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

Toxicity to reproduction

Administrative data

Endpoint:

extended one-generation reproductive toxicity – with F2 generation and both developmental neuro- and immunotoxicity (Cohorts 1A, 1B with extension, 2A, 2B, and 3)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 February 2019 - 09 September 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Justification for study design:

Test System
CD Rat (virgin); accepted by regulatory agencies, historical control data available.

Route of Administration
Oral (gavage), an appropriate route to conduct a human risk assessment.

Treatment Groups and Doses
Group Dose levels (mg/kg/day)
1 0
2 5
3 15
4 40

Rationale for Dose Level Selection
The dose levels selected for investigation in this study (0, 5, 15 and 40 mg/kg/day) were selected in conjunction with the Sponsor based on the results obtained from a preliminary study of reproductive performance in the CD rat (Envigo/Covance Study number YR11TG), in which dose levels of 0, 15, 50 and 75 mg/kg/day were employed.

Although initially tolerated, administration at 75 mg/kg/day was not tolerated by pregnant F0 females from Day 20 of gestation onwards, with 3/8 of the females in the group observed to have prolonged parturition on Day 22/23 of gestation with blood apparent in the cage but no evidence of any pups born and 2/8 females showing either total litter loss or very high levels of pup mortality on Day 1 of lactation; one female in the 50 mg/kg/day group also showed prolonged parturition on Day 23 of gestation. As a consequence of these cases of dystocia/litter death, the female given 50 mg/kg/day and all females given 75 mg/kg/day were killed for reasons of animal welfare during Days 22 of gestation and Day 1 of lactation.

At 50 mg/kg/day, extended gestation length was apparent, and the mean number of implantation sites and total litter size, along with post-implantation survival and live birth indices were low when compared to Controls. Following weaning on Day 21 of age, male offspring showed a 13% reduction in mean body weight gain and low mean food intake compared to Controls, along with a 2.3-day delay in the mean age of attainment of sexual maturation.

At scheduled termination increased adjusted mean kidney weights were seen in all groups of treated F0 males, in F0 females given 50 mg/kg/day and in F1 females given 15 or 50 mg/kg/day. In addition, adjusted mean liver weights were increased in all groups of treated F0 males, in F0 females given 50 mg/kg/day and in both sexes of F1 animals given 15 or 50 mg/kg/day. F0 males given 50 or 75 mg/kg/day also showed increased mean adjusted thyroid and parathyroid weights. Histopathological evaluation identified the liver and thyroid glands as target organs, although the findings were considered non-adverse: F0 animals showed centrilobular hypertrophy in the liver at 75 mg/kg/day, and periportal vacuolation in the liver and follicular cell hyperplasia in the thyroid glands at 15 mg/kg/day in males and both sexes at 50 mg/kg/day or above; F1 animals showed follicular cell hyperplasia in the thyroid glands in males at 15 mg/kg/day and in both sexes at 50 mg/kg/day.

Based on these results, it was considered that dose levels of 50 mg/kg/day and above were unsuitable for investigation in this extended one generation study. The high dose level was consequently set at 40 mg/kg/day, with the low and intermediate dose levels set at 5 and 15 mg/kg/day respectively, to achieve a dose response and/or aid in the determination of a No Observed Adverse Effect Level.

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

262-872-0

EC Name:

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

Cas Number:

61617-00-3

Molecular formula:

C8H8N2S.1/2Zn

IUPAC Name:

Zinc bis[4(or 5)-methyl-2-thioxo-2,3-dihydrobenzimidazol-1-ide]

Details on test material:

methyl-2-mercaptobenzimidazole, zinc salt, purity ~ 95%

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.
Batch number: RA605102T6_Dor
Expiry date: 07 March 2020
Purity: 95.2%

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Details on species / strain selection:

Species: Rat
Strain: Crl:CD(SD)
Age at the start of treatment:
Males: 76 to 82 days
Females: 69 to 75 days
Weight range at the start of treatment:
Males: 342 to 461 g
Females: 223 to 279 g
Specification: Males and females unrelated (no male/female siblings)
Duration of acclimatization
for F0 animals: Six days before commencement of treatment.
Number of animals ordered:
110 males and 110 females (220 in total), (100 animals per sex allocated to study; 10 animals per sex spare)
Supplier: Charles River (UK) Limited.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animals - Housing, Diet and Water Supply, and Environmental Enrichment
Environmental Control
Animal facility: Limited access - to minimize entry of external biological and chemical agents.
Air supply: Filtered, not recirculated.
Temperature: 20-24ºC.
Relative humidity: 40-70%.
Monitored daily: Excursions outside these ranges documented in the study data.
Lighting: 12 hours light : 12 hours dark.
Alarm systems: Activated on ventilation failure and when temperature/humidity limits exceeded.

Cages
Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals.
Solid (polycarbonate) bottom cages were used throughout the study except during pairing.
Grid bottomed cages were used during pairing. These were suspended above absorbent paper which was changed daily.
The cages were distributed on the racking to equalize, as far as possible, environmental influences amongst the groups.

Bedding, Diet and Water Supply
Bedding
Bedding type: Softwood based bark-free fiber, sterilized by autoclaving.

Diet supply
Diet name: SDS VRF1 Certified, pelleted diet.
Availability: Non-restricted.
Certification: Before delivery each batch of diet is analyzed by the supplier for various nutritional components and chemical and microbiological contaminants.
This diet contains no added antibiotic or other chemotherapeutic or prophylactic agent.

Water supply
Supply: Potable water from the public supply.
Availability: Non-restricted via polycarbonate bottles with sipper tubes.

Environmental enrichment
During the acclimatization and appropriate study periods environmental enrichment in the form of Aspen wood based products (soft white untreated wood) and a plastic shelter were available in each home cage, except when animals were separated into single housing overnight prior to functional observational battery testing and during lactation. Nesting material was provided from Day 20 after mating and throughout lactation. Environmental enrichment (wood based products and plastic shelters) was returned to F0 females on Day 21 of lactation after weaning of offspring.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: Dried corn oil

Details on exposure:

Test Item Preparation, Analysis
Formulation
Treatment Dose (mg/kg/day) Nominal concentration (mg/mL) Formulated concentration (mg/mL)*
Group 1 0 0 0
Group 2 5 1 1.05
Group 3 15 3 3.15
Group 4 40 8 8.40
* Includes conversion factor of 1.05
Dose Volume 5 ml/kg/day

Validated concentration range. 1 mg/mL and 10 mg/mL to 200 mg/mL.
Validation status. GLP (Covance Study No. WC71LQ).
Correction factor. 1.05.
Vehicle. Dried corn oil.

Method of preparation:
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.

A series of formulations at the required concentrations were prepared by dilution of individual weighings of the test item.

Frequency of preparation: Weekly.
Storage of formulation: Refrigerated (2 to 8°C).

Stability:
Formulations at 1 mg/mL were confirmed to be stable at ambient temperature (15 to 25°C) for six hours and refrigerated temperature (2 to 8°C) for eight 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.

Justification for use and choice of vehicle:
The original vehicle intended for this study was SDS VRF1 diet. An analytical method was developed and successfully validated, however, the dietary route was determined to be unsuitable after preparing numerous diets for stability trials which did not meet acceptance criteria. Therefore, it was decided that the study would be conducted using oral gavage administration, with formulations prepared in dried corn oil, as employed in the associated OECD 414 study (Covance Study No. WC71LQ).

Details on mating procedure:

Mating
F0 pairing commences After 2 weeks of treatment.
Cohort 1B F1 pairing commences After 10 weeks of formal treatment (nominal Week 14 of age).
Male/female ratio 1:1 (sibling pairing will not be permitted).
Duration of pairing Up to 2 weeks.
Daily checks for evidence of mating Ejected copulation plugs. Sperm within vaginal smear.
Day 0 of gestation When positive evidence of mating detected.
Male/female separation Day when mating evidence detected.
Pre-coital interval Calculated for each female as time between first pairing and evidence of mating.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Formulation Analysis - Liquid formulations for dose administration
Liquid formulation:
Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations were analyzed to assess the stability and homogeneity of the test item in the liquid matrix.
At specified intervals during treatment, the test formulations were analyzed for achieved concentration of the test item.

Analysis:
The method of analysis will be documented in the study data and a summary included in the final report.
The analytical method will involve the extraction of the test formulations with a suitable solvent followed by chromatographic assay.
The formulated samples will be analyzed using a method validated with respect to the determination of the specificity of analysis, limits of detection, linearity of detector response, repeatability, method accuracy and precision.

Duration of treatment / exposure:

F0 animals: For 2 weeks before pairing until termination after litters are weaned.
F1 animals: From weaning@ until termination of respective cohort.
Unselected F1 offspring: Retention of brain, spleen, thymus and mammary tissue and organ weights - no direct treatment, killed on Day 22 of age
Cohort 1A: Reproductive /developmental toxicity testing - treated from weaning to 13 weeks of age.
Cohort 1B: Reproductive /developmental toxicity testing - treated from weaning to 21/22 weeks of age following breeding at ca 14 weeks of age.
Cohort 2A: Developmental neurotoxicity testing - treated from weaning up to Day 75 of age.
Cohort 2B: Developmental neurotoxicity testing - assigned to neurohistopathology assessment at weaning.
Cohort 3: Developmental immunotoxicity testing - treated from weaning up to Day 60 of age.
@ Although direct exposure starts at weaning on Day 21 of age, all offspring have potential indirect exposure in-utero and through the milk during lactation.

Frequency of treatment:

Daily.

Details on study schedule:

Selection of Offspring to form F1 Generation
Selection On Day 18-20 of age.
Allocation - formal start of F1 generation Nominally Day 28 of age (direct dose administration will commence on Day 21 of age).
Number per group: Cohort 1A + 1B: 20 male + 20 female per dose level (0, 5, 15, 40 mg/kg/day)
Cohort 2A, 2B, 3: 10 male + 10 female per dose level (0, 5, 15, 40 mg/kg/day)
Method:
The offspring with the lowest within-litter identification per sex from each selected litter will be selected to form the F1 generation, after exclusion of grossly atypical animals.
In the event that insufficient offspring are available in a given litter, priority for populating the cohorts will be as follows: 1B, 1A, 2A, 2B and 3. For F1 Cohorts 1A and 1B where possible, one male and one female will be selected from each selected litter. If more are required, up to two males and two females may be selected from each selected litter.
For Cohorts 2A, 2B and 3: where possible, one male or one female will be selected from each selected litter. If more are required, up to one male and one female may be selected from each selected litter.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Remarks:

Control

Doses / concentrations 2

Dose / conc.:

5 mg/kg bw/day

Remarks:

Vulkanox ZMB2

Doses / concentrations 3

Dose / conc.:

15 mg/kg bw/day

Remarks:

Vulkanox ZMB2

Doses / concentrations 4

Dose / conc.:

40 mg/kg bw/day

Remarks:

Vulkanox ZMB2

No. of animals per sex per dose:

F0 generation: 25 male + 25 female per dose level (0, 5, 15, 40 mg/kg/day)
Cohort 1A + 1B: 20 male + 20 female per dose level (0, 5, 15, 40 mg/kg/day)
Cohort 2A, 2B, 3: 10 male + 10 female per dose level (0, 5, 15, 40 mg/kg/day)

Control animals:

yes, concurrent vehicle

Details on study design:

Rationale for Dose Level Selection
The dose levels selected for investigation in this study (0, 5, 15 and 40 mg/kg/day) were selected in conjunction with the Sponsor based on the results obtained from a preliminary study of reproductive performance in the CD rat (Covance Study number YR11TG), in which dose levels of 0, 15, 50 and 75 mg/kg/day were employed.

Although initially tolerated, administration at 75 mg/kg/day was not tolerated by pregnant F0 females from Day 20 of gestation onwards, with 3/8 of the females in the group observed to have prolonged parturition on Day 22/23 of gestation with blood apparent in the cage but no evidence of any pups born and 2/8 females showing either total litter loss or very high levels of pup mortality on Day 1 of lactation; one female in the 50 mg/kg/day group also showed prolonged parturition on Day 23 of gestation. As a consequence of these cases of dystocia/litter death, the female given 50 mg/kg/day and all females given 75 mg/kg/day were killed for reasons of animal welfare during Days 22 of gestation and Day 1 of lactation.
At 50 mg/kg/day, extended gestation length was apparent, and the mean number of implantation sites and total litter size, along with post-implantation survival and live birth indices were low when compared to Controls. Following weaning on Day 21 of age, male offspring showed a 13% reduction in mean body weight gain and low mean food intake compared to Controls, along with a 2.3-day delay in the mean age of attainment of sexual maturation.

At scheduled termination increased adjusted mean kidney weights were seen in all groups of treated F0 males, in F0 females given 50 mg/kg/day and in F1 females given 15 or 50 mg/kg/day. In addition, adjusted mean liver weights were increased in all groups of treated F0 males, in F0 females given 50 mg/kg/day and in both sexes of F1 animals given 15 or 50 mg/kg/day. F0 males given 50 or 75 mg/kg/day also showed increased mean adjusted thyroid and parathyroid weights. Histopathological evaluation identified the liver and thyroid glands as target organs, although the findings were considered non-adverse: F0 animals showed centrilobular hypertrophy in the liver at 75 mg/kg/day, and periportal vacuolation in the liver and follicular cell hyperplasia in the thyroid glands at 15 mg/kg/day in males and both sexes at 50 mg/kg/day or above; F1 animals showed follicular cell hyperplasia in the thyroid glands in males at 15 mg/kg/day and in both sexes at 50 mg/kg/day.
Based on these results, it was considered that dose levels of 50 mg/kg/day and above were unsuitable for investigation in this extended one generation study. The high dose level was consequently set at 40 mg/kg/day, with the low and intermediate dose levels set at 5 and 15 mg/kg/day respectively, to achieve a dose response and/or aid in the determination of a No Observed Adverse Effect Level.

In Life Sampling and Analysis
Conditions: Following overnight deprivation of food. Samples collected under light general anesthesia.

Positive control:

No.

Examinations

Parental animals: Observations and examinations:

Clinical Observations (F0 generation and F1 generation - F1 Cohorts 1, 2 & 3)
Animals and their cages: Visually inspected at least twice daily for evidence of reaction to treatment or ill-health.*
Physical examination: Once each week.
After mating of F0 and F1 Cohort 1B females: Days 0, 7, 14 and 20 after mating and Days 1, 7, 14 and 21 of lactation.

A detailed physical examination was performed at nominally the same time of day on each occasion by an observer. After removal from the home cage, animals were assessed for physical condition and behavior during handling. Particular attention was paid to possible signs of neurotoxicity such as convulsions, tremor and abnormalities of gait or behavior. Any deviations from normal will be recorded with respect to nature, and, where appropriate, degree of severity.
* Only signs which are indicative of ill-health will be routinely recorded as part of this twice daily health check. Those signs which are not indicative of ill-health will routinely be recorded, as appropriate, as part of the detailed physical examination check.

In addition detailed observations was performed on F0 and formal F1 generation animals to establish and confirm a pattern of signs associated with dosing according to the following schedule:
F0 generation and F1 generation Cohort 1B:
- Week 1 - Daily.
- Weeks 2 to 4 - twice weekly (middle and end of the week)
- Week 5 onward - once each week (Days 0, 7, 14 and 20 after mating and Days 1, 7, 14 and 20 of lactation for F0 females).

F1 generation Cohort 1A, 2 & 3:
- Week 1 - Daily.
- Weeks 2 to 4 - twice weekly (middle and end of the week)
- Week 5 onward - once each week

Detailed observations were performed in the treatment period, at the following times during the day:
Dose observations
- Pre-dose observation.
- 20 to 30 minutes after dosing each group.
- 1 to 2 hours after completion of dosing.
- As late as possible in the working day.

Clinical Signs
A detailed physical examination was performed on each animal to monitor general health according to the following schedule:
Physical examination: Once each week. After mating of F0 and F1 Cohort 1B females: Days 0, 7, 14 and 20 after mating and Days 1, 7, 14 and 21 of lactation.

Mortality
Premature sacrifice: Animals may be killed for reasons of animal welfare.
Animals found dead,
or killed for reasons
of animal welfare: A necropsy is performed as soon as possible.

Body Weight (F0 and F1 Generation)
F0 Males Day that treatment commences. Each week. Before necropsy.
F0 Females Day that treatment commences. Each week until mating detected. Days 0, 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 after mating. Days 1, 4, 7, 14, 21 and 28 of lactation. Before necropsy.
F1 selected animals Cohorts 1A and 2A and 3: From nominal Week 4 of age, twice during Week 1 of the F1 generation and weekly thereafter.Before necropsy.
Cohort 1B: From nominal Week 4 of age, twice during Week 1 of the F1 generation and weekly thereafter until mating detected then on:
Days 0, 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 after mating
Days 1, 4, 7, 14 and 21 and 28 of lactation.
Before necropsy.

Food Consumption (F0 and F1 generation)
F0 Animals Weekly.
Food consumption was not recorded for males and females during the period when paired for mating but recommenced for males once pairing of all the animals is completed.
For females after mating food consumption schedule will match body weight schedule:
Days 0-1, 2-3, 4-5, 6-7, 8-9, 10-11, 12-13, 14-15, 16-17, 18-20 after mating Days 1-4, 4-7, 7-14 and 14-21 of lactation.
F1 selected animals Cohorts 1A and 2A and 3: From nominal Week 4 of age, twice during Week 1 of the F1 generation and weekly thereafter.
Cohort 1B: From nominal Week 4 of age, twice during Week 1 of the F1 generation and weekly thereafter until paired for mating. For males, food consumption recording recommenced one week after pairing (where possible).
For females after mating, food consumption schedule was match body weight schedule:
Days 0-1, 2-3, 4-5, 6-7, 8-9, 10-11, 12-13, 14-15, 16-17, 18-20 after mating Days 1-4, 4-7, 7-14 and 14-21 of lactation.

Oestrous cyclicity (parental animals):

Estrous cycles
Dry smears (F0 females only): For 15 days before pairing, using cotton swabs.
Wet smears (F0 and F1 Cohort 1B females): After pairing until evidence of mating confirmed.

Females showing no evidence of mating - following completion of the pairing period females were separated from the male and vaginal smearing continued for up to five days or until the first estrus smear is seen. If a female showed an estrus smear during this period, she was killed as soon as practically possible and subject to macroscopic examination. For four days before scheduled termination (nominally Days 25 to 28 post partum).

Sperm parameters (parental animals):

Sperm Analysis - F0 males and Cohort 1A males
Vas deferens (from left side) Sperm sample (at least 200) was assessed for motility using a computer assisted sperm analyzer (CASA). Each animal in each group.
A manual assessment of sperm morphology was performed. Each animal in Groups 1 and 4.
Cauda epididymis (from left side) The cauda epididymis was weighed and homogenized and the number of sperm was counted using a computer assisted sperm analyzer (CASA). Each animal in Groups 1 and 4.
Testis (from left side) The testis was homogenized and the number of homogenization-resistant spermatids was counted using a computer assisted sperm analyzer (CASA). Each animal in Groups 1 and 4
Sperm analysis was not routinely performed for animals killed or dying prematurely

Litter observations:

Records Made During Littering Phase (F0 and F1B Generations)
Clinical observations Observed approximately 24 hours after birth (Day 1 of age) and then daily for evidence of ill-health or reaction to treatment.
On Day 1 of age, all offspring receive a qualitative assessment of body temperature, state of activity and reaction to handling.
Litter size Daily on Days 1-21 of lactation.
Litters culled to 10 (where possible 5 males and 5 females) on Day 4 of age.
Culled offspring on Day 4 of age were individually identified for subsequent macroscopic examination.
Sex ratio Days 1, 4 (before and after culling) and 21 of age.
Individual offspring body
weights All: Days 1, 4, 7, 14 and 21 of age.
Selected F1 generation (excluding Cohort 2B): Days 23, 25, 27 and 29 of age.
Unselected F1 offspring: Day 22 of age.
Weaning of offspring Day 21 of age.
Ano-genital distance Offspring on Day 1 of age.
Nipple count Male offspring on Day 13 of age.

Sexual Maturation (Selected F1 Generation – Cohorts 1A, 1B, 2A and 3)
Males Examined daily from Day 38 of age for the completion of balano-preputial separation. Body weight recorded on day of completion of separation.
Females Examined daily from Day 28 of age until vaginal opening occurs. Body weight recorded on day of vaginal opening. For females in Cohort 1A, a wet smear will be taken daily from the day of vaginal opening until first estrus.

Postmortem examinations (parental animals):

Time of Necropsy
F0 adult animals Females - Day 28 postpartum
Males - After weaning of the F1 animals, after confirmation that no further mating required.
F0 females failing to mate If an estrus smear is seen following completion of the pairing period animals will be terminated as soon as logistically possible.
If no estrus smear is seen, animals will be terminated on Day 25 after the last day of pairing.
F0 females failing to
produce a viable litter
and those with total
litter loss Terminated with first cohort of females with live litters.

Examinations: Abnormalities, macroscopic pathology,selected organ weights, histology.

Postmortem examinations (offspring):

Time of Necropsy
Unselected offspring Culled on Day 4 and Day 22 of age (F1) or Day 21 of age (F2 litters).
Cohort 1A At approximately 13 weeks of age.
Cohort 1B Males at approximately 21-22 weeks of age; females on Day 28 post partum.
Cohort 2A At approximately 11-12 weeks of age.
Cohort 2B At Day 21/22 of age.
Cohort 3 At Day 60 of age.

Examinations: Abnormalities, macroscopic pathology, selected organ weights, histology.

Immunophenotyping of spleen leucocytes (Cohort 1A)
Ten males and ten females per group for Cohort 1A will be selected for immunophenotyping. Where possible, one male or one female will be assigned from each selected litter.
The whole spleen is to be weighed. After weighing, a 3-5mm mid transverse section will be removed and retained for histopathological evaluation. The remaining portions of the spleen will then be weighed, placed in to a vial of chilled Hank’s Balanced Salt Solution (HBSS) and held in wet ice until processing for analysis.

Statistics:

Data-types
The following data types were analyzed at each timepoint separately, where required, in support of interpretation:-
body weight, using absolute weights and gains over appropriate study periods.
food consumption, over appropriate study periods.
estrous cycles and pre-coital interval.
mating performance and fertility.
litter size and survival indices.
pre-weaning examination (if performed)
sexual maturation, age and body weight at completion.
clinical pathology
behavioral data (arena rearing and activity counts, grip strength, landing footsplay, body weight, body temperature and motor activity)
organ weights, either absolute or adjusted for terminal body weight where appropriate. sperm analysis, motility, morphology and count.
corpora lutea and ovarian primordial follicle counts.
thyroid hormones
spleen cell immunophenotyping

Methods
For categorical data, the proportion of animals were analyzed for each treated group (as appropriate) versus the control group.
For continuous data, Bartlett’s test was first applied to test the homogeneity of variance between the groups. Using tests dependent on the outcome of Bartlett’s test, treated groups will then be compared with the control group, incorporating adjustment for multiple comparisons where necessary.

Reproductive indices:

Mating performance and fertility
Individual data tabulated. Group values calculated for males and females separately for the following:
Percentage mating [Number animals mating x 100] : [Animals paired]
Conception rate [Number animals achieving pregnancy x 100] / [Animals mated]
Fertility index [Number animals achieving pregnancy x 100] / [Animals paired]
Gestation length: Individual values tabulated for the number of days from mating to the start of parturition (inclusive), with half a day subtracted where parturition started overnight. Percentage of animals in appropriate categories tabulated for each group.
Gestation index Calculated for each group as: [Number of live litters born x 100] / [Number pregnant]

Offspring viability indices:

Survival indices (%)
Individual litter values calculated for:

Post-implantation survival index: [Total number offspring born x 100 Total] / [number uterine implantation sites]
Live birth index: [Number live offspring on Day 1 after littering x 100] / [Total number of offspring born]
Viability index: [Number live off spring on Day 4 before culling x 100] / [Number live off spring on Day 1 after littering]
Lactation index: [Number live off spring on Day 21 after littering x 100] / [Number live off spring on Day 4 (after culling)]
Sex ratio: Individual litter values tabulated for total at Day 1 and live at Days 1, 4 (before and after culling) and Day 21 of age.
Pre-weaning examination
(if performed): Surface and righting reflexes presented as the age that the reflexes are observed. Auditory and visual function presented as percentage passing or failing the test. Individual values tabulated.
Neurobehavioural examinations: Individual values tabulated. Group values calculated from individual values.
Sexual maturation: Individual values tabulated for age and body weight at completion.
Organ weights: Absolute and body weight relative.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Salivation (males given 40 mg/kg/day): Salivation is commonly observed where animals are dosed by oral gavage and the reaction is generally regarded as reflecting distaste of the dosing formulations rather than a sign of toxicity.

There were no test item-related changes in general clinical condition among males throughout the study, or among females during the 2-week pre-pairing treatment period and up to Day 20 of gestation at any dose level investigated.

Prolonged parturition/dystocia: On Day 22/23 of gestation, at the time of parturition, three females in the 40 mg/kg/day group were observed to have prolonged parturition/dystocia, with two of these females showing deteriorating clinical condition, and therefore these three females were sacrificed for reasons of animal welfare. These premature deaths were considered to be related to Vulkanox ZMB2 administration.

Respiratory distress: One Control F0 male, one Control F0 female, two F0 males given 40 mg/kg/day and one F0 female given 40 mg/kg/day were sacrificed for welfare reasons due to signs of respiratory distress; a further Control F0 male was found dead. Macroscopic and microscopic examination of these animals indicated findings commonly associated with dosing injury, such as perforated oesophagus and/or abnormal contents in the thoracic cavity, and therefore these premature deaths were incidental and unrelated to treatment.

Uterine prolapse: A further premature death in the 40 mg/kg/day group occurred on Day 1 of lactation, where one F0 female was killed for reasons of animal welfare due to uterine prolapse. This premature death had no relationship to Vulkanox ZMB2 administration.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

On Day 22/23 of gestation, at the time of parturition, three females in the 40 mg/kg/day group were observed to have prolonged parturition/dystocia, with two of these females showing deteriorating clinical condition, and therefore these three females were sacrificed for reasons of animal welfare. These premature deaths were considered to be related to Vulkanox ZMB2 administration.

Nine further premature deaths occurred in the F0 generation, however these deaths were not attributable to Vulkanox ZMB2 administration.

One Control F0 male, one Control F0 female, two F0 males given 40 mg/kg/day and one F0 female given 40 mg/kg/day were sacrificed for welfare reasons due to signs of respiratory distress; a further Control F0 male was found dead. Macroscopic and microscopic examination of these animals indicated findings commonly associated with dosing injury, such as perforated oesophagus and/or abnormal contents in the thoracic cavity, and therefore these premature deaths were incidental and unrelated to treatment.

Two F0 males (receiving 15 mg/kg/day and 40 mg/kg/day) were sacrificed for reasons of animal welfare due to physical injuries induced by their cage-mates (cannibalisation of gonads/secondary reproductive organs). These premature deaths were unrelated to treatment.

A further premature death in the 40 mg/kg/day group occurred on Day 1 of lactation, where one F0 female was killed for reasons of animal welfare due to uterine prolapse. This premature death had no relationship to Vulkanox ZMB2 administration.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Group mean body weight gain for F0 males and females receiving Vulkanox ZMB2 was essentially similar to Controls throughout all of the study phases and considered unaffected by treatment at all dose levels investigated. Mean body weight gain in a small number of recording periods attained statistical significance when compared to Controls, however the differences were minor and showed no dose response relationship, and were therefore attributed to normal biological variation.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was no effect of Vulkanox ZMB2 administration on the mean food consumption of F0 males or females throughout the study. Differences in occasional individual food consumption recording periods attained statistical significance when compared to Controls, however the differences were minor and showed no dose response relationship, and were therefore attributed to normal biological variation.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Hematology
Analysis of hematological parameters at scheduled termination of the F0 animals did not reveal any changes which were clearly attributable to Vulkanox ZMB2 administration.

All differences from control were minor, limited to one sex, lacked a dose response relationship and were within the 5-95% confidence limits of the Historical Control Data (HCD) range (4 studies) and were therefore attributed to normal biological variation; these included the statistically significant, but non-dose dependent slight increases in mean cell haemoglobin concentration in all groups of treated females (HCD range, 32.6-37.4 g/dL), and the slight decrease total white blood cell counts in males given 40 mg/kg/day (HCD range, 5.10x10^9/L -13.13x10^9/L), due to slightly low mean lymphocyte (HCD range, 3.67x10^9/L - 10.28x10^9/L) and basophil concentrations (HCD range, 0.02x10^9/L - 0.13x10^9/L), where all individual values were within the concurrent Control range.

Clinical biochemistry findings:

not specified

Description (incidence and severity):

Blood Chemistry
Biochemical analysis of the plasma at scheduled termination of the F0 animals revealed when compared to Controls, statistically significantly slightly increased mean creatinine concentrations in males receiving 40 mg/kg/day, although values were within the HCD range (22-35 µmol/L). Increased cholesterol concentrations were apparent in all groups of treated females, although in the absence of a dose response relationship and values were within the HCD range (1.33-2.30 mmol/L). Phosphorous concentrations were slightly increased in a non-dose dependent manner in males given 15 or 40 mg/kg/day and slightly decreased in females given 40 mg/kg/day, although values were within the HCD range in both sexes (1.57-2.40 mmol/L for males; 1.02-1.96 mmol/L for females).

Mean plasma total protein concentrations were slightly high and achieved statistical significance in males and females receiving 40 mg/kg/day, with an associated slight increase in mean albumin concentrations when compared to Controls; all values were within the HCD range (total protein: 60-70 g/L for males, 62-73 g/L for females; albumin: 34-38 g/L for males, 35-41 g/L for females).

All other biochemical differences from Controls observed at scheduled termination were minor, limited to one sex, lacked a clear dose relationship and were within the HCD range, and were therefore attributed to normal biological variation; this included the slight increase in gamma glutamyl transferase activity in females at 40 mg/kg/day (HCD range, 0-2 U/L).

Thyroid Hormone Analysis - T4 and TSH
Mean T4 concentrations in F0 animals given Vulkanox ZMB2 were slightly higher than those observed in Controls, with a dose response apparent in F0 males but not in F0 females. Concentrations in the Vulkanox ZMB2 groups were, however, comparable with the endogenous level (44500 pg/mL) observed in the control matrix used to prepare the QC samples. Serum T4 concentrations in Day 22 of age F1 offspring and in Cohort 1A F1 adult males and females (approximately 13 weeks of age) were similar across all groups, including Controls, and were comparable with the endogenous levels observed in the control matrix used to prepare the QC samples.

Mean serum TSH concentrations were statistically significantly higher than Control in all groups of F0 females although there was no dose response apparent; a similar difference was not apparent in F0 males. There was no effect of Vulkanox ZMB2 administration on mean serum TSH concentration in F1 offspring on Day 22 of age. In Cohort 1A, when compared to Controls, a dose dependent increase in mean serum TSH concentrations was apparent in all treated groups of males and females, with differences for males in the 15 or 40 mg/kg/day groups attaining statistical significance

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Changes related to treatment with Vulkanox ZMB2 were seen in the liver, thyroid glands and thymus of F0 males and females.

Liver
Minimal to slight hypertrophy, centrilobular or general, was present in a number of males and females given 40 mg/kg/day. Minimal hypertrophy, centrilobular was present at an increased incidence in males and females given 5 or 15 mg/kg/day.

Thyroid Glands
Minimal follicular cell hypertrophy was present in more than half of the males and females given 40 mg/kg/day and in some males given 15 mg/kg/day.

Thymus
Slight involution/atrophy was present in more than half of the males and some of the females given 40 mg/kg/day. Minimal involution/atrophy was also present in some males given 15 mg/kg/day. The occurrence in a single male given 5mg/kg/day was considered incidental.

Findings of an Uncertain Relationship to Treatment
Findings of an uncertain relationship to treatment were present in the spleen of males.

Spleen
Increased hemosiderin was present in the majority of males given 40 mg/kg/day. This was not a feature in females, thus any significance was unclear.

Spleen Cell Immunophenotyping
The administration of Vulkanox ZMB2 at doses up to and including 40 mg/kg/day was considered to have no observable effects on the immunophenotyping parameters measured in spleen leukocytes of the Cohort 1A F1 animals.

There was variation in inflammatory changes in the mammary glands of females between control and animals given 40 mg/kg/day. Review of the individual F0 female/F1 litter information did not reveal any correlation between this finding and the occurrence of premature offspring deaths where no milk was evident in the stomach, therefore this histopathological finding was considered incidental.

Histopathological findings: neoplastic:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Estrous cycle regularity of the F0 females during the 2-week pre-pairing treatment period and estrous cycles at termination were unaffected by treatment.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

No adverse effects on sperm motility, concentration, motion or morphology of F0 males were apparent following treatment with Vulkanox ZMB2 at doses up to and including 40 mg/kg/day.
Staging of Spermatogenesis
In the testes, seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No differences were observed between control and treated animals.

Reproductive performance:

no effects observed

Description (incidence and severity):

Pre-coital interval was unaffected by Vulkanox ZMB2 administration. All mating pairs showed positive evidence of mating within six days of pairing, and with the exception of one female in the 40 mg/kg/day group, all females mated at the first estrus opportunity. One female in the 15 mg/kg/day group was acyclic during the pre-pairing period following pairing and had a pre-coital interval of 6 days, indicative that positive evidence of mating was detected at the first estrus opportunity once normal cycling activity was restored.

Mating performance and fertility were unaffected by Vulkanox ZMB2. The percentage mating was 100% in all groups, and conception rate and fertility index were 92-100 % in all groups; one Control F0 female and one F0 female given 40 mg/kg/day (No. 287) were not pregnant.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

ca. 15 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

reproductive performance

2

Dose descriptor:

NOAEL

Effect level:

ca. 15 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: systemic toxicity

Target system / organ toxicity (P0)

Critical effects observed:

yes

Lowest effective dose / conc.:

15 mg/kg bw/day (nominal)

Organ:

liver

thymus

thyroid gland

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Results: P1 (second parental generation)

Effect levels (P1)

Remarks on result:

other: not applicable

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Signs associated to dose administration were limited to a single F1 Cohort 1B female in the 40 mg/kg/day group with salivation and piloerection on Day 1 of lactation. Salivation is commonly observed where animals are dosed by oral gavage and the reaction is generally regarded as reflecting distaste of the dosing formulations rather than a sign of toxicity.

Parturition/dystocia: On Day 22-24 of gestation, at the time of parturition, four F1 Cohort 1B females in the 40 mg/kg/day group were observed to have prolonged parturition/dystocia, with three of these females showing deteriorating clinical condition, and therefore these three females were killed for reasons of animal welfare. These premature deaths were considered to be related to Vulkanox ZMB2 administration.

During lactation, there were no changes in general clinical condition observed which were related to Vulkanox ZMB2.

Respiratory distress: One Control F1 Cohort 1A female and one F1 Cohort 1B female given 40 mg/kg/day were killed for welfare reasons due to signs of respiratory distress, and one F1 Cohort 1B female in the 5 mg/kg/day group was killed for welfare reasons due to a swollen area on the upper ventral surface; in addition, one F1 Cohort 1B male given 5 mg/kg/day and one F1 Cohort 1B female given 40 mg/kg/day were found dead. Macroscopic and microscopic examination of all of these animals indicated findings commonly associated with dosing injury, such as perforated oesophagus and/or abnormal contents in the thoracic cavity, and therefore these premature deaths were incidental and unrelated to treatment.

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

Two females given 15 mg/kg/day had a total litter loss, one on Day 1 of lactation and one prior to formal assignment to Day 1 of lactation.

Parturition/dystocia: On Day 22-24 of gestation, at the time of parturition, four F1 Cohort 1B females in the 40 mg/kg/day group were observed to have prolonged parturition/dystocia, with three of these females showing deteriorating clinical condition, and therefore these three females were killed for reasons of animal welfare. These premature deaths were considered to be related to Vulkanox ZMB2 administration.

Respiratory distress: One Control F1 Cohort 1A female and one F1 Cohort 1B female given 40 mg/kg/day were killed for welfare reasons due to signs of respiratory distress, and one F1 Cohort 1B female in the 5 mg/kg/day group was killed for welfare reasons due to a swollen area on the upper ventral surface; in addition, one F1 Cohort 1B male given 5 mg/kg/day and one F1 Cohort 1B female given 40 mg/kg/day were found dead. Macroscopic and microscopic examination of all of these animals indicated findings commonly associated with dosing injury, such as perforated oesophagus and/or abnormal contents in the thoracic cavity, and therefore these premature deaths were incidental and unrelated to treatment.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The group mean body weight of male and female F1 offspring on Day 1 of age, and subsequent body weight gain of the offspring to weaning on Day 21 of age, was unaffected by parental treatment with Vulkanox ZMB2 at all dose levels investigated.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was no effect of Vulkanox ZMB2 administration on the mean food consumption of F1 males or females throughout the study. Differences in occasional individual food consumption recording periods attained statistical significance when compared to Controls, however the differences were minor and showed no dose response relationship, and were therefore attributed to normal biological variation.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Hematology - Cohort 1A
Analysis of hematological parameters at scheduled termination of the F1 Cohort 1A animals revealed, when compared to Controls, a non-dose-dependent slight but statistically significant decrease in hematocrit and red blood cell concentrations in males given 40 mg/kg/day and in all groups of treated females, with an associated increase in mean cell hemoglobin concentration in both sexes given 40 mg/kg/day. All values were within the 5-95% confidence limits of the HCD range (4 studies) for both sexes (hematocrit: 0.394-0.492 L/L for males and 0.385-0.444 L/L for females; red blood cells: 7.26x10^12/L to 8.83x10^12/L for males and 7.00x10^12/L to 7.94x10^12/L for females; mean cell hemoglobin concentration: 31.9-37.7 g/dL for males and 33.4-38.7 g/dL for females). Males at 40 mg/kg/day also showed an associated statistically significant increase in red cell distribution width, although a similar difference was not evident in the treated females (add RDW HCD).
At 40 mg/kg/day, males showed slightly low eosinophil concentrations when compared to Controls; statistical significance was attained for this differences however values were within the HCD range of 0.05x10^9/L to 0.17x10^9/L.
Activated partial thromboplastin times were statistically significantly shorter than Controls in males given 40 mg/kg/day; values were within the HCD range of 13.7-21.0 secs.
All other differences from control were minor, limited to one sex or lacked a dose response relationship and were therefore attributed to normal biological variation.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Blood Chemistry - Cohort 1A
Biochemical analysis of the plasma at scheduled termination of the F1 Cohort 1A animals revealed when compared to Controls, statistically significantly slightly increased mean gamma glutamyl transferase activity and creatinine concentrations in males and females receiving 40 mg/kg/day; gamma glutamyl transferase activity values for males at 40 mg/kg/day and creatinine concentrations for females at 40 mg/kg/day were marginally above the HCD range (gamma glutamyl transferase activity: 0-1 U/L for males and females; creatinine: 20-30 mmol/L for males and 21-33 mmol/L for females). Increased cholesterol and decreased phosphorus concentrations were apparent in males given 40 mg/kg/day; the cholesterol concentrations were marginally above the HCD range (cholesterol: 0.94-1.88 mmol/L; phosphorus: 1.70-2.59 mmol/L).
Mean plasma total protein concentrations were slightly high and achieved statistical significance in all groups of treated males although in the absence of a dose response relationship and within the HCD range (total protein: 59-69 g/L). This was associated with a slight decrease in albumin/globulin ratio in males given 40 mg/kg/day although this was also within the HCD range (albumin/globulin ratio: 1.13-1.58). Conversely, females given 15 or 40 mg/kg/day showed slightly increased mean albumin concentrations when compared to Controls, but within the HCD range of 36-42 g/L.
All other biochemical differences from Controls observed at scheduled termination were minor, limited to one sex or lacked a clear dose relationship and were therefore attributed to normal biological variation.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Analysis of the clarity/colour and composition of the urine of F1 Cohort 1A animals prior to scheduled termination revealed, when compared to Controls, statistically significantly low urinary sodium concentrations in males and females in the 40 mg/kg/day group. Males in this dose group also showed slightly low urinary protein concentrations and slightly high total potassium. Microscopy of the urine sediment did not reveal any test item-related changes.

Sexual maturation:

effects observed, non-treatment-related

Description (incidence and severity):

The mean age at which F1 females in the 40 mg/kg/day group attained vaginal opening was 1.2 days earlier than observed among Controls, and these females had a lower mean body weight than Controls at attainment. These differences from Control attained statistical significance, however since the difference in age was only marginally above the degree of accuracy of the assessment (animals were assessed once per day) and since the mean values were within the Historical Control Data range (mean age 34.5 days with 5-95% confidence limits 31-38 days; mean body weight 124 grams with 5-95% confidence limits 99-151 grams), these differences were considered fortuitous and unrelated to Vulkanox ZMB2.

The age and body weight at which F1 females in the 5 or 15 mg/kg/day groups, and at which F1 males in all treated groups attained balano preputial separation was unaffected by Vulkanox ZMB2 administration.

Vaginal Opening to First Estrus - Cohort 1A
There was no effect of Vulkanox ZMB2 administration on the duration between vaginal opening and the first estrus occurring in the Cohort 1A females.

Estrous Cycles - Cohort 1A
Estrous cycle regularity of the Cohort 1A females during the last two weeks of treatment and the stage of the estrous cycle at termination for these females were unaffected by Vulkanox ZMB2 at all dose levels investigated.

Pre-Coital Interval, Mating Performance and Fertility, Gestation Length and Gestation Index - Cohort 1B
Pre-coital interval of the Cohort 1B animals was unaffected by Vulkanox ZMB2 administration. All mating pairs in the 15 or 40 mg/kg/day groups and all except one mating pair in the 5 mg/kg/day group showed positive evidence of mating within five days of pairing, i.e. at the first estrus opportunity.

Mating performance and fertility of the Cohort 1B animals were unaffected by Vulkanox ZMB2. The percentage mating was 100% in all groups, and conception rate and fertility index were 89-95 % in all groups; one Control female (No. 782), one female given 5 mg/kg/day (No. 819), two females given 15 mg/kg/day (No’s. 832 and 839) and two females given 40 mg/kg/day (No’s. 841 and 851) were not pregnant.

Stage of Estrous Cycle at Termination - Cohort 1B
The stage of the estrous cycle at termination of the Cohort 1B females was unaffected by Vulkanox ZMB2 at all dose levels investigated.

Sperm Assessment - Cohort 1A
No adverse effects on sperm motility, concentration or motion were apparent among the Cohort 1A males following treatment with Vulkanox ZMB2 at doses up to and including 40 mg/kg/day. A statistically significant slight increase in the mean number of sperm in the testis was apparent in Cohort 1A males at 40 mg/kg/day when compared to Controls. Review of the individual data showed 18/20 of these treated males had testis sperm counts within the concurrent Control range, and the difference was largely attributable to one male (No. 465) with atypically high total sperm count. In light of this, and the fact that values in the
40 mg/kg/day were higher than Control, with difference was considered incidental and unrelated to Vulkanox ZMB2 administration

Ovarian Follicle Counts and Corpora Lutea - Cohort 1A
At scheduled termination of the Cohort 1A females, a statistically significant increase in ovarian follicle counts was evident in females given 40 mg/kg/day when compared to Controls. There was no similar increase in the number of corpora lutea present.

Anogenital distance (AGD):

effects observed, treatment-related

Description (incidence and severity):

The ano-genital distance of F1 male offspring in the 15 mg/kg/day group and of both sexes of F1 offspring in the 40 mg/kg/day group on Day 1 of age was slightly, but statistically significantly, increased when compared to Controls.

Nipple retention in male pups:

effects observed, treatment-related

Description (incidence and severity):

All F1 male offspring were assessed on Day 13 of age for the presence of nipples; no nipples were observed.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Organ Weights - all F1 Cohorts
Cohort 1A
The Cohort 1A animals, when compared to Controls, has statistically significantly decreased absolute and body weight relative thymus weights were apparent in males and females given 15 or 40 mg/kg/day, with body weight relative values for males given 5 mg/kg/day also being low. Absolute lungs and bronchi weights were increased in females given 5 mg/kg/day and in both sexes given 15 or 40 mg/kg/day, with body weight relative lungs and bronchi weights also increased in males given 40 mg/kg/day. Females given 40 mg/kg/day showed increased absolute and body weight relative kidney weights, along with increased absolute liver weights.

Cohort 1A females in all treated groups showed statistically significantly reduced absolute and body weight relative uterus/cervix/oviducts weights when compared to Controls. These differences are a reflection of the stage of estrus that the individual females were in at the time of scheduled termination, and no effect of treatment was inferred.

Other inter-group differences in body weight relative organ weights which attained statistical significance were attributable to the slightly higher mean terminal body weights in treated groups when compared to Controls, and were unrelated to Vulkanox ZMB2 administration. These included decreased body weight relative brain weights, epididymal weights and heart weights in all groups of treated males, and decreased adrenal weights in females given 15 or 40 mg/kg/day.

Cohort 1B
The absolute and body weight relative weights of the primary and secondary sex organs of the Cohort 1B animals were unaffected by Vulkanox ZMB2.

Brain Morphometry
Cohorts 2A and 2B
The brain weight of Cohort 2A animals was unaffected by Vulkanox ZMB2 administration.
Absolute mean brain weights for all groups of Cohort 2A females were statistically significantly lower than Control although in the absence of a dose response relationship. There were, however, no similar differences in body weight relative brain weights indicating that the lower mean absolute weights were attributable to slightly lower mean terminal body weight, and not indicative of a direct effect.
There was considered to be no effect of treatment at 40 mg/kg/day on brain morphometry measurements. When compared to Controls, the mean width of the folia of the cerebellum was slightly but statistically significantly smaller in females given 40 mg/kg/day, however this difference was not apparent in males in this cohort, or in either sex at 11-12 weeks of age (Cohort 2A animals), and therefore this difference was considered to reflect normal biological variation.

Cohort 3
Absolute and body weight relative spleen weights of the Cohort 3 animals at scheduled termination was unaffected by Vulkanox ZMB2 administration at all dose levels investigated.

Gross pathological findings:

no effects observed

Description (incidence and severity):

The macroscopic examination performed at scheduled termination of F1 Cohort 1A, 1B, 2A, 2B and 3 animals revealed no test item-related lesions. The incidence and distribution of all findings were considered to be unrelated to treatment.

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

Cohort 2A
Examination of the nervous system of Cohort 2A males and females at 11/12 weeks of age revealed no test item-related changes.

Cohort 2B
Examination of the brain sections of Cohort 2B males and females at 21/22 days of age revealed no test item-related changes.

Cohort 1A - Treatment Related Findings
Changes related to treatment with Vulkanox ZMB2 were seen in the liver, thyroid glands and thymus of Cohort 1A males and females.

Liver
Minimal hypertrophy, centrilobular, was present in a small number of males given 15 or 40 mg/kg/day and a small number of females given 40 mg/kg/day.

Thyroid Glands
Minimal follicular cell hypertrophy was present in some of the males and females given 40 mg/kg/day and in some males given 15 mg/kg/day.

Thymus
Minimal to slight involution/atrophy was present in some of the males and females given
40 mg/kg/day.

Findings of an Uncertain Relationship to Treatment
Findings of an uncertain relationship to treatment were present in the female reproductive tract. There was a variation in stage of the estrus cycle, with an increased number of females given 40 mg/kg/day showing diestrus morphology.

Incidental Findings
No histopathological changes were noted which accounted for the reduction in adrenal weight noted in females.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

A slight, but statistically significant, shift in gestation length was apparent for females given 40 mg/kg/day compared to Controls. The expected gestation length in CD rats ranges from 22 to 23 days. In the 40 mg/kg/day group, seven females showed a 23.5-day gestation length and one female showed a 24-day gestation length. Gestation index was also statistically significantly low in the 40 mg/kg/day group, reflecting the females killed due to dystocia.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

In Cage Observations
There were no test item-related changes evident in the behaviour of the Cohort 2A animals during the in-cage observations.

In Hand Observations
During the in-hand observations there were no findings observed which represented neurological changes related to administration of Vulkanox ZMB2.

Arena Observations
The 2-minute arena observation of the Cohort 2A animals revealed, when compared to Controls, a statistically significant decrease in activity and rearing counts in males and females in the 40 mg/kg/day group; rearing counts were also low in males given 5 or 15 mg/kg/day although in the absence of a dose response relationship.

Reactivity Investigations
Reactivity investigations did not reveal any neurological changes in the Cohort 2A animals at any dose level investigated.

Auditory Startle Response Habituation - Cohort 2A
Auditory startle response habituation for F1 Cohort 2A animals at Day 24±1 of age, both in terms of latency to peak amplitude values and peak amplitude values, was unaffected by treatment with Vulkanox ZMB2 at all dose levels investigated.

The group mean percent habituation values in both sexes at all dose levels was considered to be unaffected by Vulkanox ZMB2 administration. It was noted that the percent habituation values for Cohort 2A males in the 15 or 40 mg/kg/day were statistically significantly lower than Control, although in the absence of a dose response relationship; a similar trend was not apparent in females. There was no effect on the latency to the peak response or on the amplitude of the peak response during the initial 10 trials, none of the values for the individual blocks of 10 trials attained statistical significance, and the percent habituation value for males in the 40 mg/kg/day group was not statistically significantly different to Control on the individual t-test. It was therefore considered that the statistical analysis for a monotonic trend (Williams’ test) only indicated statistical significance in the 40 mg/kg/day group due to the low mean value in the 15 mg/kg/day group.

Motor Activity - Cohort 2A
The locomotor activity of Cohort 2A F1 animals on nominal Day 65±1 of age was unaffected by Vulkanox ZMB2 administration.

Occasional differences from the Control group attained statistical significance in treated females, with a reduced number of low beam breaks during the first six minutes of the test for females given 40 mg/kg/day, and a non dose-dependent increase in the number of low and high beam breaks recorded during the last six minutes of the test in all groups of treated females. The overall total number of high and low beam breaks during the 1-hour test was unaffected, and therefore these differences were considered incidental and unrelated to Vulkanox ZMB2.

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not specified

Effect levels (F1)

1

Dose descriptor:

NOAEL

Generation:

F1 (cohort 1B)

Effect level:

ca. 15 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

clinical signs

other: reproductive performance

2

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

ca. 15 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: reduced early post-partum survival and effects on liver, thyroid gland and thymus

3

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

ca. 40 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

developmental neurotoxicity

developmental immunotoxicity

4

Dose descriptor:

NOAEL

Generation:

F1 (cohort 2A)

Effect level:

40 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

developmental neurotoxicity

Target system / organ toxicity (F1)

Critical effects observed:

yes

Lowest effective dose / conc.:

15 mg/kg bw/day (nominal)

Organ:

liver

thymus

thyroid gland

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Results: F2 generation

General toxicity (F2)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs observed among the F2 offspring that were considered to be related to parental treatment with Vulkanox ZMB2.

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

There was no effect of Vulkanox ZMB2 on the mean number of implantation sites, litter size or post-implantation survival in the Cohort 1B females. Live birth index, however, was statistically significantly lower than Control in the 40 mg/kg/day group, with offspring deaths occurring between birth and formal assignment to Day 1 of lactation in 8/13 litters compared with 2/19 litters in the Control group. There was no effect on post-implantation survival or litter size at 5 or 15 mg/kg/day, and offspring survival from Day 1 of lactation was unaffected by Vulkanox ZMB2 in all groups.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The group mean body weight of male and female F2 offspring on Day 1 of age, and subsequent body weight gain of the offspring to weaning on Day 21 of age, was unaffected by parental treatment with Vulkanox ZMB2 at all dose levels investigated.

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Sexual maturation:

not specified

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

There was no effect of treatment on the ano-genital distance of F2 offspring at any dose level investigated.

Nipple retention in male pups:

effects observed, treatment-related

Description (incidence and severity):

All F2 male offspring were assessed on Day 13 of age for the presence of nipples; no nipples were observed.

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

no effects observed

Description (incidence and severity):

The predominant macroscopic observation detected in the F2 offspring that died prior to scheduled termination was the absence of milk in the stomach.
There were no macroscopic abnormalities detected among the F2 offspring killed at scheduled termination on Day 21 of age.

Histopathological findings:

not specified

Other effects:

not specified

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not specified

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not specified

Effect levels (F2)

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

ca. 15 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: reduced early post-partum survival

Overall reproductive toxicity

1

Reproductive effects observed:

not specified

2

Reproductive effects observed:

yes

Lowest effective dose / conc.:

40 mg/kg bw/day

Treatment related:

yes

Relation to other toxic effects:

reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects

Dose response relationship:

not specified

Applicant's summary and conclusion

Conclusions:

Based on the results obtained in this study it was concluded that the 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.

Executive summary:

An EOGRTS study (OECD 443) was performed to evaluate the pre- and post-natal effects of Vulkanox ZMB2, an industrial chemical, when administered orally, by gavage, to CD rats. 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.

In the F0 generation, three groups of 25 male and 25 female CD rats received Vulkanox ZMB2 at dose levels of 5, 15 or 40 mg/kg/day at a volume dose of 5 mL/kg/day. Males were treated for two weeks before pairing, up to necropsy after litters were weaned. Females were treated for two weeks before pairing, throughout pairing up to necropsy on Day 28 of lactation. In the F1 generation, 70 males and 70 females were treated from weaning to their scheduled termination (relevant to each cohort) at the same dose levels and volume-dose as the F0 generation. A similarly constituted Control group received the vehicle, dried corn oil, at the same volume dose throughout the same period.

Clinical observations, body weight, food consumption (except Cohort 2B) and macropathology examinations were performed on all animals for signs of toxicity, with special emphasis on the integrity and performance of the male and female reproductive system and the health (including thyroid hormone analysis), growth, development and function of the offspring. 

At weaning, five cohorts of selected males and females were assigned for further investigations, including sexual maturation, reproductive organ integrity and function, neurological and behavioural endpoints, and immune functions.

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.

As well as 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.