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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline Study
Cross-reference
Reason / purpose:
reference to same study

Data source

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

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: solution
Details on test material:
- Name of test material (as cited in study report): N4-Amine N,N’-Bis-(3-Aminopropyl)-ethylenediamine
- Physical state: Clear slightly yellowish liquid
- Analytical purity: 95.9 g/100 g
- Lot/batch No.: 000STD77L0
- Stability under test conditions: stable
- Storage condition of test material: At room temperature in the dark

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: approx. 11 weeks
- Weight at study initiation: mean: females: 201 g, males: 318 g
- Housing: in groups of 5 animals/sex/cage
- Diet: free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Water: ad libitum
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 24
- Humidity (%): 40 - 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Formulations (w/w) were prepared daily within 5 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for specific gravity of the test substance. No correction was made for the purity of the test substance.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
1H-NMR spectroscopy
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: no data
- Proof of pregnancy: sperm in vaginal smear or by the appearance of an intravaginal copulatory plug referred to as day 0 of pregnancy.
- After successful mating each pregnant female was caged individually.
Duration of treatment / exposure:
Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy.
Females were exposed for 42-53 days days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy).
Pups were not treated directly, but were potentially exposed to the test substance in utero and through lactational transfer.
Frequency of treatment:
daily
Duration of test:
same as treatment
Doses / concentrations
Remarks:
Doses / Concentrations:
30, 100, 300 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on results of the dose range finding study (Project 499971; BASF Project 01R0402/04X033) where dose levels of 0, 150 and 400 mg/kg bw/day were assessed. Animals at 400 mg/kg bw/day had slightly lower body weight gains or weight loss, and slightly lower food consumption. Changes in haematology and clinical biochemistry parameters were noted, along with increased absolute and relative liver weights (both sexes) and higher relative kidney weights (males). Females at 150 mg/kg bw/day also had higher absolute and relative liver weights.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations checked: mortality, pain, distress or discomfort

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: Females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.

FOOD CONSUMPTION AND COMPOUND INTAKE : Yes

WATER CONSUMPTION AND COMPOUND INTAKE : Yes: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on lactation day 5-7
- Organs examined: Adrenal glands, Ovaries, (Pancreas), (Aorta), Peyer's patches [jejunum, ileum] if detectable, Brain - cerebellum, mid-brain, cortex, Pituitary gland, Caecum, Preputial gland, Cervix, Clitoral gland, Rectum, Colon, (Salivary glands - mandibular, sublingual), Sciatic nerve, Duodenum, Skeletal muscle, Eyes (with optic nerve (if detectable) and Harderian gland), (Skin), Spinal cord -cervical, midthoracic, lumbar, (mammary gland area), Spleen, Femur including joint, Sternum with bone marrow, Heart, Stomach, Ileum, Jejunum, Thymus, Kidneys, Thyroid including parathyroid if detectable, (Lacrimal gland, exorbital), (Tongue), (Larynx), Trachea, Liver, Urinary bladder, Lung, infused with formalin, Uterus, Lymph nodes - mandibular, mesenteric, Vagina

[Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.]
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
The following additional methods of statistical analysis were used:
Motor activity data was subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences followed by the Wilcoxon test to compare the treated groups to the control group.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Indices:
Mating index (%), Fertility index (%), Conception index (%), Gestation index (%), duration of gestation, Percentage live males at first litter check, Percentage live females at first litter check, Percentage of postnatal loss days 0 - 4 of lactation, viability index (%)

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Details on maternal toxic effects:
Gestation:
The gestation index was significantly lower for females at 300 mg/kg bw/day (50% compared to 100% for all other groups). This was attributable to the 5 females at this dose level with implantation sites only.
The duration of gestation was slightly longer (not statistically significant) for females at 300 mg/kg bw/day, which was attributable to the small number of the group and the 23 days recorded for female no. 71. This was not considered to be toxicologically relevant.
Parturition/maternal care:
No signs of difficult or prolonged parturition were noted among the pregnant females.
Female no. 80 (300 mg/kg bw/day) was seen beginning delivery though no pups were later found. It is possible they were born and immediately cannibalized. This female was later noted with 6 corpora lutea and 4 implantation sites. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Details on embryotoxic / teratogenic effects:
Early postnatal pup development:
There were significantly fewer living pups at first litter check noted for 100 and 300 mg/kg bw/day. Mean litter sizes were 13.3, 10.3, 9.0 and 3.4 pups for the control, 30, 100 and 300 mg/kg bw/day groups, respectively. In total, only 17 pups were born in the 300 mg/kg bw/day group. The significant difference seen for animals at 100 mg/kg bw/day was mostly attributable to two factors: 1. a slightly higher mean number of pups per litter for the control group (13.3, compared to the historical control mean of 11.8) and 2. to a single female with only one pup (no. 67). When discounting her data, the mean number of pups per litter for this group is 9.8. A relationship to treatment could not be completely excluded for the slightly smaller litter sizes at 100 mg/kg bw/day. However, as there was no clear or linear dose relationship between these two dose levels and as females in this group produced viable, healthy pups, a slight reduction in mean litter size was not considered adverse for either dose level. The number of dead pups at first litter check, postnatal loss, viability index and sex ratio were unaffected by treatment, and clinical signs, body weight and external macroscopy did not reveal treatment-related findings.
Mortality
One pup of the control group and two pups at 100 mg/kg bw/day went missing during the first days of lactation. These pups were most likely cannibalized. No toxicological relevance was attributed to these missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age. There were no dead or missing pups in the 30 and 300 mg/kg bw/day groups.
Clinical signs
There were no clinical signs noted for any pup.
Body weights
Pup body weights were higher for all treated groups compared to controls, though it was only statistically significant for pups at 30 mg/kg bw/day. The higher body weights for the smaller litters of females 52, 54, 56 and 58 contributed to this. The slightly smaller litters at 100 mg/kg bw/day also contributed to their higher (not statistically significant) means. The litters at 300 mg/kg bw/day all had fewer pups than controls. The five litters available had 3, 6, 4, 2, and 2 pups, and with the exception of the litter with 4 pups (female no. 75) the pups of the other litters did not have higher body weights that would be expected to coincide with litters of such small size. However, the treated pups gained a comparable or higher percentage of weight than controls from postnatal Days 1-4 (49, 63, 58 and 54% gain for pups from the control, 30, 100, and 300 mg/kg bw/day groups, respectively). As such, the differences in body weights were not considered to be toxicologically relevant.
Macroscopy
No milk in the stomach was the only macroscopic finding seen and was incidental since it was noted for animals that survived until the scheduled necropsy, indicating they were sufficiently fed the days prior to the scheduled necropsy. This was noted for a single pup (no. 2) from litter 43 (control) and the entire litter from female no. 62 (100 mg/kg bw/day).

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
> 300 mg/kg bw/day (nominal)
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

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion