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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
Study period:
Oct 2015 - Nov 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
data were derived from draft study report, final report is expected not before March 2017

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Name of test substance (as cited in study report): N,N,N´,N´-Tetramethyl-1,6-hexanediamine
- Batch identification: 000STD77L0 v. 08.12.2014
- CAS No.: 111-18-2
- Purity: 99.7 corr. area-%
- Homogeneity: given
- Physical state/appearance: Liquid / colourless to yellowish, clear
- Storage conditions: Room temperature
- Storage stability: Expiry date: 08 Dec 2016

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 10-12 weeks
- Weight at study initiation: 141.1-187.7 g
- Housing: individual
- Diet (e.g. ad libitum):
- Water (e.g. ad libitum):
- Acclimation period: 6 days (Gestation days 0-6)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
ultrapure water
Details on exposure:
The test substance preparations were administered to the animals once a day orally by gavage, from implantation to one day prior to the expected day of parturition (GD 6 to GD 19), always at approximately the same time in the morning. The animals of the control groups were treated with the vehicle (ultrapure water) in the same way. The volume administered each day was 10 ml/kg body weight. The calculation of the administration volume was based on the most recent individual body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analyses of the test substance preparations were carried out at the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology of BASF SE, Ludwigshafen, Germany.
The stability of the test substance in deionized water at room temperature over a period of 7 days had been verified prior to the start of the study.
Samples of the test substance preparations were sent to the analytical laboratory at the beginning of administration for verification of the concentrations.
Given that the test substance was completely miscible with deionized water, solutions were considered to be homogeneous without further analysis.
Details on mating procedure:
The animals were paired by the breeder and supplied on GD 0 (= detection of vaginal plug/sperm).
Duration of treatment / exposure:
Gestation days 6-19
Frequency of treatment:
once daily
Doses / concentrationsopen allclose all
Dose / conc.:
7.5 mg/kg bw/day (actual dose received)
Dose / conc.:
22.5 mg/kg bw/day (actual dose received)
Dose / conc.:
75 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale:
In a 14 day dose range finder for an OECD TG 422 study, mortality was observed in one male and two females at doses of 150 mg/kg bw/d. Therefore, the OECD TG 422 study was conducted with a high dose of 75 mg/kg bw/d (half-lethal dose). The NOAEL for general toxicity was observed at 22.5 mg/kg bw/d, the NOAEL for reproductive toxicity was 75 mg/kg bw/d. Therefore, the high dose level for this OECD TG 414 study was set at 75 mg/kg bw/d.

Examinations

Maternal examinations:
- CAGE SIDE OBSERVATIONS: at least once daily
- DETAILED CLINICAL OBSERVATIONS: During treatment period (GD 6-19) all rats were checked daily for any signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity before administration as well as within 2 hours and within 5 hours after administration.
- BODY WEIGHT: All animals were weighed on GD 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20. The body weight change of the animals was calculated based on the obtained results. Furthermore, the corrected body weight gain was calculated after terminal sacrifice (terminal body weight on GD 20 minus weight of the unopened uterus minus body weight on GD 6).
- FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): The consumption of food was recorded for the intervals GD 0-1, 1-3, 3-6, 6-8, 8-10, 10-13, 13-15, 15-17, 17-19 and 19-20.
- POST-MORTEM EXAMINATIONS: Sacrifice on gestation day # 20
- ORGANS EXAMINED: uterus, ovaries
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
Statistics:
Simultaneous comparison of all dose groups with the control group using the DUNNETT-test (two-sided) for the hypothesis of equal means was used for the following parameters:
Food consumption, body weight, body weight change, corrected body weight gain (net maternal body weight change), carcass weight, weight of unopened uterus, number of corpora lutea, number of implantations, number of resorptions, number of live fetuses, proportions of preimplantation loss, proportions of postimplantation loss, proportions of resorptions, proportion of live fetuses in each litter, litter mean fetal body weight, litter mean placental weight

Pairwise comparison of each dose group with the control group using FISHER'S EXACT test (onesided) for the hypothesis of equal proportions was used for the following parameters:
Female mortality, females pregnant at terminal sacrifice, number of litters with fetal findings

Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians was used for the following parameters:
Proportions of fetuses with malformations, variations and/or unclassified observations in each litter

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
2 of 25 females in the high dose level group showed transient salivation within 2 hours following gavage, no clinical signs were detected afterwards in any dose group.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The mean body weight (BW) and body weight gain (BWC) of the high-dose rats (75 mg/kg bw/d) were statistically significantly reduced on GD 20 (BW) and on GD 10-13 (BWC). During the treatment period (GD 6-19) the mean body weight gain was 11% below concurrent control value (attaining statistical significance). This effect was considered as test substancerelated.
The BW and the average BWC of the low- and mid-dose dams (7.5 and 22.5 mg/kg bw/d) were generally comparable to the controls throughout the entire study period.

The corrected body weight gain (terminal body weight on GD 20 minus weight of the unopened uterus minus body weight on GD 6) was statistically lower in test group 3 (75 mg/kg bw/d - about 18% below the concurrent control value).
The corrected body weight gain of test groups 1 and 2 (7.5 and 22.5 mg/kg bw/d) revealed no difference of any biological relevance to the concurrent control group.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The mean food consumption of the dams in test group 3 (75 mg/kg bw/d) was statistically significantly reduced on GD 8-10 and GD 13-15. During the treatment period (GD 6-19) the high-dose dams consumed 6% less food in comparison to the concurrent control group. The reduced food consumption was considered as test substance-related.
The mean food consumption of the dams in test groups 1 and 2 (7.5 and 22.5 mg/kg bw/d) was generally comparable to the concurrent control group throughout the whole study period. This includes the statistically significantly decreased food consumption value on GD 13-15 in test group 1.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There occurred one spontaneous finding in individual females of test groups 0 and 2 (0 and 22.5 mg/kg bw/d), i.e. dilated renal pelvis. This finding was detected in one control and one mid-dose female, respectively, and was therefore assessed as incidental. No necropsy findings which could be attributed to the test substance were seen in any dam.
The mean gravid uterus weights of the animals of test groups 1-3 (7.5, 22.5 and 75 mg/kg bw/d) were not influenced by the test substance. The differences between these groups and the concurrent control groups revealed no dose-dependency and were assessed to be without biological relevance.

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in number of pregnant:
no effects observed
Details on maternal toxic effects:
The conception rate reached 96% in the control group (0 mg/kg bw/d) and 100% in the low-, mid- and high-dose groups (7.5, 22.5 and 75 mg/kg bw/d). With these rates, a sufficient number of pregnant females were available for the purpose of the study.
No test substance-related and/or biologically relevant differences between the test groups 0-3 (0, 7.5, 22.5 and 75 mg/kg bw/d) were observed with regard to conception rate, mean number of corpora lutea and implantation sites or values calculated for the pre- and the postimplantation losses, the number of resorptions as well as viable fetuses. All observed differences were considered to reflect the normal range of fluctuations for animals of this strain and age.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
22.5 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
clinical signs

Maternal abnormalities

Abnormalities:
effects observed, treatment-related
Localisation:
not specified
Description (incidence and severity):
Evidence of maternal toxicity at the high-dose level of 75 mg/kg bw/d included decreased food consumption as well as decreased body weight/body weight gain.

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
No changes were observed in the number of viable fetuses.
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
no effects regarding the number of viable fetuses or their weight was observed.
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
One fetus, each, in test groups 1 and 3 (7.5 and 75 mg/kg bw/d) had external malformations. In one case, these external malformations, i.e. absent snout and anophthalmia, were associated with multiple skeletal malformations. None of these malformations is considered to be related to the treatment.
The total incidence of external malformations in treated animals did not differ significantly from the concurrent control group and was covered by the historical control data.
No external variations were recorded. No external unclassified observations were recorded.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Some skeletal malformations were detected in all test groups including the control (test groups 0-3; 0, 7.5, 22.5 and 75 mg/kg bw/d). One high-dose fetus with multiple skeletal malformations had associated external findings. The incidences of these malformations were neither statistically significantly different from control nor dose dependent and therefore not considered biologically relevant.
For all test groups, skeletal variations of different bone structures were observed, with or without effects on corresponding cartilages. The observed skeletal variations were related to several parts of fetal skeleton and appeared without a relation to dosing. The overall incidences of skeletal variations were covered by the historical control data.
Additionally, some isolated cartilage findings without impact on the respective bone structures, which were designated as unclassified cartilage observations, occurred in all test groups. The observed unclassified cartilage findings were related to the skull, the sternum and ribs and did not show any relation to dosing. However, the incidence of ‘branched rib cartilage’ was significantly increased in test group 2 (22.5 mg/kg bw/d). As this finding both showed no dose-dependency and could be found in the historical control data at a comparable frequency, it was assessed to be without biological relevance.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
One soft tissue malformation (situs inversus) was recorded for one high-dose fetus (75 mg/kg bw/d), which is present in the historical control data and thus considered as an incidental finding. There were no further soft tissue malformations in any of the other test groups. The overall incidences of soft tissue malformations were comparable to those found in the historical control data.
Two soft tissue variations were detected in all test groups (0, 7.5, 22.5 and 75 mg/kg bw/d), i.e. dilated renal pelvis and dilated ureter. These variations were neither statistically significantly different from control nor dose-dependent and therefore, not considered biologically relevant.
No unclassified soft tissue observations were recorded.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 75 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

Concentration control analysis of the test substance preparations

The results of the analysis of the aqueous test substance preparations confirmed the correctness of the prepared concentrations. The analytical values of the samples corresponded to the expected values within the limits of the analytical method, i.e. were always above 90% and below 110% of the nominal concentrations.

Applicant's summary and conclusion

Conclusions:
Maternal toxicity was observed at the highest dose tested . No substance-related developmental toxicity was evident at any dose level.
Executive summary:

N,N,N´,N´-Tetramethyl-1,6-hexanediamine was tested for its prenatal developmental toxicity in Wistar rats. The test substance was administered as an aqueous preparation to groups of 25 time-mated female Wistar rats by gavage at doses of 7.5, 22.5 and 75 mg/kg body weight/day (mg/kg bw/d) on gestation days (GD) 6 through 19. One control group, consisting of 25 females, was dosed with the vehicle (ultrapure water) in parallel. A standard dose volume of 10 mL/kg body weight was used for each test group. Food consumption and body weights of the animals were recorded regularly throughout the study period. The state of health of the animals was checked each day. On GD 20, all females were sacrificed by decapitation (under isoflurane anesthesia) and assessed by gross pathology (including weight determinations of the unopened uterus and the placentae). For each dam, corpora lutea were counted and number and distribution of implantation sites (differentiated between resorptions, live and dead fetuses) were determined. The fetuses were removed from the uterus, sexed, weighed and further investigated for external findings. Thereafter, one half of the fetuses of each litter were examined for soft tissue findings and the remaining fetuses for skeletal (inclusive cartilage) findings.

At terminal sacrifice on GD 20, 24 - 25 females per group had implantation sites.

The following test substance-related, adverse effects/findings were noted:

Test group 3 (75 mg/kg bw/d):

Dams

- Reduced food consumption during treatment (GD 6-19), overall about 6% below control

- Reduced body weight on GD 20, about 4% below control

- Reduced body weight gain during treatment (GD 6-19), about 11% (gross) or 18% (net) below control

Fetuses

- No test substance-related adverse effects on fetuses.

Test group 2 (22.5 mg/kg bw/d):

- No test substance-related adverse effects on dams, gestational parameters or fetuses.

Test group 1 (7.5 mg/kg bw/d):

- No test substance-related adverse effects on dams, gestational parameters or fetuses.

Under the conditions of this prenatal developmental toxicity study, the oral administration of N,N,N´,N´-Tetramethyl-1,6-hexanediamine to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) caused evidence of maternal toxicity at the high-dose level of 75 mg/kg bw/d, such as decreased food consumption as well as decreased body weight/body weight gain. Thus, the no observed adverse effect level (NOAEL) for maternal toxicity is 22.5 mg/kg bw/d. No developmental toxicity was evident at any dose level. In conclusion, the no observed adverse effect level (NOAEL) for prenatal developmental toxicity is 75 mg/kg bw/d.