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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 September 2015 - 20 November 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
performed under GLP

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
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
(January 2001)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
Official Journal of the European Union No. L142 (May 2008)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
(August 1998)
Deviations:
no
Principles of method if other than guideline:
Blood samples were analyzed for sodium bromide and triethanolamine content.The analyses were based on the following guidelines:- Guideline on Bioanalytical Method Validation, European Medicines Agency (EMA), EMEA/CHMP / EWP/192217/2009 (21 July 2011);- Guidance for industry: Bioanalytical Method Validation, U.S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER) and Center for Veterinary Medicine (CVM) (May 2001).
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Reference substance name:
Reaction Mass of CXN1-55
IUPAC Name:
Reaction Mass of CXN1-55
Test material form:
other: solid
Details on test material:
- Name of test material (as cited in study report): CXN 1-55- Substance type: Organic- Physical state: Off-white to brownish solid- Storage condition of test material: In refrigerator (2-8°C) desiccated

Test animals

Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS- Source: Charles River Deutschland, Sulzfeld, Germany- Age at study initiation: approximately 11-15 weeks- Weight at study initiation:- Fasting period before study: No- Housing: Individually housed in Macrolon plastic cages (MIII type, height 18 cm) with cage-enrichment/ nesting material- Diet: pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum- Water: tap water, ad libitum- Acclimation period: at least 5 daysENVIRONMENTAL CONDITIONS (set conditions)- Temperature (°C): 18-24- Humidity (%): 40-70- Air changes (per hr): at least 10- Photoperiod (hrs dark / hrs light): 12/12IN-LIFE DATES: From: 14 September 2015 To: 20 November 2015

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:Formulations (w/w) were prepared daily within 6 hours prior to dosing and 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. Formulations were stored at room temperature. Dose volume 5 mL/kg bw/day. 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:
Analyses were conducted on a single occasion for samples collected on first day of treatment, according to validated methods for triethanolamine and sodium bromide. 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:
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:
16 days (from days 6 to 21 post-coitum, inclusive)
Frequency of treatment:
Daily
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 only)
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. Four groups of 6 females were exposed to 0, 100, 300 and 1000 mg/kg/day for days 6 to 21 post-coitum inclusive by oral gavage. Test system, procedures and techniques were in general identical to those used during the main study. At termination, blood samples were collected for expsoure control bioanalysis. Organ weights of liver and kidney were determined. A gross necropsy was performed on the dams. Fetuses were externally examined, sexed, and weighed. Weight of fetuses was not determined.

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. From start of treatment onwards (day 6 post-coitum), detailed clinical observations were initially done immediately after dosing and at 3 hr (± 30 min) after dosing towards the end of the study. The time of onset, grade and duration of any observed signs were recorded.BODY WEIGHT: Yes - Time schedule for examinations: Days 2, 6, 9, 12, 15, 18 and 21 post-coitum. FOOD CONSUMPTION: Yes- Days 2-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post-coitum.WATER CONSUMPTION: No- 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: Liver and kidney weights (and terminal body weight) were recorded from all animals killed at the scheduled sacrifice.- All macroscopic abnormalities were recorded.OTHER:On day 21 post-coitum, a blood sample was taken at necropsy from pregnant females (10/group) and litters (10/group) at 3 hours after dosing for determination of sodium bromide and triethanolamine content. For each dose, the group mean plasma concentrations of sodium bromide and triethanolamine were calculated from individual animals and a dose/response was evaluated.
Ovaries and uterine content:
Each ovary and uterine horn was 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 (early and late resorptions).- 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 examinations: Yes: all per litter (Each viable fetus was examined in detail, weighed and sexed). Blood was drawn from 10 litters/ group and pooled to determine sodium bromide and triethylamine content. Late resorptions were subjected to a gross external examination.- Soft tissue examinations: Yes: Approximately one-half of the fetuses (live and dead) in each litter (all groups)- Skeletal examinations: Yes: Approximately one-half of the fetuses (live and dead) in each litter (all groups)- Head examinations: Yes: Approximately one-half of the fetuses (live and dead) in each litter (all groups), heads from fetuses selected for visceral 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 group 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) 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:
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;Viable fetuses affected/litter (%) = ((number of viable fetuses affected/litter)/ (number of viable fetuses/litter)) x 100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment related clinical signs of toxicity were noted up to 1000 mg/kg bw/day. Incidental findings that were noted included alopecia, focal erythema, chromodacryorrhoea and scales. Since these signs occurred within the range of background findings observed in rats of this age and strain under the conditions in this study and did not show a dose-dependent relationship, they were considered not to be related to treatment. On day 20 post-coitum, red colored matter was seen in the bedding material of the cage of a low dose female. The identity and origin of this matter could not be established, but on visual inspection it was considered to be blood.
Dermal irritation (if dermal study):
no effects observed
Mortality:
no mortality observed
Description (incidence):
No mortality occurred.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Mean body weights, body weight gain and weight gain corrected for uterus weight of treated animals remained in the same range as controls over the study period. Slightly lower mean body weight gain, accompanied by a higher standard deviation for these mean values, was noted for low dose females during the progress of pregnancy, in comparison with the other groups. These values were affected by the results of three individuals in this group, which showed a low uterus weight as a result of a high number of resorptions and no or a few viable fetuses in their litters. The slightly higher body weight gain, achieving a level of statistical significance in high dose females on day 9 post-coitum, when compared to controls, was considered a fortuitous finding and not related to treatment.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption before or after allowance for body weight was similar between treated and control animals.
Food efficiency:
no effects observed
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):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Mean liver and kidneys weights, absolute as well as relative to body weights, of treated females were within the same range as controls.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no gross observations.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

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:
effects observed, non-treatment-related
Description (incidence and severity):
Relatively high mean values for pre- and post-implantation loss were noted in low dose females in comparison with these in the other three groups. These high mean values were primarily the result of high values for both these parameters in three individual low dose females. A relation to treatment for pre-implantation loss can be ruled out since start of treatment on day 6 postcoitum was after implantation. The subsequent high values for post-implantation loss and low number of (viable) fetuses may indicate an impairments of fetal development in these three animals. The pre- and post-implantation values for the other females in mid dose group were in the same range as noted in the other three groups. Moreover, since this phenomenon was observed in a few low dose animals only and showed no dose dependent relationship, it was considered an incidental finding and not related to treatment
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on litter size for any group. Mean litter sizes were 10.6, 9.4, 10.9 and 10.8 fetuses/litter for the vehicle control, 100, 300 and 1000 mg/kg bw/d treated groups, respectively.
Early or late resorptions:
effects observed, non-treatment-related
Description (incidence and severity):
One low dose group female showed one early and one late resorption only.
Dead fetuses:
no effects observed
Description (incidence and severity):
No dead fetuses were observed in any of the groups.
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
Description (incidence and severity):
All females were pregnant.
Other effects:
not examined

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: No adverse effects seen at highest dose tested.

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
Mean male (female) fetal body weights were 5.4, 5.5, 5.5, and 5.5 (5.1, 5.2, 5.2, and 5.2) grams for the vehicle control, 100, 300 and 1000 mg/kg bw/day treated groups, respectively.
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:
not examined
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Mean sex ratios (males:females) were 52:48, 51:49, 50:50 and 55:45 for the vehicle control, 100, 300 and 1000 mg/kg bw/day treated groups, respectively.
Changes in litter size and weights:
not examined
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
One fetus from the low dose group and one fetus from the high dose group were externally malformed. The low dose fetus had an omphalocele and the fetus in the high dose group had astomia, no jaw and only one eye bulge present. At skeletal examination of the latter fetus, all the skull findings substantiated the malformations observed externally. Since each type of malformation occurred in a single occasion and no clear dose response relationship was apparent, they were considered not related to treatment. There were no other external malformations and external variations were not seen in any group.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
Skeletal malformations were found in all dose groups, and included vertebral anomaly with or without associated rib anomaly (one mid dose group fetus and a high dose fetus, for which one several external head abnormalities were noted), a vertebral centra anomaly (two low dose group fetuses and a high dose group fetus), a rib anomaly (one high dose group fetus) and a skull anomaly (one control fetus). Due to the absence of a dose response, the single occurrence and/or occurrence in a control fetus, these malformations were not considered to be treatment related. Within the observed skeletal variations, increased incidences of bent ribs and reduced ossification of the skull were seen at the high dose level of 1000 mg/kg bw/day, achieving statistical significance for bent ribs in comparison with controls. Mean litter incidences of these variations for bent ribs were 22.3%, 13.8%, 21.4%, 45.7% and for reduced ossification of the skull 11.5%, 15.1%, 13.6% and 23.5% per litter at 0, 100, 300 and 1000 mg/kg bw/day, respectively. The other skeletal variations noted were not considered treatment related as they occurred in the absence of a dose-related incidence trend, occurred infrequently or were observed in control fetuses only. The variation of 14th full ribs was noted at an incidence of 13.1%, 5.6%, 5.6% and 4.2% per litter at 0, 100, 300 and 1000 mg/kg bw/day, respectively. Compared with the current control incidence, the incidences in all treated groups were statistically significantly reduced. In five antecedent studies, the incidence of 14th full ribs ranged from 5.2% to 12.1% per litter. In comparison with this, the current control value was slightly above and the high dose value slightly below that range. Taken into account the evidently wide historical control incidence range for 14th full ribs, it was considered that the striking group distribution of 14th full ribs in this study occurred by chance and was not related to treatment.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Three fetuses, one in each CXN 1-55 treated group, had situs inversus whereby all organs were laterally transposed. The affected fetus from low dose group also showed other visceral malformations, namely abnormal lung lobation, interrupted aortic arch, enlarged heart and ventricular septum defect. Because the group distribution of these malformations does not denote a dose response, they were considered to be chance findings and not attributed to treatment.The variations that were noted in this study were small supernumerary lobe(s) and appendix of the liver, discolored liver and dilated ureter. These variations occurred at low incidences and/or in the absence of a dose-related incidence trend and therefore were not considered to be treatment related.
Other effects:
not examined

Effect levels (fetuses)

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

Fetal abnormalities

Key result
Abnormalities:
effects observed, treatment-related
Localisation:
skeletal: skull
skeletal: rib
Description (incidence and severity):
Increased incidence of bent ribs and reduced ossification of the skull at 1000 mg/kg bw/day.

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects in the absence of maternal toxicity effects
Dose response relationship:
no
Relevant for humans:
not specified

Any other information on results incl. tables

Formulation analyis based on triethanolamine:

No test item was detected in formulations of the control group. The concentrations analysed in the formulations of groups exposed to the test item were in agreement with target concentrations (i.e. mean accuracies between 101% and 110%). The formulations of low and high dose groups were homogeneous (i.e. coefficient of formulation ≤ 3.9%).

Formulation analyis based on bromide:

No test item was detected in formulations of the control group. The concentrations analysed in the formulations of groups exposed to the test item were in agreement with target concentrations (i.e. mean accuracies between 96% and 106%). The formulations of low and high dose groups were homogeneous (i.e. coefficient of formulation≤ 1.5%).

Results exposure control bioanalysis

The group mean triethanolamine and bromide concentrations (μg/mL ± st.dev.) in plasma of pregnant females and litters (pooled from several foetuses per litter) of the main study were as follows:

Group

Triethanolamine concentration

Bromide concentration

 

Females

Litter

Females

Litter

Control

Below detection limit

Below detection limit

100 mg/kg bw/day

5.63±1.13

2.02±0.41

117.3±14.21

106.4±7.35

300 mg/kg bw/day

16.32±2.05

6.09±2.23

324.3±20.18

311.6±17.93

1000 mg/kg bw/day

44.84±11.18

20.42±5.30 *

1019.5±46.85

1018.2±50.04

 

* One relatively high value of 59.2 μg/mL was observed for the plasma concentration for triethanolamine in one litter of the high dose group, which was even higher than in the pregnant female (38.5 μg/mL). Exclusion of the value of this litter resulted in a mean litter value of 16.11±5.30 μg/mL.

The mean plasma triethanolamine concentration in litters was approximately one third of that found in the pregnant females in each dose groups. The mean plasma bromide concentration in litters was similar to that found in the pregnant females in each dose groups.

Results dose-range finding study:

No mortality and no clinical signs were observed in any of the animals of all dose groups. Mean body weights and body weight gain of treated animals in all dose groups remained in the same range as controls over the study period. Mean food consumption before or after correction for body weight for treated animals remained similar to the control level over the study period. One or two females per group had delivered on day 21 post-coitum shortly before scheduled necropsy. No macroscopic findings were observed in these females, including examinations of the ovaries and uterine horns, and its pups. All fetuses were born alive and no early and late resorptions were found in the maternal females. No treatment related macroscopic abnormalities were noted in the animals, and its fetuses, that had not delivered prior to necropsy. The liver and kidney weights, absolute as well as relative to body weight, of treated animals were in the same range as that in controls. Litter sizes were within normal limits for all groups. No treatment related effects were observed in the male: female ratios of all groups. External examination of the fetuses did not show any variations and malformations.

Results bioanalysis:

The group mean triethanolamine and bromide concentrations (μg/mL ± st.dev.) in plasma of pregnant females and litters (pooled from several foetuses per litter) were as follows:

Group

Triethanolamine concentration

 Bromide concentration

 

Females

Litter

Females

Litter

Control

Below detection limit

Below detection limit

100 mg/kg bw/day

5.37±1.56

2.22±0.56

121.8±3.59

110.5±9.29

300 mg/kg bw/day

15.84±2.55

5.06±0.57

371.8±34.28

300.0±24.14

1000 mg/kg bw/day

40.74±7.50

13.94±4.01

1167.5±79.32

937.0±78.50

 

The mean plasma triethanolamine concentration in litters was approximately one third of that found in the pregnant females in each dose groups. The mean plasma bromide concentration in litters was similar to that found in the pregnant females in each dose groups.

Applicant's summary and conclusion

Conclusions:
In a prenatal developmental toxicity study with CXN 1-55 in rats by oral gavage, a developmental NOAEL of 300 mg/kg bw/day was found based on skeletal variations (increased incidence of bent ribs and reduced ossification of the skull). Maternal NOAEL was 1000 mg/kg bw/day in absence of maternal toxicity at highest dose tested.
Executive summary:

A prenatal developmental toxicity study was performed according to OECD/EC guidelines and following GLP principles. Pregnant Wistar rats (20 animals/ dose group) were exposed to CXN 1 -55 from days 6 to 21 post-coitum by oral gavage to 0, 100, 300 and 1000 mg/kg bw/day. Accuracy and homogeneity of the formulations were demonstrated by analyses. No maternal toxicity was observed during the study or at sacrifice, and no effects were observed on pregnancy duration and/or outcome. No developmental toxicity was observed in the groups exposed to 100 and 300 mg/kg bw/day. In the fetuses of the high dose group, increased incidences in skeletal variations (bent ribs and dreduced ossification of the skull) were observed, achieving statistical significance for bent ribs when compared to controls. No further signs of developmental toxicity were observed in this dose group. Based on these results, a developmental NOAEL of 300 mg/kg bw/day was established in absence of maternal toxicity. In absence of any adverse effects, the maternal NOAEL was set as 1000 mg/kg bw/d.