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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
December 2002 - February 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Conducted to GLP and OECD Guidelines.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Date received: 14 August 2006
Description: off-white powder
Storage conditions: ambient temperature and humidity in the dark.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
A sufficient number of male and female Sprague-Dawley Crl:CD (SD) IGS BR strain rats were obtained from Charles River UK. On receipt the animals were examined for signs of ill health or injury. The animals were acclimatised for 16 days during which time their health status was assessed. A total of eighty animals (forty males and forty females) were accepted into the study. At the start of the treatment the males weighed 298 to 375g and the females weighed 200 to 248g.
Upon arrival, the animals were housed in groups of four in polypropylene cages with stainless steel grid floors and tops, suspended over paper-lined polypropylene trays. During the mating period animals were transferred to a similar type cage on a one male to one female per cage basis.
Following evidence of successful mating, the males were returned to their original cages and transferred to a separate animal room of comparable conditions. The females were housed, individually, in polypropylene cages with solid floors and stainless steel tops. Mated females were given softwood chips, as bedding, throughout gestation and lactation.
The animals were allowed free access to food and water. A ground diet PMI 5002 was offered ad libitum. Main water was supplied from polycarbonate bottles attached to the cage. The diet and drinking water were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.
The animals were housed in air conditioned rooms. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerised system. Both animal rooms used for this study were maintained to operate within a target temperature range of 21 +/-2 degC and a relative humidity range of 55 +/- 15%. On isolated occasions the room temperature and/or humidity fell outside the protocol limites but this was not considered to have affected the purpose or integrity of the study.
The animals were allocated to dose groups using a randomisation procedure based on stratified bodyweights and the group mean bodyweights were then determined to ensure similarity between the dose groups.
The animals were uniquely identified within the study, by an ear punching system routinely used at the laboratory. Color coded cage labels were used to assist recognition of dose groups.

Administration / exposure

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.

Examinations

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.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

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.

Effect levels (maternal animals)

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

Results (fetuses)

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.

Effect levels (fetuses)

open allclose all
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

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

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.