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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:
July-October 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and EPA guidelines and according to GLP principles.

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)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: clear brown liquid
Details on test material:
- Name of test material (as cited in study report): EXP1503090

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Age at pairing: approximately 90 days
- Age at initiation of dose administration: approximately 14 weeks
- Weight on gestation day 0: 226-288 g
- Fasting period before study: no
- Housing: rats were paired for mating in the home cage of the male; afterwards, individually in clean, solid-bottom cages with bedding material
- Diet: ad libitum LLC Certified Rodent LabDiet 5002
- Water: ad libitum; reverse osmosis-purified drinking water
- Acclimation period: minimal 11 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.4-22.3
- Humidity (%): 44.2-55.5
- Air changes (per hr): min. 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 17 Aug 2015 To: 04 Sept 2015

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: mixture of mineral oil and corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The vehicle control was mineral oil, USP, and corn oil, NF, mixture. The concentration of mineral oil in the control was 32 mg/ml, based on the concentration of mineral oil in the high-dose formulation: (30 mg/ml x 2.85 correction factor) x 37% mineral oil content of the bulk EXP1503090.
The vehicle suspension was prepared approximately weekly for administration to the control group (Group 1); aliquots were prepared for daily dispensation to the control group and stored at room temperature, protected from light. The vehicle was mixed throughout the preparation, sampling, and dose administration procedures.
The test substance formulations were prepared with the diluent every 1-3 days as single formulations for each dosage level, divided into aliquots for daily dispensation, and stored at room temperature, protected from light. The test substance formulations were stirred continuously throughout the preparation, sampling, and dose administration procedures.
The first test substance dosing formulations were visually inspected by the Study Director and were found to be visibly homogeneous and acceptable for administration.

VEHICLE
- Justification for use and choice of vehicle (if other than water): not given
- Concentration in vehicle: 3, 10 and 30 mg/mL
- Amount of vehicle (if gavage): 10 ml/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Test substance formulations ranging in concentration from 1 to 100 mg/mL were previously assessed for homogeneity, resuspension homogeneity, and 5-day room temperature storage stability, and met WIL Research acceptance criteria (Hoobler, 2015, WIL-537042). Therefore, no further stability assessments were conducted as part of this study.
Samples for homogeneity and/or concentration analyses were collected from the top, middle, and bottom stratum of the first, second, and last test substance dosing formulations and from the middle stratum of the first and last control group dosing formulations.
Details on mating procedure:
- Impregnation procedure: cohoused with resident, untreated male
- M/F ratio per cage: 1 to 1
- Length of cohabitation: not given
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
days 6-19 of gestation
Frequency of treatment:
daily
Duration of test:
till gestation day 20 (laparohysterectomy)
No. of animals per sex per dose:
25 females
Control animals:
other: yes, concurrent mineral oil from EXP1503090 in corn oil
Details on study design:
- Dose selection rationale: Dosage levels were selected based on the results of a previous range-finding study in rats (Lambert, 2015, WIL-537040). In the previous study, male and female Sprague Dawley rats were administered EXP1503090 for 14 consecutive days at dosage levels of 0, 10, 30, 100, 300, and 450 mg/kg bw/day. In the females, there were no significant clinical signs and no effects on mean body weights, body weight gains, or food consumption at any dose level tested. Significant increases in ALP and ALT were observed in the 300 and 450 mg/kg/day groups when serum chemistry was evaluated at the time of necropsy.
Additional support comes from 3 other studies (listed below) for the Calcium Salicylate (CAS 114959 46-5) which is a good read across for the Magnesium Salicylate.
A 14-day range-finding study done at Calvert Laboratories (Zeiders, 2004, Calvert Study No. 0437RI38.001) was conducted at dosage levels of 0, 50, 125, 250, 500, and 1000 mg/kg bw/day to both male and female Sprague Dawley rats. Test substance was dosed, uncorrected, from material supplied at 43% w/v Active Ingredient (AI). Minor effects were observed with body weight or body weight changes only at the highest dose. Serum chemistry (primarily the liver) was affected in both sexes at doses =125 mg/kg bw/day.
A 28-day, repeat oral dose study (OECD 407) was conducted at WIL Research (Kirkpatrick, 2006, WIL-186047). Based on results of the 14-day range-finding study, dosage levels were set at 0, 50, 150, and 500 mg/kg bw/day uncorrected, from material supplied at 43% w/v AI. Slight changes were observed with slightly lower body weight changes, longer prothrombin times, and several elevated liver enzymes all in male animals. There were also increased liver weights (in both males and females) and increased thyroid weights in males. All findings resolved during the 14-day recovery period.
A reproduction/developmental toxicity screening study of CAS #114959-46-5 in rats was conducted at WIL Research (Knapp, 2006, WIL-186048). Dosage levels were set at 0, 50, 150, and 500 mg/kg bw/day uncorrected, from material supplied at 43% w/v AI. Slight changes occurred in body weights, but there was no impact on reproductive performance. Pup growth and survival was unaffected by the test substance at all dosage levels; therefore, the NOAEL for F1 neonatal toxicity was considered to be 500 mg/kg bw/day (uncorrected for AI).

- Route of exposure rationale: The selected route of administration for this study was oral (gavage) because this is a potential route of exposure for humans. Historically, this route has been used extensively for studies of this nature.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS:
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: prior to dose administration during the treatment period and approximately 1 hour following dose administration

BODY WEIGHT:
- Time schedule for examinations: daily recorded on gestation day 0 and 6-20

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg bw/day: Yes, on gestation days 0 and 6-20 (daily)

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS:
- Sacrifice on gestation day 20
- Organs examined: thoracic, abdominal and pelvic cavities were examined macroscopically

OTHER:
CLINICAL CHEMISTRY:
- Time schedule: at scheduled necropsy (animals were not fasted)
- Parameters examined: albumin, total protein, globulin, albumin/globulin ratio, total bilirubin, urea nitrogen, creatinine, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, gamma glutamyltransferase, glucose, total cholesterol, calcium, chloride phosphorus, potassium, sodium, triglycerides and appearance (degree of hemolysis, lipemia and and icterus)
Ovaries and uterine content:
The ovaries and uterine content was examined after termination.
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: postimplantation loss was calculated and placentae were also examined.
Fetal examinations:
- External examinations: all per litter
- Soft tissue examinations: all per litter, including heart, major blood vessels and kidney
- Skeletal examinations: all per litter
- Head examinations: half per litter were examined by the Wilson sectioning technique and half were examined by a midcoronal slice
- Fetal weight and sex: all per litter
Statistics:
All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance treated group to the control group. Each mean was presented with the standard deviation (S.D.), standard error (S.E.), and the number of animals (N) used to calculate the mean. Data obtained from nongravid animals were excluded from statistical analyses. Due to the use of significant figures and the different rounding conventions inherent in the types of software used, the means, standard deviations, and standard errors on the summary and individual tables may differ slightly.
Maternal body weights (absolute and net), body weight changes (absolute and net), and food consumption, clinical pathology parameters, gravid uterine weights, numbers of corpora lutea, implantation sites, and viable fetuses, and fetal body weights (separately by sex and combined) were subjected to a parametric one way ANOVA (Snedecor and Cochran, 1980) to determine intergroup differences. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test substance-treated groups to the control group. Mean litter proportions (percent per litter) of prenatal data (viable and nonviable fetuses, early and late resorptions, total resorptions, pre and postimplantation loss, and fetal sex distribution), total fetal malformations and developmental variations (external, visceral, skeletal, and combined) and each particular external, visceral, and skeletal malformation or variation were subjected to the Kruskal Wallis nonparametric ANOVA test (Kruskal and Wallis, 1952) to determine intergroup differences. If the nonparametric ANOVA revealed significant (p<0.05) intergroup variance, Dunn’s test (Dunn, 1964) was used to compare the test substance treated groups to the control group.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
All dams survived to the scheduled euthanasia on gestation day 20. Nonadverse, test substance-related increased incidences of clear, yellow, and/or red material around the mouth were noted in the 100 and 300 mg/kg bw/day groups approximately 1 hour following dose administration. These findings were also noted sporadically in the 30 mg/kg bw/day group, but at a much lower frequency. No test substance-related clinical findings were noted at the daily examinations at any dosage level.
Test substance-related lower mean body weight gains during gestation days 15-20 and lower mean food consumption during gestation days 6-9 and 9-12 were noted in the 300 mg/kg bw/day group; however, these differences did not impact absolute mean body weights and were not considered adverse. Mean body weights, body weight gains, and food consumption in the 30 and 100 mg/kg bw/day groups and mean net body weights, net body weight gains, and gravid uterine weights in the 30, 100, and 300 mg/kg bw/day groups were unaffected by test substance administration.
Test substance-related, significantly higher mean alkaline phosphatase values (195% and 422% of control) were noted in the 100 and 300 mg/kg bw/day groups and significantly higher alanine aminotransferase (200% of control) and triglyceride (242% of control) levels were noted in the 300 mg/kg bw/day group. Based on the magnitude of change, these increases in enzyme activity in the 300 mg/kg bw/d group were considered adverse. There were no other test substance related effects on serum chemistry parameters at any dosage level. Significantly (p<0.01) higher mean A/G ratios and cholesterol levels were noted in the 300 mg/kg/day group. These changes were likely attributed to high values noted in a single animal. In addition, significantly (p<0.05 or p<0.01) lower globulin, glucose, calcium, chloride, and sodium were noted in the 300 mg/kg/day group. These changes were of minimal magnitude and not considered related to the test substance.
There were no test substance-related macroscopic findings noted in females at any dosage level.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day
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:
Intrauterine growth and survival were unaffected by maternal test substance administration at all dosage levels. External malformations were limited to 2(2) fetuses (litters) in the control group. Fetus no. 8366 10 in the control group had vertebral agenesis with a filamentous tail. Skeletally, all vertebrae posterior to sacral vertebra no. 3 were absent. Fetus no. 8418 05 had a misshapen lower jaw, an absent naris, and cheilognathopalatoschisis (cleft upper lip and jaw, and cleft palate [entire length]). Skeletally, misshapen lower jaw consisted of fused mandibles, and cheilognathopalatoschisis consisted of fused nasal bones, small, misshapen premaxillae bones, and palatine plates that were not joined (entire length).
Fetus no. 8393-15 in the 30 mg/kg/day group had situs inversus (lateral transposition of the liver, stomach, pancreas, spleen, kidneys, adrenals, and intestine), a stenotic pulmonary trunk, lobular dysgenesis of the lungs (4 lobes present, bilateral), and an intraventricular septal defect (an opening in the anterior portion of the septum). Fetus no. 8360-14 in the 30 mg/kg/day group had retroesophageal aortic arch (the aortic arch coursed retroesophageal immediately following left carotid and returned to normal position adjacent to the ductus arteriosus; right carotid and right subclavian arose independently from the aortic arch [no brachiocephalic trunk]). Because these visceral malformations were noted in single fetuses and the mean litter proportions of these malformations were not statistically significantly different from the concurrent control group; these findings were not attributed to the test substance. Fetus no. 8322-01 in the control group had situs inversus (lateral transposition of the trachea, esophagus, heart, great and major vessels, lungs, liver, stomach, pancreas, spleen, kidneys, adrenals, and intestine).
A single fetus (no. 8322-01) in the control group had vertebral anomaly without associated rib anomaly that consisted of absent, fused, and/or malpositioned centra and arches.
There were no test substance-related fetal malformations or developmental variations noted in this study.

Effect levels (fetuses)

Key result
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: consideration of all potential effects - none observed
Remarks on result:
not determinable due to absence of adverse toxic effects

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Key result
Developmental effects observed:
no
Lowest effective dose / conc.:
300 mg/kg bw/day
Treatment related:
no
Relation to maternal toxicity:
not specified
Dose response relationship:
no
Relevant for humans:
yes

Any other information on results incl. tables

The analyzed dosing formulations were within WIL Research SOP range for suspensions (85% to 115%) and were homogeneous. Based on these results, the protocol-specified dosages of test substance were administered to the animals. The test substance was not detected in the vehicle formulation that was administered to the control group.

Applicant's summary and conclusion

Conclusions:
Based on higher alkaline phosphatase, alanine aminotransferase and triglyceride levels at 300 mg/kg bw/d, the NOAEL for
maternal toxicity was established at 100 mg/kg bw/d for EXP1503090. Based on the absence of effects on intrauterine
growth, survival and fetal morphology at any dose level, the NOAEL for developmental toxicity for EXP1503090 is
300 mg/kg bw/day
Executive summary:

EXP1503090 was administered by gavage to groups of female Sprague Dawley rats from gestation days 6 through 19 at 30, 100 and 300 mg/kg bw/d. A concurrent control group receiving vehicle control (mineral oil and corn oil) was included. Dams showed no test substance-related effects for clinical signs, body weight, food consumption or macroscopic examination. Statistically significant, higher alkaline phosphatase, alanine aminotransferase and triglyceride levels were noted at 300 mg/kg bw/d.

No treatment-related effect on intrauterine growth and survival, fetal malformations or developmental variations were noted in this study.

Based on these observations, the oral NOAEL in rat for maternal toxicity was established at 100 mg/kg bw/d and the NOAEL for developmental toxicity was set at 300 mg/kg bw/d.