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

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

No reproductive toxicity study is available for 2-mercaptomethylbenzimidazole MB2 (CAS 53988-10-6). Based on the instability of ZMB2 in aqueous solution, i.e. the quantitative dissociation within seconds, a read-across from ZMB2 to MB2 is considered fully valid and justified. Such read-across data have already been accepted by ECHA in the context of a compliance check on the registered substance (MB2, ECHA communication number:CCH-C-2114429239-45-01/F).

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.

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

No reproductive toxicity study is available for 2-mercaptomethylbenzimidazole MB2 (CAS 53988-10-6). Based on the instability of ZMB2 (CAS 61617-00-3) in aqueous solution, i.e. the quantitative dissociation within seconds, a read-across from MB2 and vice versa is fully valid and justified (ECHA communication number: TPE-C-2114492370-50-01/F).

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 withVulkanox 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.

Link to relevant study records

Reference

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

Justification for classification or non-classification

No reproductive toxicity study is available for 2-mercaptomethylbenzimidazole MB2 (CAS 53988-10-6). Based on the instability of ZMB2 in aqueous solution, i.e. the quantitative dissociation within seconds, a read-across from ZMB2 to MB2 is considered fully valid and justified. Such read-across data have already been accepted by ECHA in the context of a compliance check on the registered substance (MB2, ECHA communication number:CCH-C-2114429239-45-01/F).

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 414the 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).