Registration Dossier

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

According to an OECD 422 study, conducted to GLP, the NOAEL (rats) for reproductive performance and offspring viability of Methylenebis (dibutyldithiocarbamate), is greater than 20,000 ppm (1,225 mg/kg diet) (Safepharm Laboratories Limited, 2006).

A two generation study does not need to be conducted becuase there are no results from available repeated dose toxicity studies that indicate an adverse effect on reproductive organs or tissues, or reveal other concerns in relation with reproductive toxicity.

Link to relevant study records
Reference
Endpoint:
one-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in accordance with relevant guideline and under GLP conditions.
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Manston Road, Margate, Kent
- Weight at study initiation: At the start of the treatment the males weighted 289 to 355 g and the females weighted 195 to 238 g.
- Housing: Upon arrival, the animals were housed in groups of four in polypropylene cages with stainless steel grid floors and tops, suspended over paper-lined polypropylene trays.
- Diet (e.g. ad libitum): A ground diet 5002 was offered ad libitum.
- Water (e.g. ad libitum): The animals were allowed free access to water. Main water was supplied from polycarbonate bottles attached to the cage.
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2 ℃
- Humidity (%): (55 ± 15)%
- Air changes (per hr): at least fifteen air changes per hour
- Photoperiod (hrs dark / hrs light): The low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness.

IN-LIFE DATES: From: 04 March 2003 To: 01 May 2003

No additional data
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was prepared as direct dietary admixture. For each dose level, an appropriate aliquot of test substance was weighted and then added to approximately one third of the required amount of untreated diet in a mixing bowl. An initial mix of the test material and diet was performed using a Hobart QE200 mixer for ten minutes. Further untreated diet was then added to the initial mix to give the required concentration and the entire amount was then mixed for ten minutes using the Hobart H800 mixer.

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): untreated diet

No additional data
Details on mating procedure:
- M/F ratio per cage: one male to one female
- Length of cohabitation: basis for a period to eighteen days
- Proof of pregnancy: The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating.
- After successful mating each pregnant female was caged (how): Mated females were then separated from the male and housed individually during the period of gestation and lactation.

No additional data
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Prior to the start of the study, samples of the test diet preparation were analysed for achieved concentration, homogeneity and stability of test material in diet. The results of analysis of the original preparation showed the test substance to be stable for at least fourteen days.
Duration of treatment / exposure:
The test substance was administered throughout maturation (dosed for two weeks prior to pairing), mating, gestation and up to Day 5 of lactation.
Frequency of treatment:
Once daily
Details on study schedule:
None stated
Remarks:
Doses / Concentrations:
0, 1000, 5000, 20000 ppm
Basis:
nominal in diet
Initial dose level
Remarks:
Doses / Concentrations:
0, 900, 4500, 18000 ppm
Basis:
nominal in diet
Adjusted dose level
No. of animals per sex per dose:
Ten animals per sex per dose
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: The dose levels were chosen based on the results of a dietary range-finding study.

No additional data
Positive control:
None stated
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily during the normal working week and once daily at weekend/bank holiday.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: During the maturation and mating period the parental generation animals were weighted weekly. Following mating the parental males were weighted weekly until termination. Parental generation females showing evidence of mating were weighted on Day 0, 7, 14 and 20 post coitum. Parental generation females with a liver litter were weighted on Day 1 and 4 post partum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes. During the maturation period, food consumption was recorded for each cage of adults weekly. For females showing evidence of mating, food consumption was recorded for the periods covering Days 1 to 7, 7 to 14 and 14 to 20 post coitum. For females with live litters, food consumption was recorded for the period covering Days 1 to 4 post partum.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

OTHER:
Functional Observations
Prior to the start of treatment and on Days 8, 15 and 22, all animals were observed for signs of functional/behavioural toxicity. Functional performance tests were also performed on five selected males and five selected females per group on Day 22 for males and Day 4 of lactation for females, together with an assessment of sensory reactivity to difference stimuli.

Haematological and blood chemical investigations were performed on five males and five females selected from each test and control group on Study Day 13 (prior to mating). Blood samples were obtained from the lateral tail vein. Animals were not fasted prior to sampling.

Pregnancy and Parturition: Each pregnant female was observed at 0830, 1230 and 1630 hours at or around the period of expected parturition. At weekends, observations were carried out at 0830 and 1230 hours only. The following was recorded for each females:1) Data of mating; 2) Data and time of observed start of parturition; 3) Data and time of observed completion parturition.
Oestrous cyclicity (parental animals):
None stated
Sperm parameters (parental animals):
None stated
Litter observations:
At the observation of completion of parturition, the number of live and dead offspring was recorded.

The following observations were recorded for all individual offspring alive on the particular day of observation.
Bodyweight: Individual offspring weights were recorded on Day 1 and 4 post partum.
Offspring Numbers and sex: The number of offspring was recorded daily, up to weaning, and reported for Day 1 and 4 post partum.
Clinical signs: The clinical condition of individual offspring was observed daily and any findings recorded.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals at Day 5 of lactation
- Maternal animals: All surviving animals at Day 5 of lactation

GROSS NECROPSY
- Gross necropsy consisted of both internal and external abnormalities.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues listed next were examined from five males and five females selected from the control and high dose groups. Histopathology was extended to examination of the spleen for five males and five females selected from both the low and intermediate dose groups.
Adrenals, Ovaries, Aorta (thoracic), Pancreas, Bone and bone marrow (femur including stifle joint), Pituitary, Bone and bone marrow (sternum), Brain (including cerebrum, cerebellum and pons), Rectum, Caecum, Salivary glands (Submaxillary), Colon, Sciatic nerve, Duodenum, Seminal vesicles, Epididymides, Thyroid/parathyroid, Eyes, Trachea, Gross Lesions, Vagina, Heart, Coagulating gland, Ileum, Skin (hind limb), Jejunum, Spinal cord (cervical), Kidneys, Spleen, Liver, Stomach, Lungs (with bronchi), Testes, Lymph nodes (cervical and mesenteric), Thymus, Mammary gland, Urinary bladder, Muscle (Skeletal), Uterus, Oesophagus.
The following list of tissues were examined from the remaining unselected males and females from the control and high dose groups: Epididymides, Seminal vesicles with coagulating gland, Ovaries, Uterus and cervix, Pituitary, Vagina, Prostate, Testes.
The following list of organs were weighted for all adult males and females at necropsy where applicable: Adrenals, Liver, Brain, Ovaries, Epididymides, Spleen, Heart, Testes, Kidney and Thymus, paired organs were weighted together.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals were sacrificed at Day 5 of age.
- These animals were subjected to postmortem examinations macroscopically.

GROSS NECROPSY
- Gross necropsy consisted of internal and external abnormalities.

No additional data
Statistics:
The following parameters were analysed statistically, where appropriate using the test methods as follows:
Adult male and female bodyweight during the maturation, gestation and lactation periods, adult male food consumption, female food consumption during maturation, gestation and lactation, litter size, litter weight, individual offspring bodyweight offspring landmarks of physical development, haematology, blood chemistry and organ weights.
Values were analysed to establish homogeneity of group variance using Levene’s test followed by one-way analysis of variance. If the variances were unequal subsequent comparisons between control and treated groups were performed using a pairwise “T” test Comparison method. If variances were equal subsequent comparisons between control and treated groups were performed using Dummett’s Multiple Comparison Method. For haematology and blood chemistry values an additional regression analysis of all values was also performed.

Adult pre-coital intervals, female gestation lengths, offspring reflexological responses and litter sex ratio, relative organ weights. Individual values were compared using the Kruskal-Wallis non-parametric rank sum test. Where significant differences were seen, pairwise comparison of control values against treated group values was performed using performed using Mann-Whitney “U” test.

Histopathology: For those tissues from selected animals only.
Chi-squared analysis for differences in the incidence lesions occurring with an overall frequency of 1 or greater.
Kruskal-Wallis one-way non-parametric analysis of variance for the comparison of severity grades for the more frequently observed conditions.
Probability values were calculated as follows: P<0.001, +++, ---***; p<0.01, ++, --**; p<0.05, +, -*; p<0.1, (+), (-)(*); p≥0.1, NS, (not significant), Where plus signs indicate positive differences from the control group, and minus signs negative differences. Asterisks refer to overall between group variation.
Reproductive indices:
For each group the following were calculated:
Mating Index (%)= (Number of animals mated)/(Number of animals paired) × 100;
Pregnancy Index (%) = (Number of pregnant females)/(Number of animals mated) × 100
Offspring viability indices:
Viability Index (%) = (Number of pups alive on Day 4)/(Number of pups alive on Day 1) × 100
Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
no effects observed
Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
There were no mortalities during the course of the study. There were no clinical signs of toxicity throughout the course of the study.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
At 20000 ppm, there were inconsistent differences in bodyweight gain for males throughout, and females prior to pairing, compared to controls. Female bodyweight gain during gestation was lower than controls resulting in significant bodyweight differences during the final week of gestation and early lactation. There were no significant effects upon food consumption except for a statistically significant difference in female food consumption during the final week of gestation.
At 5000 ppm, a similar pattern of reduction in bodyweight gain during gestation for females was observed but no significant effect upon female food consumption.
At 1000 ppm, there were no significant differences in female bodyweight gain and food consumption during the study period.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
The results showed that the highest dose level of 20000 ppm achieved a test substance intake excess of 1000 mg/kg/day for males and females and this was maintained even after the reduction to 1000 ppm.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
There were no significant treatment-related effects upon reproductive performance.

ORGAN WEIGHTS (PARENTAL ANIMALS)
An increase in absolute and relative liver weight for females only when compared to control at 20000 ppm. An increase in liver weight for females only in 5000 ppm.

GROSS PATHOLOGY (PARENTAL ANIMALS)
There were no treatment-related macroscopic post mortem findings for adult males or females.

HISTOPATHOLOGY (PARENTAL ANIMALS)
At 20000 ppm, histopathology showed a reduction in the severity grades for splenic extramedullary haemopoiesis for both males and females compared to controls but this is of questionable toxicological relevance; At 5000 nppm and 1000 ppm, histopathology showed lower severity grades of splenic extramedullary haemopoiesis for male only.

OTHER FINDINGS (PARENTAL ANIMALS)
At 20000 ppm, Laboratory investigation showed a significant increase in both Activated Partial Thromboplastin Time and Clotting time for male only when compared to control values, There were no significant blood chemistry changes. There were no significant effects on blood chemistry or haematology at 5000 and 1000 ppm.
Dose descriptor:
NOAEL
Effect level:
61 mg/kg diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Dose descriptor:
NOAEL
Effect level:
> 1 225 mg/kg diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Histopathological findings:
not specified
VIABILITY (OFFSPRING)
There were no significant treatment-related effects upon offspring from birth to Day 4 post partum.

CLINICAL SIGNS (OFFSPRING)
There were no significant treatment-related effects upon offspring clinical condition.

BODY WEIGHT (OFFSPRING)
There were no significant treatment-related effects upon offspring bodyweight gain from birth to Day 4 post partum.

GROSS PATHOLOGY (OFFSPRING)
There were no significant treatment-related macroscopic post mortem findings for offspring that died from birth to Day 4 post partum. At scheduled termination of offspring, there were no significant treatment-related findings.

OTHER FINDINGS (OFFSPRING)
Offspring Sex Ratio: There were no significant treatment-related effects upon intra and inter litter sex ratios.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
> 1 225 mg/kg diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
Reproductive effects observed:
not specified
Conclusions:
At dose levels of 5000 ppm and above there was evidence of treatment-related effects upon the adult. At a dose level of 1000 ppm and above the only significant findings was lower grades of severity of splenic extramedullary haemopoiesis which is considered not to be an adverse finding. No evidence of effects upon reproductive performance or subsequent offspring viability in utero and early lactation as seen. Offspring development was comparable to control values.
The “No Observed Adverse Effect Level” for effects upon adults was 1000 ppm and for reproductive performance and offspring viability was in excess of 20000 ppm.
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
20 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
1 (reliable without restriction)
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Short description of key information:
The NOAEL (rats) for reproductive performance and offspring viability of Methylenebis (dibutyldithiocarbamate), is greater than 20,000 ppm (1,225 mg/kg diet) (Safepharm Laboratories Limited, 2006).

Justification for selection of Effect on fertility via oral route:
This study was carried out with rat based on OECD 422.

Effects on developmental toxicity

Description of key information

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.

Link to relevant study records
Reference
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
Reason / purpose:
reference to other study
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
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
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.
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
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.
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.
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.
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.
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
Abnormalities:
not specified
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.
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
Abnormalities:
not specified
Developmental effects observed:
not specified
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.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
1 (reliable without restriction)
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

Reproductive toxicity:

Based on a NOAEL of greater than 20,000ppm (1,225 mg/kg diet), in accordance with Regulation No 1272/2008, Methylene bis (dibutyldithiocarbamate) is not classified for reproductive toxicity.