Registration Dossier

Administrative data

Endpoint:
one-generation reproductive toxicity
Remarks:
based on generations indicated in Effect levels (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 July 2012 to 12 September 2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: 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:
2014
Report Date:
2014

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
Details on test material:
- Name of test material (as cited in study report): JEFFAMINE RFD 270
- Physical state: liquid
- Lot/batch No.: #880289
- Storage condition of test material: in a specific room at room temperature protected from humidity and light

Test animals

Species:
rat
Strain:
other: Wistar Hannover
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: BIOAGRI Laboratorios
- Age at study initiation: 11-12 weeks
- Weight at study initiation: 304-446 g (males) ; 179 to 267 g (females)
- Fasting period before study: not applicable
- Housing: Each animal was housed individually, except during cohabitation. After acclimation, one male was placed into females cages for pairing (up to 2 females:1 male). 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 a week 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: A larger number of animals than necessary were ordered in order to permit the selection and/or replacement of individual animals before the start of treatment. Accordingly, a total of 70 males and 70 females were received for acclimation and were 10-11 weeks of age. Upon receipt from the supplier, animals were placed into cages (1 animal/cage/sex) examined and acclimated for 8 days. All animals were observed daily for morbidity and mortality and the estrous cycle was checked. At the end of this period, the animals were weighed and a detailed clinical examination was performed. Animals showing abnormal signs or irregular estrous cycle were not used in the study. Only animals with weights within ± 20 % from mean body weight were used in this study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.2 - 24.5 °C
- Humidity (%): 40.5-69.9 %
- Air changes (per hr): 10-20
- Photoperiod (hrs dark / hrs light): 12h/12h

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
deionized water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: For each dosage group, the appropriate amount of Jeffamine RFD 270 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 identitified. A sufficient quantity of the vehicle was similarly dispensed for administration to control animals. The prepared solutions were stored at room temperature.
Test solutions were prepared daily at the testing facility and were administered within 2 hours after preparation. The test solutions were stirred continuously during the administration to maintain the homogeneity.

The volume administered each day was 4 mL/kg body weight.
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 (1male: up to 2 females) from the same dose level until evidence of copulation was observed. 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. Males were euthanized after completing a dosing period of 39 days.

Gestation:
During gestation period all females were housed individually in the cages.

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, starting at 11-12 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 occurence 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 / concentrations
Remarks:
Doses / Concentrations:
0, 10, 75 and 150 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
12 animals 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 groups
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dosage levels were selected in agreement with the sponsor.
10 mg/kg bw/day as the expected dose which causes no signs;
75 mg/kg bw/day as the intermediate dose level;
150 mg/kg bw/day as the expected dose which causes signs of systemic toxicity, but no death or severe suffering.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS/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 behavioural 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 post natal).

FOOD CONSUMPTION :
Food consumption was determined on the same day of body weight determination during premating and lactation periods, except on day 0. During gestation period, food consumption was determined on days 3, 6, 9, 12, 15, 18 and 20. After mating period, food consumption of males was determined weekly. Food consumption was not determined 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. Animals found dead or euthanized moribund during the study were evaluated by gross examination as soon as possible after death. 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), peripheral nerve (sciatic), salivary gland, 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. TThe examination of the lung, liver, brain and kidneys was extended to animals of other dosage groups.
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 the non-parametric test of Kruskal-Willis, according to the results of tests for normaility 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 Fisher's Exact Test or the 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, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related

Details on results (P0)

CLINICAL SIGNS AND MORTALITY
Most of male and female rats exposed to 150 mg/kg bw/day presented piloerection, apathy, respiratory sounds, dyspnea and rinocromodacryorrhea periodically from day 8 to day 44 of the test item administration. Males No 86, 93 and 108 at 150 mg/kg bw/day were found dead on days 12, 19 and 19 of treatment, respectively; while males rats No 84, 88, 89, 90 and 109 were sacrificed by animal welfare on days 12 and 13, after severe episodes of limited mobility and prostration. Females N° 96, 99, 101, 104, 105 and 115, at the same dose level were found dead on days 38, 12, 17, 25, 41 and 28 of treatment, respectively. These effects were dose related and could be attributed to treatment with the test item.

BODY WEIGHT, WEIGHT GAIN AND FOOD CONSUMPTION
Marked effects (decreases) on body weight, body weight gain and food consumption were observed in males and females in the high dose groups and in the high dose satellite male and female groups (150 mg/kg/day). Furthermore, even during the recovery period, a clear recovery in body weights was not observed in the satellite animals. Accordingly, changes observed in body weight, body weight gain and food consumption of males and females at 150 mg/kg/day, were considered to be treatment-related with toxicological difference.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Decreases in mating and fertility indices were observed in females at the high dose group. In addition, the high dose dams had a reduction in the number of corpora lutea implantations and the number of total pups at postnatal days 0 and 4. Also, the live birth index and viability index on day 4 were moderately lower than control and considered to be treatment related with toxicological relevance.

ORGAN WEIGHTS (PARENTAL ANIMALS)
No important alterations in organ weights were observed in treated male and female rats compared to the control groups. Only numerical differences were found and they were considered as normal biological variations.

GROSS PATHOLOGY (PARENTAL ANIMALS)
Most of males exposed at 150 mg/kg bw/day presented slight in brain, liver and lung; while the most frequent macroscopic findings in females at the same dose level were moderate congestion in liver, kidneys and brain. These findings were dose related and although they were not statistically significant, should be attributed to treamtent with the test item.

MICROSCOPIC OBSERVATIONS (PARENTAL ANIMALS)
Mild congestion in brain, liver, lung and kidney were the most common histopathological findings in both male and female rats exposed to 150 mg/kg bw/day, being statistically significant to the control group. These effects were dose related and should be considered treatment related.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects - systemic toxicity

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
maternal toxicity
Histopathological findings:
not examined

Details on results (F1)

VIABILITY (OFFSPRING)
Markedly higher mortality occurred in pups from dams exposed to 150 mg/kg/day at postnatal day 0 (33.9%) and day 4 (16.2%), being statistically significant when compared to the control group. This effect was considered to be related to the treatment with the test item. No other signs or mortality treatment related were observed in the other dose levels.

BODY WEIGHT (OFFSPRING)
Statistically significant lower body weight was observed in pups from dams exposed to 150 mg/kg bw/day compared to the control group on postnatal day 4 (-20%). Mean body weight on postnatal day 0, although without statistical significance, was moderately lower than control (-16.4%). These differences were dose related and therefore considered to treatment related.

GROSS PATHOLOGY (OFFSPRING)
Body trauma (67.7%), cyanosis (41.9%) and cannibalized pups (12.9%) were findings statistically higher at the highest dose level and dose-related. These effects were associated with maternal toxicity observed at this highest dose and should be attributed to treatment with the test item.

Effect levels (F1)

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

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions of this study, the No Observed Adverse Effects Level of the test item JEFFAMINE RFD 270 in Wistar rats was 10 mg/kg/day for male systemic toxicity and maternal systemic toxicity, and 75 mg/kg bw/day for embryo-fetal toxicity.