Registration Dossier

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 04 March to 01 May 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in accordance with relevant guideline and under GLP conditions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
Batch no.: BVL 232B023
Purity: not specified

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
Male and female Sprague-Dawley Crl:CD (SD) IGS BR
- Source: Charles River (UK) Limited, Manston Road, Margate, Kent
- A total of eighty animals (40 males and 40 females) were accepted into the study and acclimated for eight days.
- 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

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral 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
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. The dose levels were reduced during the final week of gestation (Study Day 29) onwards.
Frequency of treatment:
Once daily
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 1000, 5000, 20000 ppm (Initial dose level)
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
0, 900, 4500, 18000 ppm (Adjusted dose level )
Basis:
nominal in diet
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.

- Groups of 10 male and ten female rats were dosed according to dose group for fourteen days prior to pairing.

-All animals were given a detailed clinical examination once every week for four weeks to observe functional/behavioural canges in an open arena.

-One day proir to pairing (Day 13) five males and five females, randomly selected, were sampled for clinical chemistry and haematology.

-On Day 14 all animales were paired on a one male:one female basis within each dose group for a maximum of fourteen days.

-At the observation of mating or at the end of the fourteen day mating period males were returned to their original cages and females were transferred to individual cages/

-At the end of the mating phase five males per dose group were evaluated for functional/sensory responses to various stimuli. The males used were those selected for haematology/clinical chemistry.

-Pregnant females were allowed to give birth and maintain their offspring until Day 4 post partum. Evaluation of each litter size and weight were performed during this period.

-At Day 4 post partum, five females per dose group were evaluated for functional/sensory responses to various stimuli. The females used were those selected for haematology/clinical chemistry.

-At Day 5 post partum following completion of functional assessments all females and offspring were killed and examined macroscopically.

-Following completion of the female gestation and lactation phases, the males were killed and examined macroscopically.
Positive control:
None stated

Examinations

Observations and examinations performed and frequency:
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.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Gross necropsy consisted of both internal and external abnormalities.

HISTOPATHOLOGY: Yes
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.
Other examinations:
None stated
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.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not specified
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not specified
Details on results:
CLINICAL SIGNS AND MORTALITY
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 WEIGHT GAIN
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.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
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.

HAEMATOLOGY
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.

CLINICAL CHEMISTRY
There were no significant treatment-related effects upon chemistry values for either sex.

NEUROBEHAVIOUR
There were no significant treatment-related differences in the results of the neurotoxicity assessments. There were no significant signs of behaviour changes from the arena-based observation. There was no significant treatment-related differences in the functional test results and no significant differences in the sensory responses to various stimuli.

ORGAN WEIGHTS
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
There were no treatment-related macroscopic post mortem findings for adult males or females.

HISTOPATHOLOGY: NON-NEOPLASTIC
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 ppm and 1000 ppm, histopathology showed lower severity grades of splenic extramedullary haemopoiesis for male only.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
>= 61.3 - <= 79.9 mg/kg diet
Based on:
test mat.
Sex:
female
Basis for effect level:
other: 1000 ppm
Dose descriptor:
NOAEL
Effect level:
>= 276.6 - <= 485.8 mg/kg diet
Based on:
test mat.
Sex:
male
Basis for effect level:
other: 5000 ppm
Dose descriptor:
LOAEL
Effect level:
>= 314 - <= 425.2 mg/kg diet
Based on:
test mat.
Sex:
female
Basis for effect level:
other: 5000 ppm. LOAEL based upon increase in liver weight and inconcistent bodyweight gains.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Group Mean Chemical Intake (mg/kg/day) Before Pairing and During Gestation - Females

Group Dose Level (ppm) Study Week Gestation Day
1 2 Mean 1-7 7-14 14-20
2 1000 79.9 61.3 70.6 78.7 74 6.5
3 5000 396.2 314 355.1 425.2 398.6 320.1
4 20000 1548.7 1225.6 1387.1 1575.9 1483.5 1224.9

Group Mean Chemical Intake (mg/kg/day) - Males

Group Dose Level (ppm) Study Week Dose Level (ppm) Study week
1 2 3 4 5 * 6 Mean
2 1000 98.8 73.3 - - 900 66.9 58.1 74.3
3 5000 485.8 369.2 - - 4500 276.6 276.6 361.8
4 20000 1957 1492.3 - - 18000 1370 1370 1524.5

*One day at 1000, 5000 and 20000, six days at lowered dose

-Animals paired, no food consumption recorded

Applicant's summary and conclusion

Conclusions:
At dose levels of 5000 ppm and above with Methylenebis (dibutyldithiocarbamate) there was evidence of treatment-related effects upon females (liver weight and bodyweight). 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 treatment related.
The lowest “No Observed Adverse Effect Level” was set at 1000 ppm for females.