Registration Dossier

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:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study report): 3,3’-iminodi(propylamine)
- Physical state: Clear colourless to yellowish liquid
- Analytical purity: 99.1 area-%
- Lot/batch No.: 000STD77L0
Specific details on test material used for the study:
Purity: 99.1%; clear colourless to yellowish liquid

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: Females were approximately 10 to 14 weeks (age at maiting)
- Weight at study initiation: 188-191 g (mean)
- Housing: Females were individually housed in Macrolon plastic cages
- Diet (e.g. ad libitum): Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Water (e.g. ad libitum): Free access to tap-water
- Acclimation period: At least 5 days prior to treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 24°C
- Humidity (%): 40 - 70%
- Air changes (per hr): at least 10
- 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 the specific gravity/density of the test substance. No correction was made for the purity/composition of the test substance.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of dose preparations were taken at the test facility on a single occasion during the treatment period (formulations were prepared and sampled on 24 August 2015). The samples were dispatched on dry ice to ABL where they were analyzed to assess accuracy of preparation (all groups), homogeneity (lowest and highest concentration) and stability in vehicle over 5 hours at room temperature (lowest and highest concentrations).
Details on mating procedure:
Untreated females were mated at the Supplier, and were at Day 0 or 1 post-coitum on arrival at the Test Facility (Day 0 post-coitum was the day of successful mating; confirmed by vaginal plug).
Duration of treatment / exposure:
From Days 6 to 20 post-coitum, inclusive. Females no. 33 and 77: from Days 6 to 19 post-coitum, inclusive.
Frequency of treatment:
once daily
Duration of test:
On day 21 pos-coitum all survived animals were sacrificed.
Females with early delivery on day 21 (nos. 7, 10, 28, 29, 30, 31 and 73): Within 24 hours of early delivery.
Females with early delivery on Day 20 (nos. 33 and 77): Within 24 hours of early delivery
Doses / concentrations
Remarks:
Doses: 0, 5, 15 and 50 mg/kg bw
Dose selection rational: see "Details on study design".

No. of animals per sex per dose:
22 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were based on data from a 14-day range finding study and an OECD 422 study. Within the range finding study with 3 Wistar rats per sex and dose, reduced food consumption (males: up to 22%; females: up to 33%), reduced body weight change in males (up to 51%) and body weight loss in females was observed after treatment by oral gavage at 100 mg/kg bw/day. At 200 mg/kg bw/day the animals showed tremors and piloerection and 1/3 males was found dead on day 1. On study day 3, 2/3 males and 3/3 females were sacrificed moribund. On the basis of the range finding study, doses of 5, 15 and 50 mg/kg bw were used for the subsequent OECD 422 study: decreased food consumption (females: up to 13% from gestation day 14-20), decreased body weight (females: up to 7%), decreased body weight gain (females: up to 29% from gestation day 7-14), decreased white blood cell count (males: 35%), decreased lymphocyte count (males: 38%), decreased monocyte count (males: 45%), decreased eosinophil count (males: 50%), decreased absolute and relative thymus weight (males: 44%/42%), decreased cellularity in cortex and medulla of thymus (males: 9/10) and increased relative liver weight (males: 11%) was observed in the animals of high dose. The body weight of pups was significantly decreased on postnatal day one (males: 13%, females: 12%) and four (males: 21%, females: 22%), the body weight gain of pups was significantly decreased on postnatal days 1-4 (males: 35%, females: 39%). The NOAEL was observed at 15 mg/kg bw.
Based on the findings observed in the range finding and subsequent OECD 422 study and due to an obviously steep dose response relationship, doses of 5, 15 and 50 mg/kg bw were selected for the OECD 414 study.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: al least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from Day 2 post-coitum onwards up to the day prior to necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: Days 2, 6, 9, 12, 15, 18 and 21 post-coitum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption: Days 2-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post-coitum.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: 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 day 21 post-coitum

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: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: Yes: [half per litter]
Statistics:
Dunnett-test, Steel-test, Fischer Exact-test, Mann Whitney test, AVON test, Dunn`s test
Indices:
For each litter the following calculations were performed:
Pre-implantation loss (%) = (number of corpora lutea - number of implantation sites) / (number of corpora lutea) x 100

Post-implantation loss (%) = (number of implantation sites - number of live fetuses) / (number of implantation sites) x 100

The fetal developmental findings were summarized by: 1) presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the group; and 2) considering the litter as the basic unit for comparison, calculating the number of affected fetuses as a
mean litter proportion on a total group basis, where:
Viable fetuses affected/litter (%) = (number of viable fetuses affected/litter / number of viable fetuses/litter) x 100
Historical control data:
This species and strain of rat has been recognized as appropriate for developmental toxicity studies. WIL Research Europe B.V. has historical data on the background incidence of fetal malformations and developmental variations in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of developmental toxicants.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs were noted that were considered to be related to treatment with the test substance. One female (no.48) at 15 mg/kg bw/day showed alopecia from Day 13 of post coitum onwards. This remained within the range of background findings to be expected for rats of this age and strain.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean body weight, body weight gain and weight gain corrected for uterus weight remained within the same range as controls over the complete study period.
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Description (incidence and severity):
No necropsy findings were noted that were considered to be treatment related. Alopecia in the chest region was recorded in one female (no. 48) at 15 mg/kg bw/day and confirmed the in-life observation in this animal. This remained within the range of background findings to be expected for rats of this age and strain and was considered not to be of toxicological relevance.

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There were no effects on the number of implantation sites or pre- or post-implantation loss noted with treatment up to 50 mg/kg bw/day.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
All females, including the females that delivered early, were pregnant with viable fetuses.
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
Mortality
No mortality occurred.
Nine females delivered early (two vehicle controls, five 5 mg/kg bw/day-treated females and two 50 mg/kg bw/day- treated females), on Day 20 or 21 post coitum. As these incidences did not show a relationship with the dose, they were considered not to be treatment related.
Clinical Signs
No clinical signs were noted that were considered to be related to treatment with the test substance.
One female (no.48) at 15 mg/kg bw/day showed alopecia from Day 13 of post coitum onwards. This remained within the range of background findings to be expected for rats of this age and strain.
Body Weights
Mean body weight, body weight gain and weight gain corrected for uterus weight remained within the same range as controls over the complete study period.
Food Consumption
No toxicologically relevant treatment related effect on food consumption was noted.
At 50 mg/kg bw/day absolute food consumption was slightly lower on Days 18-21 post coitum (statistically significant). As this was very minimal and food consumption corrected for body weight appeared unaffected by treatment, this change was not considered toxicologically relevant.
Macroscopic Examination
No necropsy findings were noted that were considered to be treatment related.
Alopecia in the chest region was recorded in one female (no. 48) at 15 mg/kg bw/day and confirmed the in-life observation in this animal. This remained within the range of background findings to be expected for rats of this age and strain and was considered not to be of toxicological relevance.
Maternal Pregnancy Data
There were no effects on the number of pregnant females, corpora lutea, implantation sites or pre- or post-implantation loss noted with treatment up to 50 mg/kg bw/day. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. All females, including the females that delivered early, were pregnant with viable fetuses.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Mean combined (male and female) fetal body weights were slightly lower (6%) at 5 mg/kg bw/day and 50 mg/kg bw/day (not statistically significant) compared to the concurrent control. As this did not show a relation with dose and the difference was only slight, it was considered not to be treatment related. Mean combined (male and female) fetal body weights were 5.1, 4.8, 5.2 and 4.8 grams for the vehicle control, 5, 15 and 50 mg/kg bw/day, respectively.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
External malformations:
no effects observed
Description (incidence and severity):
The numbers of fetuses (litters) available for morphological examination were 226 (22), 232 (22), 224 (22) and 232 (22) in Groups 1, 2, 3, and 4, respectively. External examination was done for all fetuses. There were no external developmental malformations or variations for any of the fetuses following treatment up to 50 mg/kg/day.
Skeletal malformations:
no effects observed
Description (incidence and severity):
There were no treatment related effects on skeletal morphology following treatment up to 50 mg/kg bw/day. Skeletal malformations were noted in one Group 3 fetus and two control fetuses. The Group 3 fetus
(no. A046-04) had bent scapulae and the control fetuses either had fused sternebrae (no. A020-06) or a vertebral centra anomaly (absence of a cervical hemicentrum; no. A007-05). The latter fetus was delivered early. As these malformations were limited to individual fetuses and/or occurred in control fetuses, these were considered chance findings and thus not considered treatment related. The variation of unossified metacarpals and/or metatarsals occurred in 6.3%, 29.4%, 12.3% and 21.6% of fetuses per litter in Groups 1, 2, 3 and 4, respectively. This group distribution would indicate retarded skeletal ossification in Groups 2 and 4, which corresponds with the slightly lower fetal weights in these groups (fetal weights were 5.1, 4.8, 5.2 and 4.8 grams in Groups 1, 2, 3 and 4, respectively). However, retarded ossification in Groups 2 and 4 was not unambiguous substantiated by other ossification parameters such as reduced ossification of the skull and unossified sternebrae nos. 5 and 6. Mean litter percentages for these respective findings were 6.1%, 4.3%, 11.6% and 6.5%, and 0.6%, 5.9%, 0.0% and 0.8% per litter in Groups 1, 2, 3 and 4, respectively. Therefore, and due to absence of a dose-relationship, the finding unossified metacarpals and/or metatarsals was not considered to be related to treatment. The variation of 14th full ribs was observed in 11.7%, 10.8%, 10.0% and 3.5% of fetuses per litter in Groups 1, 2, 3 and 4, respectively. The mean litter proportion in Group 4 was lower (not statistically significant) than in the other groups, but this was not accompanied by a higher incidence for 14th rudimentary ribs in this group (viz.. 56.0%, 70.4%, 70.1% and 68.5% of fetuses per litter in Groups 1, 2, 3 and 4, respectively). Therefore, and due to absence of statistical significance, the lower litter incidence of 14 full ribs in Group 4 was considered to be a change finding and not treatment related. Remaining skeletal variations were not considered treatment related as they occurred infrequently or were observed in control fetuses only.
Visceral malformations:
no effects observed
Description (incidence and severity):
There were no treatment related effects on visceral morphology following treatment up to 50 mg/kg bw/day. Visceral malformations were not observed for any of the fetuses and the variations that were noted, namely small supernumerary lobe(s) or appendix of the liver and partially undescended thymus horn(s), occurred at low incidences and/or in the absence of a dose-related trend.
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Litter Size
There were no treatment-related effects on litter size for any group.
Mean litter sizes were 10.4, 10.4, 10.2 and 10.7 fetuses/litter for the vehicle control, 5, 15 and 50 mg/kg bw/day groups, respectively.
Sex Ratio
The male:female ratio was unaffected by treatment up to 50 mg/kg bw/day.
Fetal Body Weight
Mean combined (male and female) fetal body weights were slightly lower (6%) at 5 mg/kg bw/day and 50 mg/kg bw/day (not statistically significant) compared to the concurrent control. As this did not show a relation with dose and the difference was only slight, it was considered not to be treatment related. Mean combined (male and female) fetal body weights were 5.1, 4.8, 5.2 and 4.8 grams for the vehicle control, 5, 15 and 50 mg/kg bw/day, respectively.
Placenta Weights
Mean placental weights were slightly lower at 50 mg/kg bw/day for both males and females (only statistically significant in males).
Mean male/female placenta weights were 0.47/0.44, 0.48/0.47, 0.47/0.43 and 0.42/0.40 grams for the vehicle control, 5, 15 and 50 mg/kg bw/day, respectively.
Fetal Morphological Examinations
Note: In order to enter animal numbers into WTDMSTM an adjustment in the numbering was made, for example: animal 1 was reassigned as animal A001, animal 2 as A002 etc. Also numbering of fetuses was changed; Fetus 1 of animal 1 was reassigned as A001-01 etc.
The numbers of fetuses (litters) available for morphological examination were 226 (22), 232 (22), 224 (22) and 232 (22) in Groups 1, 2, 3, and 4, respectively. External examination was done for all fetuses, visceral examination was done for approximately half of the fetuses of all groups, and skeletal examination was done for the other half of fetuses. Nine dams had an early delivery.
External Malformations and Variations
There were no external developmental malformations or variations for any of the fetuses following treatment up to 50 mg/kg/day.
Visceral Malformations and Variations
There were no treatment related effects on visceral morphology following treatment up to 50 mg/kg bw/day.
Visceral malformations were not observed for any of the fetuses and the variations that were noted, namely small supernumerary lobe(s) or appendix of the liver and partially undescended thymus horn(s), occurred at low incidences and/or in the absence of a dose-related trend.
Skeletal Malformations and Variations
There were no treatment related effects on skeletal morphology following treatment up to 50 mg/kg bw/day.
Skeletal malformations were noted in one Group 3 fetus and two control fetuses. The Group 3 fetus (no. A046-04) had bent scapulae and the control fetuses either had fused sternebrae (no. A020-06) or a vertebral centra anomaly (absence of a cervical hemicentrum; no. A007-05). The latter fetus was delivered early. As these malformations were limited to individual fetuses and/or occurred in control fetuses, these were considered chance findings and thus not considered treatment related.
The variation of unossified metacarpals and/or metatarsals occurred in 6.3%, 29.4%, 12.3% and 21.6% of fetuses per litter in Groups 1, 2, 3 and 4, respectively. This group distribution would indicate retarded skeletal ossification in Groups 2 and 4, which corresponds with the slightly lower fetal weights in these groups (fetal weights were 5.1, 4.8, 5.2 and 4.8 grams in Groups 1, 2, 3 and 4, respectively).
However, retarded ossification in Groups 2 and 4 was not unambiguous substantiated by other ossification parameters such as reduced ossification of the skull and unossified sternebrae nos. 5 and 6. Mean litter percentages for these respective findings were 6.1%, 4.3%, 11.6% and 6.5%, and 0.6%, 5.9%, 0.0% and 0.8% per litter in Groups 1, 2, 3 and 4, respectively. Therefore, and due to absence of a dose-relationship, the finding unossified metacarpals and/or metatarsals was not considered to be related to treatment.
The variation of 14th full ribs was observed in 11.7%, 10.8%, 10.0% and 3.5% of fetuses per litter in Groups 1, 2, 3 and 4, respectively. The mean litter proportion in Group 4 was lower (not statistically significant) than in the other groups, but this was not accompanied by a higher incidence for 14th rudimentary ribs in this group (viz.. 56.0%, 70.4%, 70.1% and 68.5% of fetuses per litter in Groups 1, 2, 3 and 4, respectively). Therefore, and due to absence of statistical significance, the lower litter incidence of 14 full ribs in Group 4 was considered to be a change finding and not treatment related. Remaining skeletal variations were not considered treatment related as they occurred infrequently or were observed in control fetuses only.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
50 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
external malformations
skeletal malformations
visceral malformations

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

For result tables see "Attached background material"

Applicant's summary and conclusion

Conclusions:
Based on the results in this prenatal developmental toxicity study the maternal and developmental No Observed Adverse Effect Level (NOAEL) for 3,3’-iminodi(propylamine) was established as being at least 50 mg/kg bw/day.