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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-05-15 to 2012-07-08
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Well documented GLP study performed according to OECD Guideline 422. However, dose formulations were not analyzed.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

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:
yes
Remarks:
no analytical verification of the dose is performed
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- State of aggregation : liquid
- Appearance : colourless to yellow brown
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: # 1D403

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature protected from humidity and light

OTHER SPECIFICS:
- Name of test material (as cited in study report): Jeffcat DMDEE

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: BIOAGRI Laboratorios
- Age at study initiation: 10 - 11 weeks old
- Weight at study initiation: males: 324.4 - 341.5 grams; females: 204.3 - 217.3 grams; satellite males: 336.0 - 371.0 grams; satellite females: 207.4 - 208.0 grams
- Fasting period before study: no
- Housing: Each animal was housed individually, except during cohabitation. After acclimation, one male was placed into each female cage for pairing. After pairing, females that presented vaginal smears with the presence of sperm were considered mated and housed individually. The rats were housed in polypropylene cages (41x34x19 cm) with wire mesh tops and bedding material (wood shavings). Clean cages were provided twice weekly for all animals. The cages with the test animals were arranged on the racks in such a way that uniform experimental conditions (ventilation and light) were ensured.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.2-22.6°C
- Humidity (%): 40.5-69.9%
- Air changes (per hr): 10-20 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
unchanged (no vehicle)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
For each dosage group, the appropriate amount of Jeffcat DMDEE was weighed into a pre-calibrated beaker. The vehicle (deionized water) was added in sufficient quantity to achieve the desired concentration. Each solution was stirred and dispensed into individual containers properly identified. A sufficient quantity of the vehicle was similarly dispended for administration to control animals. The prepared solutions were stored at room temperature.
Details on mating procedure:
Premating:
At the end of the acclimation period, animals were randomly assigned to the experimental groups, housed and the treatment started.

Mating:
After a premating period of 2 weeks, females were cohabited with an assigned male (1 female: 1 male) from the same dose level until evidence of copulation was observed, or 2 weeks had elapsed. Care was taken to avoid sibling mating. Vaginal smears were collected daily during mating period and examined for the presence of sperm. Day 0 of gestation was defined as the day a sperm is found in the vaginal smear. One female that showed no evidence of copulation was assumed pregnant at the end of the mating period, and housed accordingly. Males were euthanized after completing a dosing period of 41 to 42 days.

Gestation:
During gestation period all females were housed individually in the cages. One female without copulation date was housed in an individual cage at the end of the mating period. Female showing no evidence of copulation was euthanized 24-25 days after the last day of mating period.

Lactation:
The day when delivery is completed was designated day 0 of lactation (postnatal day 0). On day 0 of lactation the number of alive and dead pups/sex were recorded. Dams with offspring were euthanized on day 4 postnatal. All pups were euthanized at day 4 postnatal.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
Parental animals (males and females) were treated with a volume of 4 mL/kg bw, starting at 10-11 weeks old and ending when the animals were euthanized. Satellite animals (5 animals per sex of the control and 5 animals per sex of the high dose group) were kept for 14 days after the scheduled necropsy of parental animals without treatment, for observation of reversibility, persistence or delayed occurrence of toxic effects. Satellite animals were not mated and, consequently, were not used for assessment of reproduction/developmental toxicity.
Frequency of treatment:
7-day per-week-basis
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
No. of animals per sex per dose:
12 in the main study
5 animals per sex of the control group and 5 animals per sex of the high dose group for the satellite group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dosage levels (in mg/kg body weight/day) were selected following consultation with the Sponsor.

Examinations

Parental animals: Observations and examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
All animals underwent a daily clinical observation for overt signs of ill health. These include, but are not limited to, changes in skin and fur, eye and mucous membranes, respiratory, circulatory, autonomic and central nervous system, motor activity and behavioral patterns.

BODY WEIGHT: Yes
Males were weighed on the first day of dosing and weekly thereafter (including mating and post-mating periods). Females were weighed on first day of dosing and once a week during premating and mating periods, on days 0, 7, 14 and 20 of gestation, and during lactation on the same days as the weighing of litters (on days 0 and 4 postnatal).

FOOD CONSUMPTION
Food consumption was determined on the same day of body weight determination during premating and lactation periods, except on day 0. During the gestation period food consumption was determined on days 3, 6, 9, 12, 15, 18 and 20. After the mating period, food consumption of males was determined weekly. Food consumption was not determined for exposed animals during the mating period.

OTHER:
Organ weights: At scheduled necropsy, testes and epididymides of all males were weighed. Organ weights were obtained for the following organs from 5 animals/sex/group:
liver, kidneys, adrenals, thymus, spleen, brain, heart
Sperm parameters (parental animals):
testis weight, epididymides weight
Litter observations:
Live pups were counted, sexed and weighed on days 0 and 4 postnatal.

Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes
At termination, all parental animals were examined macroscopically for any abnormalities or pathological changes. The animals were euthanized in a carbon dioxide chamber. The number of implantation sites and corpora lutea were recorded. The animals were disposed in biological garbage and then incinerated. All pups were grossly examined for abnormalities of the oral, thoracic and abdominal cavities.

HISTOPATHOLOGY: Yes
At the scheduled necropsy, the following organs of all animals were preserved:
testes, epididymides, ovaries, prostate, semincal vesicle and coagulating gland, bulbourethral gland, organs showing alterations

The following organs and tissues of 5 animals/sex/group were preserved:
adrenals (right and left), bone marrow (femur), brain (cerebrum, cerebellum and pons), esophagus, heart, intestine (duodenum, jejunum, ileum - including Peyer's patches, cecum, colon, rectum/anus), kidneys (right and left), liver (3 lobes), lungs, lymph nodes (mesenteric and submaxillary), pancreas, peripheral nerve (sciatic), spinal cord (cervical, midthoracic and lumbar sections), spleen, stomach (glandular and non-glandular), trachea, thymus, thyroid/parathyroid, urinary bladder, uterus, all gross lesions

Full histopathology of the preserved organs and tissues listed above were performed in highest dose and control animals. The examination of the thymus was extended to females of other dosage groups and satellite females, because changes were observed in the highest dose group.
Postmortem examinations (offspring):
SACRIFICE
All pups were euthanized at day 4 postnatal

GROSS NECROPSY
All pups were grossly examined for abnormalities of the oral, thoracic and abdominal cavities.
Statistics:
Quantitative variables such as body weights, food consumption and organ weights were analyzed by One Way Analysis of Variance (ANOVA), followed by Dunnett's test if significance is detected, or by non-parametric test of Kruskal-Wallis, according to the results of tests for normality and homogeneity of variance. For qualitative or non-parametric data such as clinical findings, macroscopic and microscopic findings and fetal findings, comparison between means were carried out using the Fisher's Exact Test or Chi-Square Test. The level of significance was set at 5%.
Reproductive indices:
The precentage of pre-implantation loss, post-implantation loss, mating index, fertility index, gestation index on day 4 post-partum were calculated (for each pregnant animal) according to the following:
% Pre-implantation loss = (Number of corpora lutea - Number of implantation sites x 100)/Number of corpora lutea
% Post-implantation loss = (Number of implantations - Number of live fetuses x 100)/Number of implantations
% Mating index = (Number of females mated x 100)/Number of females paired
% Fertility index = (Number of females pregnant x 100)/Number of mated pairs
% Gestation index = (Number of females with live pups at birth x 100)/Number of females pregnant
Offspring viability indices:
The percentage of live birth index and viability index on day 4 post-partum were calculated (for each pregnant animal) according to the following:
% Live birth index = (Number of live born pups x 100)/Number of delivered pups
% Viability index on day 4 post-partum = (Number of surviving pups on day 4 post-partum x 100)/Number of live born pups

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Two females and one male at 300 mg/kg/day presented clinical signs between treatment days 25 to 41. These clinical signs were variable in nature, transitory, occurred in only a few animals, and were not observed in the high dose group satellite animals. The significance of these findings is not clear, but they are unlikely to be specific to the test article.
Mortality:
no mortality observed
Description (incidence):
The test substance did not cause mortality during the study
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weight, body weight gain and food consumption were unaffected at all dose levels.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Body weight, body weight gain and food consumption were unaffected at all dose levels.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Decreased diffuse lymphocyte cellularity in thymus was observed in five females exposed to 150 mg/kg/day (animals 71, 74, 76, 77 and 82), while in females at 300 mg/kg/day was noted a thymus involution in rats 95, 98, 102, 104 and 106. These findings were not observed in treated and satellite males or satellite females, terminal thymus weights were substantially decreased in comparison to the female satellite control group. This observation is most likelly due to the nanimals' physiological response to, and following, pregnancy, and is not considered test article related.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
None of the mating or gestation parameters were considered to have been affected by treatment with the test item.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects

Target system / organ toxicity (P0)

Critical effects observed:
not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs were observed in pups until day 4 postnatal.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
On post-natal day 0, two pups at 150 mg/kg/day and three pups at 300 mg/kg/day were found dead. These findings were considered incidental and not related to the test item since they were observed only in a few pups and were not statistically significant. Pup mortality on post-natal day 4 was higher in control than treated groups.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The mean body weight of pups on days 0 and 4 postnatal was similar in all groups
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not specified
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
The necropsy evaluation did not reveal any treatment-related findings in pups.
Histopathological findings:
not examined
Other effects:
not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not specified

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not specified

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: embryo-fetal toxicity

Target system / organ toxicity (F1)

Critical effects observed:
not specified

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions of the study, the No Observed Adverse Effect Level (NOAEL) of the test item in Wistar rats was 300 mg/kg/day for males and females and 300 mg/kg/day for maternal-embryo-fetal toxicity.