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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:
Jan. 2017 - Sep. 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: BYK Chemie LS-15-109A, LS-15-109B, LS-15-109C
- Expiration date of the lot/batch: 1-Nov-2018
- Purity test date: UVCB

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient temperature in the dark
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: stable

Test animals

Species:
rat
Strain:
other: RccHan; WIST rat
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS Ltd
- Age at study initiation: 77 - 84 days (at Day 0 of gestation)
- Weight at study initiation: 180-218 g (at Day 0 of gestation)
- Fasting period before study: no
- Housing: 1 animal per cage
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days before commencement of pairing

DETAILS OF FOOD AND WATER QUALITY: SDS VRF1 Certified pelleted diet; potable water from public supply

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 40-70%
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12 h / 12 h

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The required amounts of test material were weighed. Approx. 50% of the final volume of vehicle was added and magnetically stirred until the test item was uniformly mixed. The remaining vehicle was added to achieve the required volume and the formulation was mixed using a magnetic stirrer until homogeneous.

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle: 0, 16, 40, 100 mg/ml
- Amount of vehicle (if gavage): 5 ml / kg bw
- Lot/batch no. (if required):
- Purity:
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentrations of dose formulations were analysed by GC with flame ionisation detector. Mean concentrations were within an acceptable range of the nominal concentrations.
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1 : 1
- Length of cohabitation: until evidence of mating
- Verification of same strain and source of both sexes: yes, a colony of stud males was maintained
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
from day 6 till day 19 of gestation
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
80 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
20 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels selected for investigation in this study (0, 80, 200 and 500 mg/kg/day) were chosen in conjunction with the Sponsor based on the results of a preliminary rat embryo-fetal study conducted at these laboratories (Envigo study No. PW46CS), in which dose levels of 300, 500 or 700 mg/kg/day were employed. At 700 mg/kg/day there were marked effects on maternal weight gain, mean food intake was low throughout the treatment period and a spectrum of post-dosing signs were apparent including tremors, piloerection, reduced body temperature (cold to touch), underactive behaviour, partially closed eyes, abnormal/unsteady gait, repetitive movements, pallor and hunched posture. In addition, one female experienced a short-lasting convulsion at approximately 10-15 minutes after dosing on Day 11 of gestation and although this event did not impact upon subsequent maternal body weight gain, food intake or litter data (compared to the other females in the group), placental and fetal weights in this litter were very low. Based on all of these effects, the dose level of 700 mg/kg/day was considered unsuitable for further investigation in pregnant rats.

At 500 mg/kg/day, although maternal body weight gain and food intake were lower than Control and some post dosing observations were apparent, the magnitude, nature and incidence of the effects were lower than observed at 700 mg/kg/day, and since the OECD414 guideline states that “the highest dose level should be chosen with the aim to induce some developmental and/or maternal toxicity”, 500 mg/kg/day was considered suitable for selection as the high dose level in this current study. The low and intermediate dose levels of 80 and 200 mg/kg/day were chosen to achieve a dose response and/or aid in the determination of a No-Observed-Adverse-Effect-Level (NOAEL).

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations : visual inspection for ill-health or reaction to treatment

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: days 0, 5, 12, 18, and 20

BODY WEIGHT: Yes
- Time schedule for examinations: days 0, 3, and daily from 6-20

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: full macroscopic examination of tissues
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: number of fetuses (live and dead)
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations external: Yes: all per litter
Statistics:
The following sequence of statistical tests was used for body weight, gravid uterus weight, food consumption, corpora lutea, implantations, live young, fetal, placental and litter weight data:
A parametric analysis was performed if Bartlett's test for variance homogeneity (Bartlett 1937) was not significant at the 1% level. For pretreatment data, analysis of variance was used to test for any group differences. Where this was significant (p<0.05) inter group comparisons using t-tests, with the error mean square from the one-way analysis of variance, were made. For all other comparisons t/The F1 approximate test was applied. This test is designed to detect significant departure from monotonicity of means when the main test for the comparison of the means is a parametric monotonic trend test, such as Williams’ test (Williams 1971, 1972). The test statistic compares the mean square, NMS, for the deviations of the observed means from the maximum likelihood means, calculated under a constraint of monotonicity with the usual error mean square, EMS. The null hypothesis is that the true means are monotonically ordered. The test statistic is F1 = NMS/EMS which can be compared with standard tables of the F-distribution with 1 and error degrees of freedom. If the F1 approximate test for monotonicity of dose-response was not significant at the 1% level, Williams' test for a monotonic trend was applied. If the F1 approximate test was significant, suggesting that the dose response was not monotone, Dunnett's test (Dunnett 1955, 1964) was performed instead.

A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. For pretreatment data, Kruskal-Wallis’ test (Kruskal and Wallis 1952, 1953) was used to test for any group differences. Where this was significant (p<0.05) inter group comparisons using Wilcoxon rank sum tests (Wilcoxon 1945) were made.
Indices:
Prenatal losses were separated into pre- and post-implantation phases. Pre-implantation loss was considered to reflect losses due to non-fertilization of ova and failure to implant. It was calculated from the formula:
Pre-implantation loss (%) = (Number of corpora lutea – Number of implantations) x 100 / Number of corpora lutea

Where the number of implantations exceeded the number of corpora lutea observed, pre implantation loss was assumed to be zero (i.e. no pre-implantation loss was considered to have occurred).
Post-implantation loss was calculated from the formula:
Post-implantation loss (%) = (Number of implantations – Number of live fetuses) x 100 / Number of implantations

All group values and SD (as appropriate) were calculated from the individual litter values
Historical control data:
Historical control data from 7 studies performed during 2016 with the same rat strain are provided in the report.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
There were no signs observed at routine physical examination that were related to treatment with BYK-LP X 20954.
Test item-related signs in relation to dose administration were primarily restricted to animals given 500 mg/kg/day and comprised a low incidence of underactive behaviour, reduced body temperature (cold to touch), piloerection, body tremors, reduced body tone, unsteady gait and flat or hunched posture. A very low incidence of piloerection was also observed at 200 mg/kg/day.

At 500 mg/kg/day there was also a low incidence of salivation, post-salivation coat staining and chin rubbing; a single incidence of salivation was recorded in the 200 mg/kg/day group. These signs are commonly observed with oral gavage administration and are considered to represent distaste of the formulations and not an effect of treatment.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There was one premature death during the course of the study which was unrelated to treatment with BYK-LP X 20954. Female No. 38 in the 80 mg/kg/day group was killed for reasons of animal welfare on Day 19 of gestation due to signs of piloerection, a firm area on the ventral abdomen and a prominent vagina. Macroscopic examination revealed the uterus and vagina were distended with a large amount of yellow fluid; this female was pregnant with 14 live and grossly normal fetuses.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
During Days 6-8 of gestation, mean body weight stasis or slight mean body weight loss (-2 grams) was apparent among females given 200 or 500 mg/kg/day respectively when compared to the performance of Controls (+3 grams); these differences from Control attained statistical significance. In the 500 mg/kg/day group, mean body weight gain was 26% lower than Control during Days 8-15 of gestation, with statistical significance attained, but was similar to Control thereafter. Overall mean weight gain from Day 6-20 of gestation in the 500 mg/kg/day group was statistically significantly lower than Control (-13%), but was unaffected by treatment at 80 or 200 mg/kg/day.
The mean gravid uterine weight on Day 20 of gestation was similar in all groups. When overall mean body weight gain was adjusted for the contribution of the gravid uterus, net mean body weight gain was 45% lower than Control at 500 mg/kg/day, with statistical significance attained, but was similar to Control at 80 or 200 mg/kg/day.

see "overall remarks, attachments" : Table Body weight and body weight change
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Mean food consumption was statistically significantly lower than Control in a dosedependent manner (ranging from 12% to 29%) for females given 200 or 500 mg/kg/day during Days 6-9 of gestation. At 500 mg/kg/day, mean food consumption remained 21% lower than Control during Days 10-13 of gestation. From Day 18 of gestation, mean food consumption was marginally lower than Control at 200 or 500 mg/kg/day, although in the absence of a dose response relationship.
see "overall remarks, attachments" : Table Food consumption
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
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
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not specified

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
see "overall remarks, attachments" : Table Litter data
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
see "overall remarks, attachments" : Table Litter data
Early or late resorptions:
no effects observed
Description (incidence and severity):
see "overall remarks, attachments" : Table Litter data
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
not examined

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks on result:
other: NOAEL = highest dose tested

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
see "overall remarks, attachments" : Table Litter and fetal weights
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:
no effects observed
Description (incidence and severity):
see "overall remarks, attachments" : Table Litter data
Changes in sex ratio:
no effects observed
Description (incidence and severity):
see "overall remarks, attachments" : Table Litter data
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
see "overall remarks, attachments" : Table Litter and fetal weights
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Incidental fetal findings were unrelated to maternal treatment.
see "overall remarks, attachments" : Table Fetal examinations
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Incidental fetal findings were unrelated to maternal treatment.
see Table Fetal examinations
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Incidental fetal findings were unrelated to maternal treatment.
see "overall remarks, attachments" : Table Fetal examinations
Other effects:
not examined

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other:
Remarks on result:
other: NOAEL = highest dose tested

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
At the highest dose level tested, 500 mg/kg bw/day, maternal toxicity was observed. No developmental toxicity was observed at the highest dose level.