg/kg/day group and until Day 18 of gestation in the 100/75 mg/kg/day group. Litter parameters and fetal weights were considered unaffected at all dose levels investigated.
The data obtained from the preliminary embryo-fetal development study clearly demonstrated a steep dose response for Vulkanox ZMB2 administration to pregnant female rats, with little evidence of maternal toxicity at 50 mg/kg/day but with doses of 100 mg/kg/day and above exceeding the maximum tolerated dose. In the current main embryo-fetal development study, the high dose level was therefore set at 70 mg/kg/day, a 30% reduction of the lowest dose level considered to exceed the maximum tolerated dose in the preliminary study. This dose level was expected to show some evidence of maternal toxicity in terms of transient post dosing signs during the first few days of dose administration, slight initial mean body weight loss and slight reductions in food consumption. An approximate 3-fold decrease was applied for selection of the intermediate and low dose levels (25 mg/kg/day and 8 mg/kg/day) and these dose levels were chosen to achieve a dose response and/or aid in the determination of a No Observed Adverse Effect Level.

Route: Oral gavage using a suitably graduated syringe and a rubber catheter inserted via the mouth.
Treated at: Constant doses in mg/kg/day.
Volume dose: 5 mL/kg body weight.
Individual dose volume Calculated from the most recently recorded scheduled body weight.
Control (Group 1) Vehicle at the same volume dose as treated groups.
Frequency Females were treated from Day 6 to Day 19 (inclusive) after mating, once daily at approximately the same time each day.
Formulation A daily record of the usage of formulation was maintained based on weights. This balance was compared with the expected usage as a check of correct administration. No significant discrepancy was found. Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.

Maternal examinations:

Clinical Observations
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.
During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

Signs Associated with Dosing
Detailed observations were recorded daily during the treatment period at the following times in relation to dose administration:
Dose observations on Days 6-9 after mating occurred as follows:
Pre-dose
20 to 30 minutes after dosing each group
One to two hours after completion of dosing
Three to four hours after completion of dosing
As late as possible in the working day.

Dose observations from Day 10 after mating onwards occurred as follows:
Pre-dose
One to two hours after completion of dosing
As late as possible in the working day.

Clinical signs:
A detailed physical examination was performed on each animal on Days 0, 5, 12, 18 and 20 after mating to monitor general health.

Body Weight
The weight of each adult was recorded on Days 0, 3 and 6-20 after mating.

Food Consumption
The weight of food supplied to each adult, that remaining and an estimate of any spilled was recorded for the periods 0-2, 3-5, 6-9, 10-13, 14-17 and 18-19 after mating inclusive.

Terminal Investigations:
Method of kill: Carbon dioxide asphyxiation.

Necropsy:
All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.
Schedule Animals were killed on Day 20 after mating.
Sequence To allow satisfactory inter-group comparison.

Ovaries and uterine content:

Reproductive Assessment:
The following were recorded for all animals:
Uterus: Gravid uterine weight (including cervix and ovaries).
For each ovary/uterine horn Number of: Corpora lutea.
Implantation sites.
Resorption sites (classified as early or late).
Fetuses (live and dead).

Apparently non pregnant animals The number of uterine implantation sites were checked after staining with ammonium sulphide (modification of the Salewski staining technique (Salewski, E, 1964).

Fetal examinations:

Examination of all viable fetuses and placentae:
Dissected from the uterus, individually weighed and identified within the litter using a coding system based on their position in the uterus. Examined externally with abnormalities recorded. The sex of each fetus was recorded.

Examination of nominally 50% of fetuses in each litter:
Sexed internally and eviscerated

Fixation:
Fetuses eviscerated were fixed in Industrial Methylated Spirit (IMS). Remaining fetuses were fixed whole in Bouin’s fluid.

Processing:
Bouin’s fixed fetuses were subject to free-hand serial sectioning. IMS fixed fetuses were processed and stained with Alizarin Red.

Method of kill for fetuses:
Chilling on a cool plate (approximately 0°C).

Fetal Pathology Examination:
Bouin’s fixed fetuses:
Serial sections were examined for visceral abnormalities.

Alizarin Red stained fetuses:
Assessed for skeletal development and abnormalities.

Statistics:

The following data types were analyzed at each timepoint separately:
Body weight, using absolute weights and gains over appropriate study periods
Gravid uterine weight and adjusted body weight
Food consumption, over appropriate study periods
C-section litter data (corpora lutea, implantations, pre/post implantation loss, live young and sex ratio - percentage male)
Placental, litter and fetal weights

The following comparisons were performed:
Group 1 vs 2, 3 and 4

The following sequence of statistical tests was used for body weight, gravid uterus weight, food consumption, corpora lutea, implantations, pre/post implantation loss, live young, sex ratio - percentage male, placental, litter and fetal weights:

A parametric analysis was performed if Bartlett's test for variance homogeneity (Bartlett 1937) was not significant at the 1% level. For pre-treatment data, analysis of variance was used to test for any group differences. Where this was significant (p<0.05) inter group comparisons using t-tests, with the error mean square from the one-way analysis of variance, were made. For all other analyses the F1 approximate test was applied.

A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. For pre treatment data, Kruskal-Wallis’ test (Kruskal and Wallis 1952, 1953) was used to test for any group differences. Where this was significant (p<0.05) inter group comparisons using Wilcoxon rank sum tests (Wilcoxon 1945) were made.

Significant differences between the groups compared were expressed at the 5% (p<0.05) or 1% (p<0.01) level.

Indices:

Prenatal losses are separated into pre- and post-implantation phases. Pre-implantation loss was considered to reflect losses due to non-fertilization of ova and failure to implant. It was calculated from the formula:
Pre-implantation loss (%) = (Number of corpora lutea – Number of implantations) / Number of corpora lutea x 100

Where the number of implantations exceeded the number of corpora lutea observed, pre implantation loss was assumed to be zero (i.e. no pre-implantation loss was considered to have occurred).
Post-implantation loss was calculated from the formula:
Post-implantation loss (%) = (Number of implantations – Number of live fetuses) / Number of implantations x 100

All group values and SD (as appropriate) were calculated from the individual litter values.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

On Day 6 of gestation (Day 1 of treatment), decreased activity and/or unsteady gait was evident in several females shortly after the administration of Vulkanox ZMB2 at 70 mg/kg/day. Additional signs observed shortly after the first dose administration comprised piloerection in one female only (No. 68) and splayed hindlimbs in one female (No. 64).
From Day 7 to 20 of gestation, there were no signs clearly associated with administration of Vulkanox ZMB2. Whole body pallor and piloerection was observed for one female receiving 25 mg/kg/day (No. 58) however, as these observations were evident only on Days 10 and 11 of gestation and in the absence of similar findings at either 8 or 70 mg/kg/day, this was considered to have arisen by chance.

Please see tables 1 and 2 in the any other information on results section. The tables have also been attached within the attached background material section of this robust study summary.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths during the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Overall body weight gain during Days 6-20 of gestation for females receiving Vulkanox ZMB2 at 25 or 70 mg/kg/day was slightly lower than Control (94 and 90% of control, respectively). Differences in body weight performance were considered not to be adverse and were only apparent from Days 6-8 of gestation (greater mean weight loss followed by low weight gain) for females receiving 70 mg/kg/day and low weight gain on Days 7-8 of gestation for females receiving 25 mg/kg/day.
There was no effect on mean gravid uterine weights. For females receiving 25 or 70 mg/kg/day when the contribution of the gravid uterus was taken into account, a dose dependent and statistically significant decrease in adjusted maternal body weight gain was apparent (29 grams and 23 grams, respectively, compared to 36 grams in Controls).

Please see tables 3 and 4 in the any other information on results section. The tables have also been included within the attached background material section of this robust study summary. Please also see figure 1 within the illustrations section.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Following the start of treatment on Day 6 of gestation and throughout gestation up until termination on Day 20, the food consumption of females at 70 mg/kg/day was slightly consistently lower than that of Controls and attained statistical significance at each scheduled recording. For females receiving 25 mg/kg/day, food consumption was slightly lower than that of Controls from Days 10-14 of gestation only, and attained statistical significance.

Please see table 5 in the any other information on results section. The tables have also been included within the attached background material section of this robust study summary.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not specified

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not specified

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macropathology: There were no test item-related macroscopic abnormalities detected among the parent females at scheduled termination on Day 20 of gestation.
There were no test item-related macroscopic abnormalities detected among the parent females or fetuses at scheduled termination.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

not examined

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

There was no effect of maternal treatment on the mean numbers of implantations, resorptions (early or late), live young, sex ratio or the levels of pre or post implantation losses.

Please see table 6 in the any other information on results section. The tables have also been included within the attached background material section of this robust study summary.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

There was no effect of maternal treatment on the mean numbers of implantations, resorptions (early or late), live young, sex ratio or the levels of pre or post implantation losses.

Please see table 6 in the any other information on results section. The tables have also been included within the attached background material section of this robust study summary.

Early or late resorptions:

no effects observed

Description (incidence and severity):

There was no effect of maternal treatment on the mean numbers of implantations, resorptions (early or late), live young, sex ratio or the levels of pre or post implantation losses.

Please see table 6 in the any other information on results section. The tables have also been included within the attached background material section of this robust study summary.

Dead fetuses:

no effects observed

Description (incidence and severity):

All females receiving Vulkanox ZMB2 at 8, 25 or 70 mg/kg/day were pregnant with live young at scheduled termination on Day 20 of gestation. One control female (No. 20) was non-pregnant.

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

There was no effect of maternal treatment on the mean numbers of implantations, resorptions (early or late), live young, sex ratio or the levels of pre or post implantation losses. All females receiving Vulkanox ZMB2 at 8, 25 or 70 mg/kg/day were pregnant with live young at scheduled termination on Day 20 of gestation. One control female (No. 20) was non-pregnant.

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

70 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

changes in number of pregnant

clinical signs

dead fetuses

early or late resorptions

food consumption and compound intake

gross pathology

maternal abnormalities

mortality

necropsy findings

pre and post implantation loss

total litter losses by resorption

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

There was no effect of maternal treatment on mean placental or total litter weights at any dose level investigated.
When compared with Controls, mean fetal weights in the 70 mg/kg/day group were slightly but statistically significantly lower than Control (3.48 grams compared to 3.75 grams in Controls). These differences may reflect the marginally higher litter size recorded in this group (mean of 15.6 live young) compared with Controls (mean of 14.6 live young). Mean fetal weights were unaffected at 8 or 25 mg/kg/day.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

There was no effect of maternal treatment on the mean numbers of implantations, resorptions (early or late), live young, sex ratio or the levels of pre or post implantation losses.

Please see table 6 in the any other information on results section. The tables have also been included within the attached background material section of this robust study summary.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

There was no effect of maternal treatment on the mean numbers of implantations, resorptions (early or late), live young, sex ratio or the levels of pre or post implantation losses.

Please see table 6 in the any other information on results section. The tables have also been included within the attached background material section of this robust study summary.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

There was no effect of maternal treatment on mean placental or total litter weights at any dose level investigated.
When compared with Controls, mean fetal weights in the 70 mg/kg/day group were slightly but statistically significantly lower than Control (3.48 grams compared to 3.75 grams in Controls). These differences may reflect the marginally higher litter size recorded in this group (mean of 15.6 live young) compared with Controls (mean of 14.6 live young). Mean fetal weights were unaffected at 8 or 25 mg/kg/day.

Please see table 7 in the any other information on results section. The tables have also been included within the attached background material section of this robust study summary.

Changes in postnatal survival:

not examined

Description (incidence and severity):

Not applicable as animals killed on day 20 of gestation.

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

At 70 mg/kg/day there was a total of three fetuses in three litters with major abnormalities. One fetus in Litter 64 presented with misshapen/malpositioned palatine bones. One fetus in Litter 72 showed malrotated heart and retroesophageal right-sided aortic arch. One fetus in Litter 76 showed a range of skeletal abnormalities in the head, along with absent right pinna. Although the incidence of many of these major fetal abnormalities exceeded the Historical Control Data (HCD) range, since there was no correlation between the range of abnormalities seen in the individual fetuses these were considered spontaneous malformations which were unrelated to maternal treatment with Vulkanox ZMB2.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

Across all treated groups there was an increase in incidence of incompletely ossified cranial bones compared to concurrent control and outside of the historical control data (HCD) range. Delayed ossification in various skeletal elements is a minor skeletal abnormality associated with marginal developmental delay (often accompanying slight reductions in fetal weight). The assessment of ossification is, however, an evaluation at a snapshot in time, occurring at a transitory stage in fetal development, and would continue as the animals matured and therefore is considered to have no long term consequence. This minor skeletal abnormality was considered not to represent an adverse effect on embryo-fetal growth or development.

Please see tables 8, 9 and 10 in the any other information on results section. The tables have also been included within the attached background material section of this robust study summary.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Three fetuses in the 70 mg/kg/day group were noted to have a unilaterally malpositioned testis. The incidence of this minor visceral abnormality was within the HCD range, and considered incidental and unrelated to maternal treatment with Vulkanox ZMB2.

Other effects:

not examined

Key result

Dose descriptor:

NOEL

Effect level:

70 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

changes in sex ratio

fetal/pup body weight changes

changes in litter size and weights

external malformations

skeletal malformations

visceral malformations

Abnormalities:

no effects observed

Developmental effects observed:

no

TABLE 1

Signs associated with dosing - group distribution of observations

Request ID: 5270297

 

	Control	Vulkanox ZMB2
Dose Group	1	2	3	4
Dose (mg/kg/day)	0	8	25	70

 

			Number of animals affected
Category	Observation	Group/Sex:				1F	2F	3F	4F
		Initial no:				20	20	20	20
Abnormal gait	Hindlimbs splayed					0	0	0	1
	Unsteady					0	0	0	8
Behavior	Decreased activity					0	0	0	5
Coat	Piloerection					0	0	0	1

 

TABLE 2

Clinical signs - group distribution of observations

Request ID: 5270298

 

	Control	Vulkanox ZMB2
Dose Group	1	2	3	4
Dose (mg/kg/day)	0	8	25	7

 

			Number of animals affected
Category	Observation	Group/Sex:				1F	2F	3F	4F
		Initial no:				20	20	20	20
Build (Deformity)	Swollen area, Lower ventral surface					0	0	0	1
Build Conformation	Thin					1	0	1	0
Coat	Hair loss, Forelimbs					1	2	1	4
	Hair loss, Hindlimbs					1	0	0	1
	Hair loss, Ventral surface					1	0	0	0
	Piloerection					0	0	1	0
	Ungroomed					1	0	0	0
Posture	Hunched					1	0	0	0
Skin	Encrustation, Forelimbs					0	0	0	1
	Encrustation, Muzzle					0	0	1	0
Skin color	Pallor, Whole body					0	0	1	0

TABLE 3

Body weight and body weight change - group mean values (g) during gestation

Request ID: 5267315

 

	Control	Vulkanox ZMB2
Dose Group	1	2	3	4
Dose (mg/kg/day)	0	8	25	70

 

Group		Day
/Sex		0	3	6	7	8	9	10	11	12	13	14	15	16
Statistics	test	Av	Av	Av	Wi	Wi	Wi	Wi	Wi	Wi	Wi	Wi	Wi	Wi
1F	Mean	257	271	285	285	293	297	303	310	317	322	329	335	346
	SD	17.5	17.0	16.2	17.0	15.9	17.2	18.5	18.6	19.0	19.3	20.2	21.3	21.6
	N	19	19	19	19	19	19	19	19	19	19	19	19	19
2F	Mean	256	271	284	282	289	294	299	307	314	319	327	335	348
	SD	16.4	18.8	18.7	19.0	18.4	18.0	19.1	19.8	20.0	20.5	19.6	21.7	21.5
	N	20	20	20	20	20	20	20	20	20	20	20	20	20
3F	Mean	254	272	283	282	287	291	296	300	307	314	321	327	339
	SD	19.1	19.3	18.7	18.3	19.4	21.0	18.1	19.0	19.3	20.9	19.0	22.2	23.3
	N	20	20	20	20	20	20	20	20	20	20	20	20	20
4F	Mean	256	273	285	280	283	289	296	303	308	313	321	328	338
	SD	19.5	19.7	22.2	22.7	23.2	22.8	23.3	23.7	25.3	25.9	25.2	28.0	28.9
	N	20	20	20	20	20	20	20	20	20	20	20	20	20

Table 3 continued

Group		Day					Change	Change	Change	Change	Change
/Sex		17	18	19	20		0-6	6-7	7-8	8-20	6-20
Statistics	test	Wi	Wi	Wi	Wi		Av	Wi	Wi	Wi	Wi
1F	Mean	360	376	392	409		28	-1	8	116	124
	SD	23.0	24.3	25.9	27.6		8.2	8.3	5.1	16.0	16.7
	N	19	19	19	19		19	19	19	19	19
2F	Mean	362	379	394	411		28	-2	6	122	127
	SD	22.7	23.8	24.2	25.6		6.7	4.9	4.4	12.3	11.7
	N	20	20	20	20		20	20	20	20	20
3F	Mean	353	367	383	400		30	-1	5*	113	117
	SD	23.1	25.4	27.6	27.4		7.5	4.6	4.5	17.1	16.7
	N	20	20	20	20		20	20	20	20	20
4F	Mean	352	367	384	397		29	-5*	3**	114	112*
	SD	30.7	30.1	32.8	35.0		6.5	5.6	4.2	17.1	19.1
	N	20	20	20	20		20	20	20	20	20

TABLE 4

Gravid uterine weight, adjusted body weight and adjusted body weight change - group mean values (g) on day 20 of gestation

Request ID: 5267316

 

	Control	Vulkanox ZMB2
Dose Group	1	2	3	4
Dose (mg/kg/day)	0	8	25	70

 

Group		Body weight	Terminal body weight	Body weight	Gravid uterine	Adjusted body weight	Adjusted body weight
/Sex		Day 6	Day 20	change 6-20	weight	Day 20	change 6-20
Statistics	test	Av	Wi	Wi	Sh	Wi	Wi
1F	Mean	285	408	123	86.7	322	36
	SD	16.2	26.6	15.7	8.70	21.3	10.0
	N	19	19	19	19	19	19
2F	Mean	284	410	126	91.3	319	35
	SD	18.7	25.8	11.8	9.60	23.0	8.5
	N	20	20	20	20	20	20
3F	Mean	283	400	116	87.2	312	29*
	SD	18.7	28.6	18.4	18.27	21.9	10.8
	N	20	20	20	20	20	20
4F	Mean	285	396	112*	88.4	308	23**
	SD	22.2	34.7	19.3	12.35	27.8	12.8
	N	20	20	20	20	20	20

TABLE 5

Food consumption - group mean values (g/animal/day) during gestation

Request ID: 5267317

 

	Control	Vulkanox ZMB2
Dose Group	1	2	3	4
Dose (mg/kg/day)	0	8	25	70

 

Group		Day
/Sex		0-3	3-6	6-10	10-14	14-18	18-20
Statistics	test	Av	Av	Wi	Wi	Wi	Wi
1F	Mean	22	24	20	24	25	23
	SD	2.8	2.9	2.6	2.3	3.1	2.7
	N	19	19	19	19	19	19
2F	Mean	22	24	20	23	26	23
	SD	2.4	2.3	1.8	1.7	1.9	2.4
	N	20	20	20	20	20	20
3F	Mean	22	24	19	22**	24	23
	SD	2.3	2.1	1.9	2.3	2.4	2.6
	N	20	20	20	20	20	20
4F	Mean	22	25	18**	22**	23*	21**
	SD	2.3	2.5	2.1	2.6	2.6	3.1
	N	20	20	20	20	20	20

TABLE 6

Litter data - group mean values on Day 20 of gestation

Request ID: 5270090

 

	Control	Vulkanox ZMB2
Dose Group	1	2	3	4
Dose (mg/kg/day)	0	8	25	70

 

Group		Corpora	Implantations	Resorptions			Implantation loss (%)		Live young			Sex ratio
/Sex		lutea		Early	Late	Total	Pre-	Post-	Male	Female	Total	(%M)
Statistics	test	Wi	Sh				sWi	sWi			Sh	Wi
1F	Mean	17.2	15.8	1.2	0.1	1.3	8.2	7.5	7.6	7.0	14.6	52.0
	SD	1.30	1.47	1.13	0.23	1.24	6.32	7.30	1.43	1.53	1.68	8.78
	N	19	19	19	19	19	19	19	19	19	19	19
2F	Mean	17.3	16.0	0.9	0.0	0.9	7.1	5.6	7.3	7.8	15.1	47.8
	SD	2.27	1.50	0.91	0.00	0.91	8.39	5.97	2.59	2.21	1.57	14.66
	N	20	20	20	20	20	20	20	20	20	20	20
3F	Mean	16.9	15.7	0.9	0.6	1.5	8.8	9.3	7.0	7.3	14.3	48.0
	SD	1.94	2.85	1.76	2.04	2.82	12.97	18.68	2.68	2.49	4.02	12.20
	N	20	20	20	20	20	20	20	20	20	20	20
4F	Mean	17.3	16.4	0.7	0.1	0.8	5.5	4.6	7.5	8.2	15.6	47.4
	SD	2.34	2.25	0.86	0.31	1.01	7.24	6.21	2.39	2.06	2.35	11.53
	N	20	20	20	20	20	20	20	20	20	20	20

 

TABLE 7

Placental, litter and fetal weights - group mean values (g) on Day 20 of gestation

Request ID: 5270091

 

	Control	Vulkanox ZMB2
Dose Group	1	2	3	4
Dose (mg/kg/day)	0	8	25	70

 

Group		Placental	Total litter	Male fetal	Female fetal	Overall fetal
/Sex		weight	weight	weight	weight	weight
Statistics	test	Sh	Sh	Wi	Wi	Wi
1F	Mean	0.56	54.55	3.85	3.64	3.75
	SD	0.070	6.167	0.199	0.211	0.188
	N	19	19	19	19	19
2F	Mean	0.58	57.24	3.93	3.70	3.81
	SD	0.054	6.378	0.205	0.208	0.198
	N	20	20	20	20	20
3F	Mean	0.61	52.83	3.82	3.60	3.70
	SD	0.240	15.043	0.292	0.327	0.318
	N	20	20	20	20	20
4F	Mean	0.55	54.21	3.60**	3.39**	3.48**
	SD	0.057	7.530	0.212	0.163	0.175
	N	20	20	20	20	20

 

TABLE 8

Fetal examinations - major abnormality findings - group incidences

Request ID: 5269913

 

	Control	Vulkanox ZMB2
Dose Group	1	2	3	4
Dose (mg/kg/day)	0	8	25	70

 

		Fetuses					Litters
Group		1	2	3	4		1	2	3	4
Number Examined		277	301	285	312		19	20	20	20
Total Number Affected		0	0	1	3		0	0	1	3
Head
Skeletal	Absent upper incisor socket(s)	0	0	0	1		0	0	0	1
	Absent presphenoid	0	0	0	1		0	0	0	1
	Small/misshapen orbital socket(s)	0	0	0	1		0	0	0	1
	Microphthalmia	0	0	0	1		0	0	0	1
	Small/misshapen squamosal(s)	0	0	0	1		0	0	0	1
	Misshapen basisphenoid	0	0	0	1		0	0	0	1
	Absent tympanic annulus	0	0	0	1		0	0	0	1
	Misshapen/malpositioned palatine bone(s)	0	0	0	1		0	0	0	1
	Small/misshapen/fused/partially fused palatine bone(s)/mandible(s)/premaxilla/maxilla/jugal(s)/nasal(s)	0	0	0	1		0	0	0	1
	Brachygnathia	0	0	0	1		0	0	0	1
	Fused exoccipital/cervical vertebral arch(es)	0	0	1	0		0	0	1	0
External	Absent pinna(e)	0	0	0	1		0	0	0	1
Cervical/Thoracic
Skeletal	Absent cervical vertebral arch(es)	0	0	1	0		0	0	1	0
Visceral	Retroesophageal right sided aortic arch	0	0	0	1		0	0	0	1
	Malrotated heart	0	0	0	1		0	0	0	1

 

Note: Individual fetuses/litters may occur in more than one category.

  

TABLE 9

Fetal examinations - minor skeletal abnormality and variants findings - group incidences

Request ID: 5269895

 

	Control	Vulkanox ZMB2
Dose Group	1	2	3	4
Dose (mg/kg/day)	0	8	25	70

 

		Fetuses					Litters
Group		1	2	3	4		1	2	3	4
Number Examined		140	152	143	156		19	20	20	20
Minor skeletal abnormalities
Cranial	sutural bone(s)	0	1	0	0		0	1	0	0
	fissure(s)	0	1	0	0		0	1	0	0
	interparietal fissure(s)	1	0	1	0		1	0	1	0
Ribs	medially thickened/kinked	1	0	0	0		1	0	0	0
Sternebrae	bipartite ossified	0	0	1	0		0	0	1	0
	misaligned ossification sites	2	0	0	1		2	0	0	1
	misaligned hemicentres	0	0	1	1		0	0	1	1
	misshapen	0	0	1	0		0	0	1	0
Costal cartilage	misaligned	0	0	2	1		0	0	1	1
	7th not connected to sternum	1	0	0	0		1	0	0	0
Appendicular	misshapen cranial margin scapula(e)	0	1	0	0		0	1	0	0
Total affected by one or more of the above		5	3	3	2		5	3	2	2

 

Note: Individual fetuses/litters may occur in more than one category.

TABLE 9 (cont)

Fetal examinations - minor skeletal abnormality and variants findings - group incidences

Request ID: 5269895

 

	Control	Vulkanox ZMB2
Dose Group	1	2	3	4
Dose (mg/kg/day)	0	8	25	70

 

		Fetuses					Litters
Group		1	2	3	4		1	2	3	4
Number Examined		140	152	143	156		19	20	20	20
Rib and vertebral configuration
Cervical rib	short supernumerary	1	0	1	0		1	0	1	0
13th rib	short with/without costal cartilage	1	2	2	1		1	2	1	1
Number of 14th ribs	short supernumerary	14	5	13	15		7	4	8	8
	full supernumerary	0	0	0	1		0	0	0	1
	total	14	5	13	16		7	4	8	9
Pelvic girdle	unilateral cranial shift	1	0	1	3		1	0	1	1
	unilateral caudal shift	0	0	0	1		0	0	0	1

 

Note: Individual fetuses/litters may occur in more than one category.

 

TABLE 10

Fetal examinations - minor visceral abnormality and necropsy findings - group incidences

Request ID: 5269896

 

	Control	Vulkanox ZMB2
Dose Group	1	2	3	4
Dose (mg/kg/day)	0	8	25	70

 

		Fetuses					Litters
Group		1	2	3	4		1	2	3	4
Number Examined		137	149	142	156		19	20	20	20
Total Number Affected		32	24	38	37		15	16	15	17
Visceral abnormalities
Brain	dilated interventricular foramen	0	2	1	0		0	1	1	0
Eyes	folded retina	1	0	0	0		1	0	0	0
Thyroid	small lobe	0	0	0	1		0	0	0	1
	absent lobe	0	1	0	0		0	1	0	0
Thymus	partially undescended lobe	2	3	2	1		2	2	2	1
Left subclavian artery	arising from aortic arch	0	0	1	0		0	0	1	0
Caudal vena cava	anomalous confluence with left hepatic vein	1	0	2	0		1	0	2	0
Diaphragm	thinning with liver protrusion	1	3	3	3		1	3	3	2
Liver	bilobed/fissured posterior caudate lobe	0	0	1	1		0	0	1	1
	folded posterior caudate lobe	0	1	1	0		0	1	1	0
Kidney(s)	small renal papilla	2	0	0	0		2	0	0	0
Ureter(s)	dilated	2	0	0	2		2	0	0	2
Testis(es)	undescended	1	0	1	0		1	0	1	0
	malpositioned	0	0	0	3		0	0	0	3
Umbilical artery	left	1	2	0	4		1	2	0	4
	additional right	1	0	0	0		1	0	0	0

 

Note: Individual fetuses/litters may occur in more than one category.

TABLE 10 (cont)

Fetal examinations - minor visceral abnormality and necropsy findings - group incidences

Request ID: 5269896

 

	Control	Vulkanox ZMB2
Dose Group	1	2	3	4
Dose (mg/kg/day)	0	8	25	70

 

		Fetuses					Litters
Group		1	2	3	4		1	2	3	4
Number Examined		137	149	142	156		19	20	20	20
Total Number Affected		32	24	38	37		15	16	15	17
Haemorrhages
Head	brain	5	0	0	6		4	0	0	5
	vitreous humour eye	1	0	0	0		1	0	0	0
	eye lid(s)	0	1	0	0		0	1	0	0
Neck/Thorax	dorsal fat pad	3	1	4	4		3	1	4	4
Abdomen	abdominal cavity	12	9	19	13		10	7	11	9
	liver lobes	2	4	7	5		2	4	5	5
General	subcutaneous	0	0	1	1		0	0	1	1
Necropsy observations (external)
Skin	shiny	0	1	0	0		0	1	0	0

 

Note: Individual fetuses/litters may occur in more than one category.

 

 

Conclusions:

Based on the results of this study, the No-Observed-Adverse-Effect-Level (NOAEL) for maternal toxicity and embryo-fetal survival and development was concluded to be 70 mg/kg/day.

Executive summary:

The purpose of this studywas to assess the influence of Vulkanox ZMB2 on embryo-fetal survival and development when administered during the organogenesis and fetal growth phases of pregnancy in the CD rat [Crl:CD(SD)].

Four groups of 20 females received Vulkanox ZMB2 at doses of 8, 25 or 70 mg/kg/day by oral gavage administration, from Day 6 to 19 after mating, at a volume dose of 5mL/kg body weight. A similarly constituted Control group received the vehicle, dried corn oil at the same volume dose as treated groups. Animals were killed on Day 20 after mating for reproductive assessment and fetal examination.

Clinical observations, body weight and food consumption were recorded. Adult females were examined macroscopically at necropsy on Day 20 after mating and the gravid uterus weight recorded. All fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination or skeletal examination.

Results

Oral administration of Vulkanox ZMB2 to pregnant female rats from Day 6 to 19 after mating, inclusive, at 8, 25 or 70 mg/kg/day was generally well tolerated and elicited no deaths. On Day 6 of gestation (Day 1 of treatment), clinical signs comprised decreased activity and/or unsteady gait in several females shortly after the administration of Vulkanox ZMB2 at 70 mg/kg/day. In addition, piloerection was evident in one female and splayed hindlimbs was evident in a separate second female on Day 6 of gestation when administered with ZMB2 at a concentration of 70 mg/kg/day. From Days 7 to 20 of gestation, there was no toxicity-related changes in clinical condition of the adult females that could be clearly related to treatment.

For females receiving 25 or 70 mg/kg/day, overall mean bodyweight gain from Day 6 to 20 of gestation was slightly reduced when compared with Controls. These differences however, were not considered adverse and differences when compared with Controls were only evident from Days 6-8 of gestation for females receiving 70 mg/kg/day (greater mean weight loss followed by low weight gain) and Days 7 to 8 of gestation for females receiving 25 mg/kg/day (low weight gain), respectively. When compared with Controls, there was no effect of treatment on mean gravid uterine weights however, when the contribution of the gravid uterus was taken into consideration, a dose-dependent and statistically significant decrease in adjusted maternal body weight gain was evident in females administered with Vulkanox ZMB2 at 25 or 70 mg/kg/day.

Mean food consumption for females receiving 70 mg/kg/day was slightly but consistently lower than that of Controls and attained statistical significance at each scheduled recording. For those females receiving 25 mg/kg/day, mean food consumption was slightly lower than that of Controls from Days 10-14 of gestation only.

There were no test item-related macroscopic abnormalities detected among the parent females or fetuses at scheduled termination.

There was no effect of maternal treatment with Vulkanox ZMB2 on litter data, as assessed by the mean number of implantations, resorptions, live young, sex ratio and pre- and post‑implantation losses. Placental and litter weights were essentially similar in all groups but, fetal weights were slightly low at 70 mg/kg/day.

The incidence of major and minor fetal abnormalities and skeletal variants showed no dose resopnse relationship to maternal treatment with Vulkanox ZMB2. Across all treated groups there was an increase in the incidence of incompletely ossified cranial bones when compared to concurrent control, the fetal and litter incidences of which exceeded the historical control data range. The assessment of ossification is, however, an evaluation at a snapshot in time, occurring at a transitory stage in fetal development, and would continue as the animals matured, and therefore this minor skeletal abnormality is considered to have no long term consequence and is not adverse.

Conclusion:

Based on the results of this study, the No-Observed-Adverse-Effect-Level (NOAEL) for maternal toxicity and embryo-fetal survival and development was concluded to be 70 mg/kg/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

70 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Guideline study.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

In a Prenatal Developmental Toxicity Study according to OECD guideline 414 four groups of 20 females received Vulkanox ZMB2at doses of 8, 25 or 70 mg/kg/day by oral gavage administration, from Day 6 to 19 after mating, at a volume dose of 5mL/kg body weight.

There were no test item-related macroscopic abnormalities detected among the parent females or fetuses at scheduled termination.

There was no effect of maternal treatment withVulkanox ZMB2on litter data, as assessed by the mean number of implantations, resorptions, live young, sex ratio and pre- and post‑implantation losses. Placental and litter weights were essentially similar in all groups but, fetal weights were slightly low at 70 mg/kg/day.

The incidence of major and minor fetal abnormalities and skeletal variants showed no dose response relationship to maternal treatment with Vulkanox ZMB2. 

The No-Observed-Adverse-Effect-Level (NOAEL) for maternal toxicity and embryo-fetal survival and development was concluded to be 70 mg/kg/day.

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 (2012) 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.

Justification for classification or non-classification

In an extended one generation reproductive toxicity study according to OECD TG 443, Vulkanox ZMB2 was administered orally, by gavage, to CD rats at dose levels of 5, 15 or 40 mg/kg bw/day.  

Based on the results obtained in this study it was concluded that the No-Observed-Adverse-Effect-Level (NOAEL) for reproductive performance of the F0 and F1 Cohort 1B animals was 15 mg/kg/day due to the incidences of prolonged parturition/dystocia in females of both generations receiving 40 mg/kg/day.

Aside from the above mentioned instances of prolonged parturition/dystocia among females at 40 mg/kg/day, increased incidences of liver hypertrophy, thyroid gland hypertrophy and involution/atrophy of the thymus were observed at 40 mg/kg/day, therefore the NOAEL for systemic toxicity in the F0 and F1 adult animals was concluded to be 15 mg/kg/day.

The NOAEL for the F1 and F2 offspring up to weaning was concluded to be 15 mg/kg/day due to reduced early post-partum survival at 40 mg/kg/day in both generations.

In a Prenatal Developmental Toxicity Study according to OECD guideline 414 the No-Observed-Adverse-Effect-Level (NOAEL) for maternal toxicity and embryo-fetal survival and development was concluded to be 70 mg/kg/day (highest applied dose).

In 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.

According to CLP classification criteria (Regulation (EC) No 1272/2008) a classification as Repr. 1B ( H360: May damage fertility or the unborn child) is proposed (self-classification).