Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 September 2019 to XXXX
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to other study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 June 2019 to XXXX
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
other: Range-Finding Toxicity Study in the Rat, to inform dose selection in an OECD 408 and 414 study.
Version / remarks:
To bridge from a rat dietary OECD 422 study
Deviations:
no
Principles of method if other than guideline:
This non-GLP study was conducted in accordance with Good Laboratory Practice principles.
GLP compliance:
no
Limit test:
no
Specific details on test material used for the study:
Test item: 4,4’-methylene bis(dibutyldithiocarbamate)
CAS number: 10254-57-6
Appearance: Amber-green liquid.
Storage conditions: Controlled ambient temperature (15 to 25°C), in the dark
Species:
rat
Strain:
other: Han-Wistar [RccHan™:WIST ]
Details on species / strain selection:
The rat was chosen as the test species because it is accepted as a predictor of toxicity in humans and the requirement for a rodent species by regulatory agencies. The Han Wistar [RccHan™:WIST ] strain was used because of the historical control data available at this laboratory.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS Limited
- Females (if applicable) nulliparous and non-pregnant: No data
- Age at study initiation: 56 to 63 days
- Weight at study initiation:
Males: 184 to 219 g
Females: 133 to 168 g
- Fasting period before study: No data
- Housing: Polycarbonatebody cages, with a stainless steel mesh lid
- Diet (e.g. ad libitum): Non-restricted
- Water (e.g. ad libitum): Non-restricted
- Acclimation period: 14 days before commencement of treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 40-70
- Air supply: Filtered fresh air which was passed to atmosphere and not recirculated
- Photoperiod (hrs dark / hrs light): Artificial lighting, 12 hours light: 12 hours dark

There was limited access to the animal facility to minimise entry of external biological and chemical agents and to minimise the transfer of such agents between rooms.
Route of administration:
oral: gavage
Details on route of administration:
Oral, by gavage, using a suitably graduated syringe and a rubber catheter inserted via the mouth.
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

The required amount of test item for 200 mg/mL was weighed out into a suitable container. Approximately 50% of the final volume of vehicle was added to the test item and mixed with a magnetic stirrer until uniformly mixed. The solution was then made up to the required volume with vehicle. The formulation was transferred to a final container and mixed with a magnetic stirrer until homogenous.

A series of formulations at the required concentrations (66 and 20 mg/mL) were prepared by dilution of individual weighings of the test item.


Frequency of preparation: Weekly

Storage of formulation: Refrigerated (2 to 8°C) for up to eight days
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
14 days
Frequency of treatment:
Once daily at approximately the same time each day.
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Vehicle
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
330 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
3 Males; 3 Females
Control animals:
yes, concurrent vehicle
Details on study design:
Doses were selected following consultation between the Study Director and the Study Monitor for the Sponsor, using a dietary combined repeat dose toxicity study with reproduction/ developmental screening test in the rat (SafePharm Laboratories Study, 2006).
Observations and examinations performed and frequency:
Detailed observations were recorded daily at the following times in relation to dose administration:
- Pre-dose
- As each animal was returned to its home cage
- At the end of the dosing all groups
- One to two hours after completion of dosing
- As late as possible in the working day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Three days before treatment commenced, on the day that treatment commenced (Day-1), twice weekly during the treatment period, and before necropsy.

FOOD CONSUMPTION: Yes
- The weight of food supplied to each cage, that remaining and estimate of any spilled was recorded for the three days before treatment and twice weekly during the treatment period.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Daily by visual observation.
Sacrifice and pathology:
TERMINATION
- Carbon dioxide asphyxiation with subsequent exsanguination.

NECROPSY
All 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.

The retained tissues were checked before disposal of the carcass.
Statistics:
No statistical analysis of the data was performed on this study.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Isolated clinical signs of chin rubbing were observed on one occasion in one male from all treated groups and one female receiving 330 mg/kg/day. Excessive salivation was observed in one male receiving 1000 mg/kg/day.

No other clinical signs were observed.
Mortality:
no mortality observed
Description (incidence):
All animals survived to the scheduled sacrifices.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There were no differences in body weight or body weight change that were considered to be treatment related.

Group mean body weight change was lower at the end of treatment in females receiving 1000 mg/kg/day when compared with controls. However, this was due to one animal, who only gained 13 g over the treatment period, whereas the remaining animals in the group gained a similar weight to controls. Group mean body weight change was also lower at the end of treatment in males receiving 100 or 330 mg/kg/day when compared with controls. However, due to the lack of a dose relationship, the large variation in all treated groups (not seen in controls), some of which were similar or higher than controls, these were considered not to be related to treatment.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was unaffected by treatment.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
The daily visual assessment of water intake did not reveal any treatment related changes in water consumption.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no differences in any organ weight that could be positively attributed to treatment. The absolute and adjusted (relative to body weight) organ weights were within the historical control data range of this laboratory.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no macroscopic findings observed in the necropsy at termination of the study.
Details on results:
Clinical signs of chin rubbing and salivation observed in some animals were considered to be caused by the administration method and palatability of the test item, whereby the animals were able to taste the formulation.

Differences in mean body weight change at the end of treatment were considered to be incidental due to the small group sizes and short duration of the study.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
water consumption and compound intake
organ weights and organ / body weight ratios
gross pathology
Critical effects observed:
not specified
Conclusions:
Based on the results of this study, the test material was well tolerated in Han Wistar rats at oral doses up to 1000 mg/kg/day.
Executive summary:

Daily administration of the test item by oral gavage to Han Wistar rats for 14 days was well tolerated at dose levels up to 1000 mg/kg/day with no deaths and no effects seen on clinical observations, body weight, food consumption, water consumption, organ weights or macropathology.

Clinical signs of chin rubbing and salivation observed in some animals were considered to be caused by the administration method and palatability of the test item, whereby the animals were able to taste the formulation.

Differences in mean body weight change at the end of treatment were considered to be incidental due to the small group sizes and short duration of the study.

Therefore, it was concluded that 1000 mg/kg/day would be a suitable high dose level in the subsequent longer term toxicity studies in rats.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agricultural Chemicals, 12 Nousan No. 8147, Agricultural Production Bureau, (November 24, 2000)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
Test item: 4,4’-methylene bis(dibutyldithiocarbamate)
CAS number: 10254-57-6
Appearance: Amber-green liquid.
Storage conditions: Controlled ambient temperature (15 to 25 deg.C), in the dark

Test animals

Species:
rat
Strain:
other: RccHan™;WIST
Details on test animals and environmental conditions:
Animals:

Supplier: Envigo RMS Ltd (UK).
Duration of acclimatization: Six days before commencement of pairing.
Age of the animals at the start of the study (Day 0 of gestation): 78 to 84 days old.
Weight range of the animals at the start of the study (Day 0 of gestation): 183 to 217 g.


Environmental conditions:

There was limited access to the animal facility to minimise entry of external biological and chemical agents and to minimise the transfer of such agents between rooms.

Air supply: Filtered fresh air which was passed to atmosphere and not recirculated.

Diet and water suply: Not restricted.

Temperature and relative humidity was monitored and maintained within the range of 20-24C and 40-70%.

Although conditions were occasionally outside the indicated ranges, these deviations were minor and of short duration and were not considered to have influenced the health of the animals or the outcome of the study.

Lighting: Artificial lighting, 12 hours light: 12 hours dark.

Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals.

Solid (polycarbonate) bottom cages were used during the acclimatization and gestation periods and grid bottomed cages were used during pairing. Cages were suspended above absorbent paper which was changed daily during pairing.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
The study was conducted in two phases to confirm that the dose selection based upon preliminary studies was suitable while reducing the number of animals required. For Phase 1, three groups of six females received the test item at doses of 100, 330 or 1000 mg/kg/day by oral gavage administration with a dose volume of 5 mL/kg, from Day 5 to 19 after mating. A similarly constituted Control group received the vehicle, Corn oil, at the same volume dose as the treated groups. Animals were killed on Day 20 after mating for reproductive assessment and fetal examination. Phase 2 replicated the conditions for Phase 1, but with 14 additional females in the Control, 100, 330 or 1000 mg/kg/day groups, for a total of 20 females per Control and dose group.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of each formulation prepared for administration in Weeks 1 and 3 of treatment were analyzed for achieved concentration of the test material by HPLC using UV detection. The analytical procedure was validated for the test material in corn oil with respect to the specificity of chromatographic analysis, LOD, LOQ, linearity of detector response, system precision, calibration accuracy, and precision.
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 stud males was maintained specifically for the purpose of mating; these animals were not part of the study and were maintained as stock animals.
Duration of treatment / exposure:
14 days
Frequency of treatment:
Day 5 to 19 after mating
Duration of test:
20 days from confirmation of mating
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
330 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
No. of animals per sex per dose:
20 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
The study was conducted in two phases:

For Phase 1, three groups of six females received the test material at doses of 100, 330 or 1000 mg/kg/day by oral gavage administration with a dose volume of 5 mL/kg, from Day 5 to 19 after mating. A similarly constituted Control group received the vehicle, Corn oil, at the same volume dose as the treated groups. Animals were killed on Day 20 after mating for reproductive assessment and fetal examination.

For Phase 2, three groups of 14 females received the test material at doses of 100, 330 or 1000 mg/kg/day by oral gavage administration with a dose volume of 5 mL/kg, from Day 5 to 19 after mating. A similarly constituted Control group received the vehicle, Corn oil, at the same volume dose as the treated groups. Animals were killed on Day 20 after mating for reproductive assessment and fetal examination.

Examinations

Maternal examinations:
During the acclimatization period, observations of the animals were recorded at least once per day.

Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Any deviation from normal was recorded in respect of nature and severity, date and time of onset, duration and progress of the observed condition.

Detailed observations were also recorded at the following times in relation to dose administration, at a minimum:
- Pre-dose
- One to two hours after dosing

A detailed physical examination was performed on each animal on Days 0, 4, 8, 12, 18 and 20 after mating to monitor general health. The weight of each adult was recorded on Days 0, 3 and 5-20 after mating. The weight of food supplied to each adult, that remaining, and an estimate of any spilled was recorded for the periods Days 0-2, 3-4, 5-7, 8-10, 11-13, 14-16 and 17-19 after mating inclusive.

All adult animals were subject to a detailed necropsy on Day 20 after mating. 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. Gravid uterine weight and thyroid weight were recorded. Blood samples were taken for thyroid hormone analysis. The number of corpora lutea, implantation sites, resorption sites (classified as early or late), and fetuses (live and dead) was recorded for each ovary/uterine horn.
Fetal examinations:
All viable fetuses were dissected from the uterus, individually weighed and identified within the litter using a coding system based on their position in the uterus, and examined externally with abnormalities recorded. The sex of each fetus was recorded. Serial sections of fixed fetuses were examined for visceral abnormalities. Alizarin red stained fetuses were assessed for skeletal development and abnormalities. Ano-genital distance was measured post fixation for half of the fetuses in each litter.
Statistics:
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 and organ weight data:

- A parametric analysis was performed if Bartlett's test for variance homogeneity 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. If the F1 approximate test for monotonicity of dose response was not significant at the 1% level, Williams' test for a monotonic trend was applied. If the F1 approximate test was significant, suggesting that the dose response was not monotone, Dunnett's test was performed instead.

-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, the Kruskal-Wallis test was used to test for any group differences. Where this was significant (p<0.05) inter group comparisons using Wilcoxon rank sum tests were made. For all other analyses the H1 approximate test was applied. If the H1 approximate test for monotonicity of dose-response was not significant at the 1% level, Shirley's test for a monotonic trend was applied. If the H1 approximate test was significant Steel's test was performed instead.
Historical control data:
See attachment.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no signs observed at the detailed physical examinations that were attributable to treatment and there were no signs observed in any animal, in association with dosing.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Treatment at 1000 mg/kg/day was associated with minor mean body weight loss on Days 6-7 of gestation following administration of the second dose in both phases of the study (mean losses of 7 g and 5 g on phases 1 and 2 respectively). Thereafter, weight gain was similar or slightly superior to Controls.

Treatment at 330 mg/kg/day was associated with mean body weight stasis on Days 6-7 of gestation in Phase 1 of the study and mean body weight loss of 3 g in phase 2 of the study.

There was no conclusive effect on body weight gain at 100 mg/kg/day.

There was no effect of treatment on the weight of the gravid uterus in females treated with the test material. Maternal body weight gain, when adjusted for the weight of the gravid uterus did not reveal a clear effect of treatment. Adjusted weight gain was generally low in females treated with the test material when compared with Controls however the magnitude of change across the treated groups revealed no correlation to dose level.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption was marginally lower in females treated at 330 or 1000 mg/kg/day, predominantly during the first week of treatment (Day 5 - Day 11); thereafter food intake was comparable to Controls.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There was no effect of treatment on the weight of thyroids and parathyroids in females treated with the test item.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no findings observed at macroscopic examination on Day 20 after mating, that were considered related to treatment.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Microscopic examination of the thyroids of females treated with the test item revealed no treatment related changes.

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
There was no effect of treatment on corpora lutea counts.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There was no effect of treatment on implantation counts.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
There was no effect of treatment on resorptions (early or late).
Early or late resorptions:
no effects observed
Description (incidence and severity):
There was no effect of treatment on resorptions (early or late).
Dead fetuses:
no effects observed
Description (incidence and severity):
There was no effect of treatment on offspring survival.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
maternal abnormalities
number of abortions
pre and post implantation loss
total litter losses by resorption

Maternal abnormalities

Abnormalities:
not specified

Results (fetuses)

Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Mean fetal weights from females treated at 1000 mg/kg/day were marginally low when compared with Controls (4% and 7% lower in phases 1 and 2 respectively).
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
There was no effect of treatment on offspring survival.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There was no effect of treatment on sex ratio.
Changes in litter size and weights:
effects observed, non-treatment-related
Description (incidence and severity):
Mean fetal weights from females treated at 1000 mg/kg/day were marginally low when compared with Controls (4% and 7% lower in phases 1 and 2 respectively).
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
There was no effect of treatment on offspring survival.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The incidence of major abnormalities showed no relationship to treatment.

Across the treated groups there was an increase in incidence of short supernumerary cervical ribs and a decrease in incidence of ossified cervical vertebral centra compared with Controls. However the incidences observed were found to be within Historical Control Data (HCD) ranges.

At 1000 mg/kg/day there was an increase in incidence of incompletely ossified 1st to 4th sternebrae and partially undescended lobe of thymus compared to concurrent Controls; these findings are outside of the HCD range but are not considered to be adverse.

These findings (with the exception of cervical ribs) indicate slight fetal immaturity and may be associated with the slight decrease in mean fetal weight seen at this dose level. These findings are not considered adverse, but may nonetheless be related to treatment.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Mean placental weights from females treated with the test item were low when compared with Controls, however the magnitude of change across the three treated groups revealed no correlation with dose level.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
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
changes in postnatal survival
skeletal malformations

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
According to an OECD 414 study, the No-Adverse-Effect-Level for maternal toxicity and fetal development and growth is considered to be 1000 mg/kg/day.
Executive summary:

Oral administration of the test material at 100, 330 or 1000 mg/kg/day was well tolerated in pregnant females from Day 5 to Day 19 of gestation. There were no adverse effects of treatment on body weight gain or food intake and there were no in-life signs or treatment related findings at macroscopic or microscopic examination on Day 20 of gestation.

 

Treatment at 1000 mg/kg/day was associated with minor mean body weight loss on Days 6-7 of gestation and marginally low food intake during Days 5-11 of gestation in both phases of the study. At 300 mg/kg/day, mean body weight performance was reduced on Days 6-7 of gestation (stasis or minor weight loss recorded) along with marginally low food intake during Days 5-11 of gestation on both phases of the study.Thereafter, body weight gain and food intake were comparable to Controls.

 

Reproductive performance was generally unaffected by treatment. Mean placental weights from females treated with the test material were low when compared with Controls, however the magnitude of change across the three treated groups revealed no correlation with dose level. Mean fetal weights from females treated at 1000 mg/kg/day were marginally low when compared with Controls in both phases of the study, which correlates with an increase in incidence of incompletely ossified 1st to 4th sternebrae and partially undescended lobe of thymus at this dose level when compared to concurrent Controls; these findings are suggestive of slight fetal immaturity. These findings observed in fetuses from litters treated at 1000 mg/kg/day are considered to be a response to treatment, but are not considered to be adverse. 

 

In conclusion, based on the results of this study, to No-Adverse-Effect-Level for maternal toxicity and fetal development and growth is considered to be 1000 mg/kg/day.