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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:
From June 10, 2013 to July 18, 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): N,N,N’,N’,N’-Pentamethyl-N-C16-18 (even numbered) C18 unsat.-alkyl-1,3-propanediammonium chloride (Pure), solvent free sample
- Substance type: organic
- Physical state: Light yellow waxy powder
- Purity: 96.0 %m/m
- Lot/batch No.: 20130125JB
- Expiration date of the lot/batch: 25 January 2023
- Storage condition of test material: At room temperature in the dark under nitrogen
- Purity/composition correction factor required: No
- Hygroscopic: Yes, store in well-sealed container
- Volatile: No
- Reactivity: Reactive to oxygen
- Test substance handling: Flush container with nitrogen after handling
- Stability at higher temperatures: Yes, maximum temperature: 80°C, maximum duration: 60 min
- Stability in water: Stability for at least 5 hours at room temperature under normal laboratory light conditions and 8 days in the refrigerator in the dark under nitrogen is confirmed over the concentration range 1 to 10 mg/mL.

Test animals

Species:
rat
Strain:
other: Wistar (Han)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Sex: females, nulliparous and nonpregnant females and untreated animals were used at initiation of the study.
- Age at study initiation: Approximately 12 weeks.
- Weight at study initiation: 206-258g.
- Fasting period before study: no
- Housing:
Acclimatization: Animals were housed in groups of 5 animals/sex/cage in Macrolon cages.
Mating: Females were caged together with stock males on a one-to-one-basis or two-to-one-basis in Macrolon cages.
Post-mating: Females were individually housed in Macrolon cages.
General: Sterilised sawdust as bedding material and paper as cage enrichment were supplied.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days

Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, approximately 15 room air changes/hour, and a 12-hour light/12-hour dark cycle. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.

IN-LIFE DATES: From: 10 June - 18 July 2013

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
- Method of formulation: Formulations (w/w) were prepared at least once every 8 days and were homogenized to a visually acceptable level. No adjustment was made for specific gravity/density of the test substance, vehicle, and/or formulation. No correction was made for the purity/composition of the test substance.
- Storage conditions of formulations: At ambient temperature when prepared 5 hour before dosing. When prepared more than 5 hours before dosing, in the refrigerator under nitrogen.
- Dose volume: 10 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%.
Details on mating procedure:
- M/F ratio per cage: 1/1 or 2/1 (one or two females were cohabitated with one stockmale).
- Age at mating of the animals in the study: Approximately 12 weeks
- Length of cohabitation: until sufficient mated females had been obtained for each dose group.
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage and/or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated.
Duration of treatment / exposure:
Duration of treatment: From Days 6 to 19 post-coitum, inclusive.
Frequency of treatment:
Once daily for 7 d/w.
Duration of test:
Administration of test substance was 14 days.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
22
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale: Dose levels were selected based on results of the dose range finding study (Project 502145)

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS
- Time schedule: At least twice daily. Animals showing pain, distress or discomfort, which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ (2000)7).

DETAILED CLINICAL OBSERVATIONS
- Time schedule: At least once daily from Day 0 post-coitum onwards up to the day prior to necropsy. The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity.

BODY WEIGHT
- Time schedule for examinations: Days 0, 3, 6, 9, 12, 15, 17 and 20 post-coitum.

FOOD CONSUMPTION
- Time schedule for examinations: Days 0-3, 3-6, 6-9, 9-12, 12-15, 15-17 and 17-20 post-coitum.

WATER CONSUMPTION
No. Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

GENERAL REPRODUCTION DATA
- Mating date and confirmation of pregnancy were recorded.
- Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of these females was examined to detect signs of abortion or premature birth.

POST-MORTEM EXAMINATIONS
- Sacrifice on gestation day 20, animals surviving to scheduled necropsy and all moribund animals were deeply anaesthetised and subsequently exsanguinated.
- Organs examined: according to guidelines
Ovaries and uterine content:
Each ovary and uterine horn of animals surviving to planned necropsy were dissected and examined
as quickly as possible to determine:
- The number of corpora lutea.
- The weight of the (gravid) uterus.
- The number and distribution of live and dead fetuses.
- The number and distribution of embryo-fetal deaths.
- The weight of each fetus.
- The sex of each fetus from the ano-genital distance (during necropsy) and also from gonadal
inspections (during further fetal examination).
- Externally visible macroscopic fetal abnormalities.
Fetal examinations:
External, visceral and skeletal fetal findings were recorded as developmental variations or malformations.
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all 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 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 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 postimplantation 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 (Ref. 10) 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. 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:
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 x 100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed

Maternal developmental toxicity

Details on maternal toxic effects:
Details on maternal toxic effects:
MORTALITY: At 100 mg/kg, one female had to be killed in extremis on Day 8 post-coitum. This animal showed lethargy (severe), flat posture, piloerection and hypothermia. At necropsy, stomach abnormalities (reddish glandular mucosa, enlarged stomach, granular red-brown contents) were noted and 16 implantations in utero.

CLINICAL SIGNS: Treatment related clinical signs were noted at 100 mg/kg, and consisted of hunched posture (two females for 6-8 days), rales (two females for 3 days), piloerection (six females for 1-4 days), and lean appearance (two females for 6 days). In addition, the female that was euthanized in extremis showed lethargy (severe), flat posture, piloerection and hypothermia. Salivation seen after dosing for one female at 10 mg/kg and sixteen females at 100 mg/kg was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). Alopecia was noted for single animals of all groups. This finding occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth.

BODY WEIGHTS: At 100 mg/kg, body weights and body weight gain were decreased during treatment (statistically significant on most occasions). Additionally, for (gravid) uterus corrected weight gain was statistically significantly decreased. Almost all females at this dose showed a body weight loss on Day 9 post-coitum showing a clear recovery during the remainder of the treatment period for all but two females. These two females showed early resorptions only at necropsy. At 10 mg/kg, five females showed reduced body weight gains when compared to controls. This was considered due to their small litter sizes, and not considered treatment related.

FOOD CONSUMPTION: At 100 mg/kg, absolute and relative food consumption was lower during treatment (statistically significant on most occasions). The main effect was noted during Days 6-9 post-coitum and showed some recovery during the rest of treatment. For two females food consumption remained very low on Days 9-15 postcoitum. At 10 and 30 mg/kg, no toxicologically relevant changes in food consumption before or after allowance for body weight were noted.

MACROSCOPIC EXAMINATION: At 100 mg/kg, there were two females that showed emaciation. In addition, the female that was euthanized in extremis showed stomach abnormalities (i.e. reddish glandular mucosa, enlarged stomach, granular red-brown contents).
The incidence of other incidental findings (alopecia and diaphragmatic hernia of the liver) was within the background range of findings that are encountered among rats of this age and strain, and did not show a dose-related incidence trend.

MATERNAL PREGNANCY DATA: There were 22, 21, 21, and 22 pregnant females in the control, 10, 30 and 100 mg/kg groups, respectively, with 22, 19, 21 and 19 litters available for evaluation. At 100 mg/kg, there were two females that had 100% post-implantation loss, consisting of early resorptions. This resulted in an high mean value for early resorptions (not statistically significant). This was considered a secondary test substance effect caused by the very low food consumption and body weight loss during post-coitum. At 10 mg/kg, the lower number of viable fetuses and implantation sites per litter were mainly due to lower number of corpora lutea (for 4/6 rats with lower number of viable fetuses). As this occurred before start of treatment, it was not considered a treatment related effect. In addition at this dose, two females showed a high rate of early resorptions and low rate of viable fetuses. These values remained within normal limits, and as this was not noted at 30 mg/kg, it was not considered toxicologically relevant.

Effect levels (maternal animals)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Key result
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:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
FETAL FINDINGS
LITTER SIZE
No treatment related effect on litter size was noted up to 100 mg/kg. The mean number of viable fetuses per litter was 12.3, 9.1, 11.9 and 11.7 in the control, 10, 30 and 100 mg/kg groups, respectively. The lower mean value for viable fetuses per litter at 10 mg/kg was mainly due to lower number of corpora lutea. As this occurred before start of treatment, it was not considered a treatment related effect.

SEX RATIO
There were no treatment-related effects on the sex ratio of the fetuses.

FETAL BODY WEIGHT
Fetal body weights were unaffected for treatment up to 100 mg/kg. Body weights of fetuses (sexes combined) were 3.5, 3.5, 3.5 and 3.4 grams for the control, 10, 30 and 100 mg/kg groups, respectively.

FETAL MORPHOLOGICAL EXAMINATIONS
EXTERNAL MALFORMATIONS AND VARIATIONS
There were no treatment related effects on fetal external morphology. One external malformation was noted in this study. One fetus at 10 mg/kg had a small lower jaw and due to singly occurrence in the low dose group, this finding was not considered to be treatment related. No external malformations and developmental variations were observed in any of the other fetuses, thus it was considered that test substance treatment at dose levels up to 100 mg/kg had no effect on fetal external morphology.

VISCERAL MALFORMATIONS
There were no treatment related effects on fetal visceral morphology. Visceral malformations were observed in one fetus at 10 mg/kg and two fetuses from two litters at 100 mg/kg. The malformations that occurred in the high dose group were situs inversus and absent eye. Both these findings were seen previously in historical control fetuses and due to singly occurrence they were not considered to be treatment related. At 10 mg/kg, the affected fetus (the one with the small jaw externally) had a multiple cardiovascular abnormality characterized by malpositioned right subclavian artery (originating from the pulmonary trunk), narrow pulmonary trunk, absent ductus arteriosus and ventricular septum defect. This is an unusual abnormality (of the individual malformations, only the ventricular septum defect was seen in historical control fetuses), however, as it was noted in one fetus of the low dose group, it was not considered to be a test substance effect.

VISCERAL VARIATIONS
Visceral variations occurred in 9.8%, 6.9%, 13.1% and 13.1% of fetuses per litter in the control, 10, 30 and 100 mg/kg groups, respectively. Those observed in the test substance treated groups were small supernumerary liver lobe, liver appendix, pale spleen, dilated ureter, convoluted ureter, supernumerary artery, small thyroid and partially undescended thymus horn. These variations were not considered to be treatment related, because they occurred at similar frequencies in the control group, occurred infrequently, occurred without dose relationship and/or occurred at frequencies within the historical control range.

SKELETAL MALFORMATIONS
There were no treatment related effects on fetal skeletal morphology. Skeletal malformations were observed in 3(3) fetuses (litters) of both the control and 100 mg/kg group. In the high dose group, two fetuses from two litters had a vertebral anomaly with or without associated rib anomaly. The one fetus with situs inversus viscerally, missed a lumbar centrum and arch and the finding in the other fetus was characterized by absence of a hemicentrum, arch and head of a rib. The latter fetus also had another skeletal malformation, namely severely malaligned sternebrae. The third skeletally malformed fetus in this group, had a rib anomaly (fusion of ribs). None of the above malformations were noted in concurrent controls, but two out of three (vertebral anomaly with or without associated rib anomaly, and severely malaligned sternebrae) were seen previously within the range of historical controls. Therefore, and due to the single occurrence of the rib anomaly, all skeletal malformations noted at 100 mg/kg were not considered to be treatment related.
Other skeletal malformations in this study only affected control fetuses. Bent limb bones in one fetus, malpositioned metatarsals in one fetus and costal cartilage anomaly in one fetus. Due to occurrence in the control group, these malformations were not related to treatment.

SKELETAL VARIATIONS
Skeletal variations occurred in 89.7% and 86.2% of fetuses per litter in the control and 100 mg/kg group, respectively. Of the variations at 100 mg/kg, statistically significantly decreased mean litter proportions were noted, when compared to concurrent control values, for reduced ossification of the skull (2.8% versus 7.5% per litter) and bent ribs (1.0% versus 9.9% per litter). However, for both groups, the incidences of reduced ossification of the skull and bent ribs were within their historical control ranges (2.7-21.2% per litter and 1.6-27.4% per litter, respectively). Consequently, these findings were considered not to be treatment related. Moreover, the decrease in the number of fetuses with reduced ossification of the skull and bent ribs at 100 mg/kg indicates that a greater proportion of fetuses in this group had complete ossification of the skull bones and ribs that were not bent, and this is not considered to be an adverse developmental effect.
Other variations noted in the high dose group were 14th rudimentary rib, 14th full rib, 7th cervical rudimentary rib, unossified sternebrae nos. 5 and/or 6, unossified sternebrae nos. 1,2,3 and/or 4, entire sternum unossified, slightly to moderately malaligned sternebrae, partially fused zygomatic arch, unossified hyoid, ossified vertebral centrum no. 1, reduced ossification of vertebral centra, reduced ossification of vertebral arches, unossified vertebral centra, unossified or reduced ossification of pubis, caudal shift of pelvic girdle and unossified metacarpals and/or metatarsals. These variations were not considered to be treatment related, because they occurred at similar frequencies in the control group, occurred infrequently and/or occurred at frequencies within the historical control range.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No abnormalities

Fetal abnormalities

Key result
Abnormalities:
not specified

Overall developmental toxicity

Key result
Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Under study conditions, prenatal developmental toxicity maternal No Observed Adverse Effect Level (NOAEL) for test substance, solvent free sample was established to be 30 mg/kg bw/day. The developmental NOAEL is considered to be at least 100 mg/kg/ kg bw/day.
Executive summary:

A study was conducted to determine the developmental toxicity / teratogenicity of the test substance, N,N,N',N',N''-Pentamethyl-N-C16-18 (even numbered) and C18 unsat.-alkyl-1,3-propanediammonium chloride, according to a method similar to OECD Guideline 414, in compliance with GLP. Rats were exposed to the test substance once daily by oral gavage from Days 6 to 19 post-coitum at doses of 10, 30 and 100 mg/kg bw/day. Accuracy and homogeneity of formulations were demonstrated by analyses. On Day 8 post-coitum, one female had to be euthanized in extremis. This animal showed lethargy (severe), flat posture, piloerection and hypothermia. At necropsy, stomach abnormalities (reddish glandular mucosa, enlarged stomach, granular red-brown contents) were noted. Treatment-related clinical signs noted for eight surviving animals at 100 mg/kg bw/day consisted of hunched posture, piloerection and/or lean appearance. At 100 mg/kg bw/day, almost all females showed a body weight loss on Day 9 post-coitum with recovery during the remainder of the treatment period for all but two females; these two females showed early resorptions only at necropsy. For (gravid) uterus, corrected weight gain was also statistically significantly decreased. No substance-related toxicity was observed in the groups at 10 and 30 mg/kg bw/ day. No developmental toxicity was observed at any of the dose levels. Under the study conditions, the maternal systemic toxicity No Observed Adverse Effect Level (NOAEL) of the test substance was determined to be 30 mg/kg bw/day and the developmental toxicity NOAEL was considered to be 100 mg/kg/ kg bw/day (Wil research, 2013).