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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:
June - November 2016
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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
Jan 2001
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
May 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
August 1998
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
- Name of test material (as cited in study report): di-C16-C18 (evennumbered) alkyl tripropylenetetramine
- Substance type: Viscous liquid
- Physical state: Liquid
- Storage condition of test material: At room temperature container flushed with nitrogen
- Batch/Lot number: B1; batch inspection lot no.:890000394200
- Expiration date of the lot/batch: 22 October 2017
- Purity: 100% (UVCB substance)
- pH: 8.5-10.5 (1% solution)
- specific gravity: 0.83 at 60°C
Specific details on test material used for the study:
Stability in corn oil: stable for at least 5 hours

Test animals

Species:
rat
Strain:
other: Crl:WI(Han)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 10-14 weeks
- Weight at study initiation: 155 - 254 g
- Fasting period before study: no
- Housing: individually housed in Macrolon plastic cages. Sterilized sawdust as bedding material and paper as cage enrichment/nesting material.
- Diet: free access to pelleted rodent diet.
- Water: free access to tap-water.
- Acclimation period: at least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.6 – 21.9
- Humidity (%): 51 - 80
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 07 June 2016 To: 30 June 2016

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Formulations (w/v) were prepared daily within 5 hours prior to dosing and were homogenized to a visually acceptable level. No correction was made for the purity/composition of the test item.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at Charles River Den Bosch.
- Amount of vehicle (if gavage): 5 ml/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of dose preparations were taken on a single occasion during the treatment phase (13 June 2016), and were stored and dispatched on dry ice to the test site for formulation analysis. Samples of formulations were taken for accuracy (all groups) and homogeneity (100 and 1000 mg/kg) of the preparations.
Stability of formulations over 5 hours at room temperature under normal laboratory light conditions (concentration range 20-200 mg/mL) was determined in Test Facility Study No. 509313.
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.
Frequency of treatment:
once daily for 7 days per week
Duration of test:
until day 21 post-coitum
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
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 a combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test of di-C16-C18 (evennumbered) alkyl tripropylenetetramine in rats by oral gavage (OECD Test Guideline 422). In this study, the rats received the test item at dose levels of 0, 100, 300 and 1000 mg/kg in corn oil. No relevant toxicity was observed up to 1000 mg/kg.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at 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: Yes
- Time schedule: days 2-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post-coitum

WATER CONSUMPTION: 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 gestation day 21
- Organs examined: external, thoracic and abdominal examination, with special attention being paid to the reproductive organs
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:
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 group.
• 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 Mann Whitney test was used to compare mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and post-implantation loss, and sex distribution.
• Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test was used to compare the compound-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. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations might be rounded off before printing. Therefore, two groups might display the same printed means for a given parameter, yet display different test statistics values.
No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and post-implantation loss.
Indices:
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
Viable fetuses affected/litter (%) = ((number of viable fetuses affected/litter)/number of viable fetuses/litter) x 100
Historical control data:
see under "any other information on materials and methods".

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No treatment related clinical signs were noted up to and including 300 mg/kg.
At 1000 mg/kg, signs of ill health were observed in two females, shortly before they were found dead on Days 14 or 15 post-coitum, respectively. On Day 13 post-coitum lethargy, uncoordinated movement and piloerection were noted before dosing of one female. For the other female hunched posture, piloerection and/or rales were noted between Days 12 and 14 post-coitum. Furthermore, piloerection was noted in a third female between Days 12 and 14 post-coitum. No other treatment related clinical signs were observed in this dose group.
Mortality:
mortality observed, treatment-related
Description (incidence):
Two unscheduled deaths occurred in the 1000 mg/kg group. One female was found dead on Day 14 post-coitum. Additionally, another female was found dead on Day 15 post-coitum, shortly after the decision was made to euthanize the animal for animal welfare reasons. This female showed approximately 20% body weight loss between Days 9 and 15 post-coitum, combined with limited to no food consumption in this period. Both females showed signs of ill health shortly before they were found dead.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Overall, body weight, body weight gain and body weight gain corrected for weight of the gravid uterus were unaffected by treatment up to 1000 mg/kg.
One female treated at 1000 mg/kg showed a significant body weight loss (i.e. 20%), which led to the decision to euthanize her on Day 15 post-coitum. Additionally, in two animals, a slight body weight loss was noted between Days 18 and 21 post-coitum (one female in the 300 and one in the 1000 mg/kg group). Body weight corrected for weight of the uterus revealed a corrected body weight loss for these individual females. In addition, fetal weights in their litters were also low compared to other litters in these groups and the vehicle control group. As these effects were limited to two females only, these were considered incidental and not toxicologically relevant.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
No treatment related effects on food consumption were noted in any of the groups.
The statistically significant increase in absolute and relative food consumption in the 1000 mg/kg group between Days 18 and 21 post-coitum, was considered incidental and not toxicologically relevant.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
At necropsy, no treatment related macroscopic findings were noted in any of the groups.
The incidence of macroscopic findings among control and treated animals was within the background range of findings that are encountered among rats of this age and strain. These necropsy findings were therefore considered to be of no toxicological relevance.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined

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 pregnancy duration:
not examined
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined
Changes in number of pregnant:
no effects observed
Details on maternal toxic effects:
Maternal pregnancy data:
One female in the 300 mg/kg group had only one dead fetus and an early resorption. As this was a single occurrence in the mid dose group only, this was considered not to be treatment related. All other females at scheduled necropsy were pregnant and had litters with viable fetuses.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
mortality

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
There was no treatment related effect on mean fetal body weights. Mean combined (male and female) fetal body weights were 5.4, 5.3, 5.3 and 5.4 grams for the vehicle control, 100, 300 and 1000 mg/kg groups, respectively.
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
Description (incidence and severity):
Mean litter sizes were 10.4, 9.5, 10.2 and 10.3 for the control, 100, 300 and 1000 mg/kg groups, respectively.
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The only malformation that occurred in this study was observed in 100 mg/kg fetus A031-06. This fetus had a small lower jaw for which at skeletal examination also the mandibles appeared to be fused with presence of one instead of two sockets. In historical control fetuses, a small or absent lower jaw was seen previously (skeletally) and because it occurred singly at the low dose level, it was considered a chance finding.
External variations were not seen in any group.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Only two different malformations were revealed at skeletal examination. Bent limb bones were observed in one 1000 mg/kg fetus and two 300 mg/kg fetuses, of which the latter also had a vertebral anomaly with associated rib anomaly and
several visceral malformations. The low incidence and fact that these are the two most common skeletal malformations among historical controls do not suggest any treatment relationship.
Skeletal variations that were noted occurred in the absence of a dose-related incidence trend, infrequently and/or at frequencies that were within the range of available historical control data. Therefore, they were not considered treatment related.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The malformation “absent and/or small eyes” was noted at soft tissue cephalic examination in 3 (2) and 6 (3) fetuses (litters) in the low and mid dose groups, respectively, and not in the control and high dose group. Previously among 2061 (295) historical control fetuses (litters), this eye finding was seen in 3 (3) fetuses (litters).
The group distribution is remarkable, but the lack of cases in the high dose group indicates that it is not a test item related effect. Furthermore, the presence of small eyes in two or more litter mates more strongly indicates a genetic origin, rather than another cause. Therefore, the eye findings in the low and mid dose groups were not considered to be treatment related.
Remaining visceral malformations were observed in one 300 mg/kg fetus (situs inversus, abnormal lobation of the lung, narrow pulmonary trunk and ventricular septum defect), one 100 mg/kg fetus (abnormal lobation of the lung and right-sided aortic arch) and one control fetus (situs inversus). These were considered to be chance findings, because they occurred singly and also in a control fetus.
The variations that were noted in this study occurred at low incidences and in the absence of a dose-related trend and therefore were not considered to be treatment related.
Details on embryotoxic / teratogenic effects:
The numbers of fetuses (litters) available for morphological examination were 228 (22), 209 (22), 225 (21) and 206 (20) in the control, 100, 300 and 1000 mg/kg groups, respectively.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 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

Developmental effects observed:
no

Any other information on results incl. tables

Formulation analysis: No test item was detected in the control group formulations. The concentrations analysed in the formulations of 100, 300 and 1000 mg/kg were in agreement with

the target concentrations i.e. mean accuracies between 85% and 115% (87.7 -101.3%).

The formulations 100 and 1000 mg/kg prepared were homogeneous with a coefficient of variation of 6.7 and 1.0%, respectively (i.e. coefficient of variation ≤ 10%).

Developmental toxicity table:

Dose level (mg/kg bw/day)

0

100

300

1000

Pregnant/total dams

22/22

22/22

22/22

22/22a

-early resorptions

-late resorptions

(% per litter)

2.4

0.4

5.0

0.0

5.6

0.0

4.0

0.0

Dams with abortion, early deliveries, stillbirths and/or resorptions only

Dams with dead fetuses

0

 

 

1

0

 

 

0

0

 

 

1

0

 

 

0

Pre-implantation loss (number and percent)

21 (7.7%)

34 (13.9%)

21 (8.8%)

17 (6.8%)

Post-implantation loss (number and percent)

8 (3.3%)

12 (5.0%)

10 (7.9%)

8 (4.0%)

Body weight on day 21

317

313

315

321

Body weight gain day 6-21 (%)

49

46

47

48

Gravid uterine weight (g)

74.5

68.0

72.4

74.3

Mean live offspring (number)

10.4

9.5

10.2

10.3

Live offspring (percent)

96.7

95.0

92.1

96.0

Mean fetal body weight males (g)

5.5

5.4

5.4

5.6

Mean fetal body weight females

5.3

5.2

5.1

5.3

Mean fetal body weight (sexes combined)

5.4

5.3

5.3

5.4

Malformations (including runts) number and percent of fetuses per litter

 

 

1 (0.8%)

 

 

5 (3.3%)

 

 

8 (6.7%)

 

 

1 (0.8%)

Variations (% per litter)

-external

-soft tissue

-skeletal

 

0

9.9

70.1

 

0

10.7

77.5

 

0

9.1

73.4

 

0

8.8

82.4

atwo dams died

Applicant's summary and conclusion

Conclusions:
Based on the results in this prenatal developmental toxicity study the maternal NOAEL for di-C16-C18 (evennumbered) alkyl tripropylenetetramine was established as being 300 mg/kg. The developmental NOAEL for di-C16-C18 (evennumbered) alkyl tripropylenetetramine was established as being at least 1000 mg/kg.
Executive summary:

In a prenatal developmental toxicity study in rats (22/group) performed according to OECD 414 and GLP guidelines, 0, 100, 300 or 1000 mg/kg bw/d of di-C16-C18 (evennumbered) alkyl tripropylenetetramine was administered by oral gavage from days 6 to 20 post-coitum.

At 1000 mg/kg, two unscheduled deaths occurred on Days 14 and 15 post-coitum. Both females showed signs of ill health 1 resp. three days before they died, including piloerection, hunched posture, lethargy, uncoordinated movements and/or rales. For one female of these females severe body weight loss was observed between Days 9 and 15 post-coitum. Although these effects were limited to these two females, a treatment related effect could not be excluded. No maternal toxicity was observed in the 100 and 300 mg/kg bw/d groups.

 

No developmental toxicity was observed in the 100, 300 and 1000 mg/kg bw/d groups.

Based on the results in this prenatal developmental toxicity study the maternal NOAEL for di-C16-C18 (evennumbered) alkyl tripropylenetetramine was established as being 300 mg/kg bw/d.

Considering that no death or symptoms were seen in the earlier OECD 422 covering an even more extended duration of dosing at 1000 mg/kg bw/day than in this OECD 414, and that additionally in the current study basically no clinical signs except piloerection on 3 days in 1 HD animal were observed (besides for the two animals that died), it is conceivable that the deaths are coincidental. However, without a clear cause of death that is not substance related (eg gavage error) the arguments are not considered sufficient to change the conclusions.

 

The developmental NOAEL for di-C16-C18 (evennumbered) alkyl tripropylenetetramine was established as being at least 1000 mg/kg bw/d. The substance does not have to be classified for developmental toxicity according to Regulation 1272/2008 and amendments.