Registration Dossier

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:
Experimental Starting Date: 24th October, 2018 Experimental Completion Date: 22nd November, 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guideline
Qualifier:
according to
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. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
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.

Correction factor 1.05 (for purity)
Expiry date: 10 May 2019


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.

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Details on test animals 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

Administration / exposure

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.
Doses / concentrationsopen allclose all
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.


Examinations

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.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

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

Maternal developmental toxicity

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

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
70 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
gross pathology
maternal abnormalities
pre and post implantation loss
total litter losses by resorption
early or late resorptions
dead fetuses
changes in number of pregnant
necropsy findings

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

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

Effect levels (fetuses)

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

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no

Any other information on results incl. tables

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.

 


 

Applicant's summary and conclusion

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.