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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:
21 June 2015 - 09 July 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was performed according to OECD/EC guidelines and GLP prinicples.

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:
January 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)
Remarks:
dd 3 November 2015
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): C12-14-alkylmorpholine
- Substance type: Organic
- Physical state: light yellow liquid
- Storage condition of test material: At room temperature
- Specific gravity/ density: Approximately 0.87 at 25°C

Test animals

Species:
rat
Strain:
other: RccHan:WIST
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories B.V., Venray, The Netherlands
- Age at study initiation: 10-14 weeks
- Weight at study initiation: 187-285g
- Fasting period before study: No
- Housing: Individual housing in Macrolon cages (MIII type)
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SNIFF®Spezialdiäten GMBH, Soest, Germany)
- Water: Free access to tap water
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0-21.6
- Humidity (%): 47-78
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 21 June 2015 To: - 09 July 2015

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for specific gravity of the test substance (0.87) and vehicle (0.92). No correction was made for the purity/ composition of the test substance.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations at testing facility and on information provided by the sponsor.
- Amount of vehicle (if gavage): 5 mL/ kg body weight
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on a single occasion during the treatment phase, according to a validated method. 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 varation was ≤10%.
Details on mating procedure:
- Impregnation procedure: purchased timed pregnant (proof: vaginal plug (day 0 of gestation))
Duration of treatment / exposure:
15 days (days 6 to 20 post coitum, inclusive)
Frequency of treatment:
once daily
Duration of test:
until day 21 post-coitum
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 10, 30, 100 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
22 (females only, main study);
9 (females only, dose range finding study)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: dose levels were selected based on results of dose range finding study (details below).
- Assignment rationale: randomized by body weight, females mated on the same day in the same subgroup.

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 1 post coitum up to the day prior to necropsy

BODY WEIGHT: Yes
- Time schedule for examinations: days 1, 3, 6, 9, 12, 15, 18 and 21 post coitum

FOOD CONSUMPTION :
- Time schedule: days 1-3, 3-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post coitum

WATER CONSUMPTION: Yes
- Subjective appraisal was maintained during the study (no quantitative investigation as no treatment related effect was expected)

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21 post coitum
- External, thoracic and abdominal examination

OTHER:
Each ovary and uterine horn of animals surviving to planned necropsy was dissected and examined throughfully.
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
- Other: weight of each fetus, sex of each fetus from the ano-genital distance and also from gonadal inspections and externally visible macroscopic fetal abnormalities
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter (incl. confirmation of sex by internal examination)
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 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). Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off. Therefore, two groups may 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

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
One rat treated at 100 mg/kg bw/day was killed in extremis on day 20 post coitum, after a rapidly declining health status from day 19 post coitum onwards (incl. clinical signs like hunched posture on day 15 post coitum). No further mortality occurred at any dose level. No clinical signs were noted amongst surviving rats that were considered related to the treatment (only incidental observations). Body weight gain (with and without correction for uterus weight) appeared slightly lower in rats treated at 100 mg/kg bw/day compared to controls (35% vs 41% body weight gain in controls (absolute), 6.5% vs 9.6% body weight gain in controls (with correction for uterus weight)), this was primarily related to three animals with marked body weight loss (not statistically significant). Relative food consumption was reduced on day 21 post coitum for rats dosed at 100 mg/kg bw/day, which was merely caused by the three rats that suffered weight loss. Body weight gain with and without correction for uterus weight at 10 and 30 mg/kg bw/day was unaffected by the treatment, food consumption (absolute and relative) in these groups was similar to control group. Macroscopic examination revealed effects in females treated at 100 mg/kg bw/day. The female killed in extremis showed black-brown foci on the glandular stomach with irregular surface of the forestomach, enlarged and dark-red discoloured adrenal glands, and a reduced size of the thymus. Irregular surface of the stomach was also recorded for 3 additional females in this group, with one of these females showing black-brown foci on the glandular mucosa of the stomach. Another female in this group showed yellowish contents of the gastro-intestinal tract, a thickened duodenal wall with gelatinous contents, black-brown foci on the ileum with gelatinous contents and reduced size of the spleen. None of the animals in the other groups showed test substance related macroscopic effects.

There were no effects on the number of pregnant females, corpora lutea, implantation sites or pre- or post-implantation loss noted up to 100 mg/kg bw/day, a summary of the outcome is included in tabular form.
Examination of cage debris of pregnant females revealed ni signs of abortion or premature birth.

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:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
There were no treatment-related effects on litter size for any group. Overview in tabular format of the results is included below. Mean litter sizes were 11.1, 11.0, 10.9 and 10.5 fetuses/litter for respectively the control, 10, 30 and 100 mg/kg bw/day groups. The male:female sex ratio and fetal body weight was unaffected by the treatment up to and including 100 mg/kg bw/day.
Three fetuses at 100 mg/kg bw/day and one at 30 mg/kg bw/day were external malformed, which consisted at 100 mg/kg bw/day of a hind limb rotated inwards (no skeletal cause found), a small lower jaw and cleft palate and a fetus with a small tail (only three caudal vertebrae present). At 30 mg/kg bw/day, one fetus had a meningocele (skeletally confirmed). Because of the single occurrences and diverse nature of these abnormalities, they were not considered treatment related.
Visceral malformations were found in one control, one fetus at 30 mg/kg bw/day and in 4 fetuses (in 4 litters) at 100 mg/kg bw/day. Two fetuses at 100 mg/kg bw/day and one control fetus had external hydrocephaly. One fetus at high dose had a small eye and internal hydrocephaly. The fourth affected fetus at high dose had a retroesophageal aortic arch. The affected fetus at 30 mg/kg bw/day was found to have situs inversus. Due to the incidental nature and previous observation of these malformations in historical control fetuses, these malformations were not considered adverse.
Distended urinary bladder and absent and/or small renal papillae occurred in 2 high dose litters at respectively 7.0% and 6.2% (in historical controls these variations occurred at respectively 0.0% and 0.2%). These findings occurred in the two litters with lowest mean fetal body weights, and marked body weight loss was recorded for both dams, suggesting influence of maternal toxicity rather than direct effect of the test substance. Other visceral variations were not considered to be treatment-related as they occurred in the absence of a dose-related incidence trend or were observed within the range of historical control data.
A statistically significant increase was attained for reduced ossification of vertebrae centra at 100 mg/kg bw/day. This variation was noted at a mean litter proportion of 0.0%, 0.0%, 2.0% and 5.9% per litter in respectively the control, 10, 30 and 100 mg/kg bw/day groups. The values at 30 and 100 mg/kg bw/day were also above the maximum historical value (1.6% per litter). The increase at 100 mg/kg bw/day was caused by 5 fetuses out of 5 litters, of which 3 fetuses had low body weights. Moreover, two of these had a vertebra anomaly as well. This variation was not considered adverse. Other skeletal variations were not considered treatment related as they occurred at frequencies that were within the range of historical control data or were observed in control fetuses only.
Skeletal malformations were noted in 1 (1), 4 (3), 1 (1) and 3 (3) fetuses (litters) in respectively the control, 10, 30 and 100 mg/kg bw/day groups. Two skeletally malformed fetuses at 100 mg/kg bw/day also had external end visceral abnormalities. One fetus additionally had fused sternebrae and bent limb bones, the other fetus also had a vertebral anomaly with associated rib anomaly and a sternal anomaly. The third skeletally malformed fetus in the high dose group had a malpositioned metatarsal. The skeletal malformations in these three fetuses occurred incidentally, therefore they were not considered treatment related. The only other skeletal malformation in this study (a rib without a head) was observed in a control fetus.
Overall it was concluded that there were no treatment-related external variations or malformations, skeletal malformations and visceral malformations. The observed visceral variations were considered to be related to maternal toxicity and rather a secondary effect to test substance exposure. The observed skeletal variation was not considered adverse.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Results dose range finding study:

Maternal effects:

At 600 mg/kg bw/day, one animal showed significant weight loss and was sacrificed in extremis. All other animals in this dose group showed clinical signs (hunched posture, piloerection, lethargy, slow breathing, faeces containing mucus and/or ptosis). Five remaining rats were sacrificed on day 20 post coitum (fetuses were not examined). Necropsy revealed irregular surface of the forestomach, and in the sacrificed animal emaciation and gelatinous contents of the gastro-intestinal tract.

At 300 mg/kg bw/day, one female was found dead on day 15 and two on day 20 post coitum, the remaining females were sacrificed in extremis on day 20 post coitum. Clinical signs of these rats included lethargy, hunched posture, piloerection, diarrhea, lean appearance, yellow mouth, wet/dirty genital region (urine) and/or low water intake). Mean body weight gain and food consumption were lower than controls throughout treatment. At necropsy, two females appeared emaciated. At 100 mg/kg bw/day, no mortality occurred. One female showed ptosis, and one female showed hunched posture, piloerection and lean appearance on days 19 and/or 20 post coitum. Body weight and food consumption appeared similar to controls. At 30 mg/kg bw/day, all animals survived until sacrifice, no clinical signs were noted. Body weight and food intake was normal.

Necropsy revealed irregular surface of the forestomach in all rats examined from group treated with 600 mg/kg bw/day (5/5), in 5/6 females at 300 mg/kg bw/day, in 2/6 females at 100 mg/kg bw/day and in 1/6 rats at 30 mg/kg bw/day.

Pregnancy/delivery/fetal findings:

Three early deliveries were recorded on day 21 post coitum for one female in different dose groups, which were considered unrelated to treatment. One control and one female at 100 mg/kg bw/day were found to be non-gravid. This was not considered related to treatment. The number of early resorptions appeared higher than controls at 100 mg/kg bw/day. Based on individual variations across the dose groups, this was considered unrelated to treatment. Up to 100 mg/kg bw/day, litter sizes were within normal limits, sex ratios were similar to controls, fetal body weights appeared unaffected by treatment, and all viable fetuses had no external abnormalities. Corpora lutea and implantation counts were normal up to 100 mg/kg bw/day. The incidence of pre- and post-implantation loss across the dose groups showed no relationship to treatment.

Results chemical analysis of formulations:

The concentrations analysed in the formulations of low, mid and high dose groups were in agreement with target concentrations (i.e. mean accuracies between 94% and 107%). No test substance was detected in the control group formulation. The formulations of low and high dose groups were homogeneous (i.e. coefficient of variation ≤ 1.9%).

Applicant's summary and conclusion

Conclusions:
Prenatal developmental toxicity study of C12-14-alkylmorpholine in rat resulted to a maternal NOAEL of 30 mg/kg based on mortality, effects on body weight gain and macroscopic findings (local irritant effects) at 100 mg/kg bw/day. Developmental NOAEL > 100 mg/kg, being the highest dose tested.
Executive summary:

A prenatal developmental toxicity study was performed with C12-14-alkylmorpholine in rats according to OECD/EC guidelines and GLP principles. Pregnant rats (22/group) were exposed by oral gavage to 0, 10, 30 and 100 m/kg bw/day from day 6 to day 20 post coitum. Accuracy and homogeneity of formulations were confirmed by chemical analyses.

No maternal toxicity was observed at 10 and 30 mg/kg bw/day. At 100 mg/kg bw/day, one rat died on day 20 post coitum. It could not be excluded that this death was related to the test substance, since necropsy findings in the gastro-intestinal tract for this female resembled those seen for several other females in the high dose group and consisted of lesions in the stomach wall and black brown foci). At high dose, the average body weight gain was affected (not statistically significant, mainly caused by two surviving females with marked body weight loss). There were no treatment-related effects on litter size for any group. The male:female sex ratio and fetal body weight was unaffected by the treatment up to and including 100 mg/kg bw/day. Overall it was concluded that there were no treatment-related external variations or malformations, skeletal malformations and visceral malformations. The observed visceral variations were considered to be related to maternal toxicity and rather a secondary effect to test substance exposure. The observed skeletal variation was not considered adverse.

In conclusion, the maternal No Observed Adverse Effect Level (NOAEL) for C12-14-alkylmorpholine was established at 30 mg/kg bw/day based on mortality, effects on body weight gain and macroscopic findings (local irritant effects) at 100 mg/kg bw/day. A developmental NOAEL of at least 100 mg/kg was established, as no adverse effects on fetal development were seen at this level.