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Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 2016 to March 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
The required amount of test item was weighed and approximately 50% of the final volume of vehicle added. This was magnetically stirred until uniformly mixed, and then made up to final volume with vehicle. The formulation was magnetically stirred until homogenous.

A series of formulations at the required concentrations were prepared by dilution of individual weighings of the test item.

Test animals

Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
Number of animals: 96 females.

Duration of acclimatization: 19 days.

Age of the animals at the start of the study (Day 0 of gestation): Approximately 19 to 23 weeks old.

Weight range of the animals at the start of the study (Day 0 of gestation): 2.64 to 4.28 kg.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, limits of detection and quantification, linearity of detector response, repeatability, method accuracy and precision.

The homogeneity and stability was confirmed for Sodium Xylene Sulphonate in Vehicle formulations at nominal concentrations of 1 mg/mL and 200 mg/mL during distribution between the bottles, during magnetic stirring for 4 hours, ambient temperature (+15 to +25°C) storage for 1 day and refrigerated storage (+2 to +8°C) for up to 15 days. Stability of discrete samples was confirmed for 15 days refrigerated storage

The mean concentrations of Sodium Xylene Sulphonate in test formulations analyzed for the study were within ±10% of nominal concentrations, confirming accurate formulation.
Details on mating procedure:
Male/female ratio: 1:1 using identified stock New Zealand White bucks.

Checks: Natural mating observed.

After mating: Each female was injected intravenously with 25 i.u. luteinizing hormone.

Day 0 of gestation: On the day of mating.

A colony of stud males was maintained specifically for the purpose of mating; these animals were not part of the study and were maintained as stock animals.
Duration of treatment / exposure:
Females were treated from Day 6 to Day 28 (inclusive) after mating
Frequency of treatment:
Once daily at approximately the same time each day
Duration of test:
28 days
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Three groups of 22 females received Sodium Xylene Sulphonate at doses of 100, 300 or 1000 mg/kg/day by oral gavage administration at a volume-dose of 5 mL/kg body weight from Day 6 to Day 28 after mating, inclusive.
Control animals:
yes, concurrent vehicle

Examinations

Ovaries and uterine content:
For females surviving to term, the following was recorded:

Uterus: Gravid uterine weight (including cervix and ovaries).

The following were recorded for all animals including those prematurely sacrificed, where possible:

For each ovary/uterine horn: Number of: Corpora lutea, Implantation sites, Resorption sites (classified as early or late), Fetuses (live and dead).

Apparently non-pregnant animals and for apparently empty uterine horns: The absence or number of uterine implantation sites was confirmed.
Fetal examinations:
Examination of all viable fetuses and placentae: Dissected from the uterus, individually weighed and identified within the litter using a coding system based on
their position in the uterus. Examined externally with abnormalities recorded, sampled as appropriate and retained in appropriate fixative. All fetuses were subject to a gross internal examination of the viscera of the neck, thorax and abdominal cavities and the sex of each fetus was also recorded.

Fixation: Nominally one half of eviscerated fetuses were decapitated; heads were initially stored in Bouin’s fluid. Remaining eviscerated fetuses and torsos were fixed in Industrial Methylated Spirit.

Processing: Bouin’s fixed fetal heads were subject to free-hand serial sectioning. Industrial Methylated Spirit fixed fetuses and torsos were processed and stained with Alizarin Red.

Bouin’s fixed heads: Serial sections were examined for soft tissue abnormalities.

Alizarin Red stained fetuses and torsos: Assessed for skeletal development and abnormalities.
Statistics:
The following data types were analyzed at each timepoint separately:
Body weight, using gains over appropriate study periods
Gravid uterine weight and adjusted body weight
Food consumption, over appropriate study periods
Litter size and survival indices
Fetal, placental and litter weight

The following comparisons were performed:
Group 1 vs 2, 3 and 4

A 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.

For litter size and survival indices and fetal, placental and litter weight and gravid uterine weight data, if 75% of the data (across all groups) were the same value, for example c, Fisher’s exact tests (Fisher 1973) were performed. Treatment groups were compared using pairwise comparisons of each dose group against the control both for i) values c, as applicable.

Pre/post implantation loss and sex ratio were analyzed by generalized mixed linear model with binomial errors, a logit link function and litter as a random effect (Lipsitz 1991). Each treated group was compared to control using a Wald chi-square test. For pre implantation loss, the numerator was number of corpora lutea - number of implantations, the denominator was Number of corpora lutea. For post-implantation loss, the numerator was number of implantations - number of live fetuses, the denominator was number of implantations. For sex ratio, the numerator was number of males; the denominator was number of live fetuses.

For resorptions, each treated group was compared to control by exact Wilcoxon rank sum test (Wilcoxon 1945).

Significant differences between the groups compared were expressed at the 5% (p<0.05) or 1% (p<0.01) level.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
Among females surviving to scheduled termination, there were no signs observed at routine physical examination or in relation to dose administration that were clearly attributable to Sodium Xylene Sulphonate administration.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There were three premature deaths during the course of the study, none of which were considered to be related to Sodium Xylene Sulphonate administration.

On Day 14 of gestation, Female No. 69 receiving 1000 mg/kg/day was killed for reasons of animal welfare. This animal had shown noisy respiration (rales) since Day 12 of gestation and due to this sign was not able to be dosed on Day 12 or 13 of gestation; the animal was despatched to necropsy due to severe respiratory impairment. Other clinical signs observed between Day 12 and Day 14 of gestation included low hay and water intake, thin build, reduced faecal and urine output and small faecal pellets; no signs had been observed in relation to dose administration. Low food consumption was evident from Day 9 of gestation along with weight loss of 0.21 kg from Day 8 of gestation. Macroscopic examination revealed poorly defined dark areas on the lungs, aerated fluid adjacent to the salivary glands and the capsule above the salivary glands was slightly distended with air; there was no evidence of any trauma to the trachea or oesophagus (i.e. no evidence of mis-dosing). The
uterus contained ten grossly normal embryos.

On Day 19 of gestation, Female No. 54 receiving 300 mg/kg/day was found dead shortly after dose administration. This female had previously shown normal food consumption and body weight performance, no ante-mortem clinical signs had been observed during the study, post-dosing observations were limited to difficulty in achieving intubation of the catheter on Day 12 of gestation and signs at despatch to necropsy were limited to abnormally coloured coat staining on the muzzle. There were no significant abnormalities detected at macroscopic examination; the uterus contained six grossly normal embryos and one early resorption.

On Day 22 of gestation, Control Female No. 8 died shortly after dose administration having experienced a prolonged convulsion. On two occasions (Days 16 and 19 of gestation) difficulty in achieving intubation of the catheter had occurred, and the female had shown clinical signs of noisy/irregular respiration on Days 16-17 of gestation. Normal body weight performance and levels of food consumption had been observed throughout the study.

Macroscopic examination revealed a perforation in the lower section of the trachea, indicating that the female had been mis-dosed; the uterus contained three grossly normal fetuses.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The body weight performance of pregnant females surviving to scheduled termination was unaffected by Sodium Xylene Administration at doses up to and including 1000 mg/kg/day. The mean gravid uterine weight on Day 29 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 loss was recorded in all groups of females and no effect of treatment with Sodium Xylene Sulphonate was inferred.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The body weight performance of pregnant females surviving to scheduled termination was unaffected by Sodium Xylene Administration at doses up to and including 1000 mg/kg/day.

The mean gravid uterine weight on Day 29 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 loss was recorded in all groups of females and no effect of treatment with Sodium Xylene Sulphonate was inferred.
Food efficiency:
no effects observed
Description (incidence and severity):
Mean food consumption during Days 1-29 of gestation was similar in all groups and unaffected by the administration of Sodium Xylene Sulphonate.
Water consumption and compound intake (if drinking water study):
not examined
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
Description (incidence and severity):
There were no test item-related macroscopic abnormalities detected among the females at scheduled termination on Day 29 after mating.

The incidence of major and minor fetal abnormalities and skeletal variants showed no relationship to maternal treatment with Sodium Xylene Sulphonate.

Across the treated groups there was a slightly increased incidence of short supernumerary cervical rib and 7th costal cartilage not connected to sternum compared to concurrent control. There was, however, no dose response apparent and all fetal and litter incidences were within the Historical Control Data range, therefore these slight increases were considered were considered to be fortuitous and not adverse.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Litter Responses: At scheduled termination on Day 29 after mating, three Control females (No’s. 5, 12 and 15), three low dose group females (No’s. 32, 40 and 41), two intermediate dose group females (No’s. 45 and 46) and five high dose females (No’s 70, 71, 73, 75 and 82) were found not to be pregnant. Therefore, a total of 18, 19, 19 and 16 litters were available for assessment at 0, 100, 300 and 1000 mg/kg/day, respectively.

Placental, Litter and Fetal Weights: There was no effect of maternal treatment on mean placental, litter or fetal weights at any dose level investigated.

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
There was no evidence that maternal treatment with Sodium Xylene Sulphonate at doses up to and including 1000 mg/kg/day had any adverse effect on litter data, as assessed by the mean numbers of implantations, resorptions, live young and pre- and post-implantation losses, at any of the doses investigated. Sex ratio, as assessed by the percentage of males per litter, was in line with expectations and unaffected by maternal treatment.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There was no evidence that maternal treatment with Sodium Xylene Sulphonate at doses up to and including 1000 mg/kg/day had any adverse effect on litter data, as assessed by the mean numbers of implantations, resorptions, live young and pre- and post-implantation losses, at any of the doses investigated. Sex ratio, as assessed by the percentage of males per litter, was in line with expectations and unaffected by maternal treatment.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
There was no evidence that maternal treatment with Sodium Xylene Sulphonate at doses up to and including 1000 mg/kg/day had any adverse effect on litter data, as assessed by the mean numbers of implantations, resorptions, live young and pre- and post-implantation losses, at any of the doses investigated. Sex ratio, as assessed by the percentage of males per litter, was in line with expectations and unaffected by maternal treatment.
Early or late resorptions:
no effects observed
Description (incidence and severity):
There was no evidence that maternal treatment with Sodium Xylene Sulphonate at doses up to and including 1000 mg/kg/day had any adverse effect on litter data, as assessed by the mean numbers of implantations, resorptions, live young and pre- and post-implantation losses, at any of the doses investigated. Sex ratio, as assessed by the percentage of males per litter, was in line with expectations and unaffected by maternal treatment.
Dead fetuses:
no effects observed
Description (incidence and severity):
There was no evidence that maternal treatment with Sodium Xylene Sulphonate at doses up to and including 1000 mg/kg/day had any adverse effect on litter data, as assessed by the mean numbers of implantations, resorptions, live young and pre- and post-implantation losses, at any of the doses investigated. Sex ratio, as assessed by the percentage of males per litter, was in line with expectations and unaffected by maternal treatment.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
There was no evidence that maternal treatment with Sodium Xylene Sulphonate at doses up to and including 1000 mg/kg/day had any adverse effect on litter data, as assessed by the mean numbers of implantations, resorptions, live young and pre- and post-implantation losses, at any of the doses investigated. Sex ratio, as assessed by the percentage of males per litter, was in line with expectations and unaffected by maternal treatment.
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): There was no evidence that maternal treatment with Sodium Xylene Sulphonate at doses up to and including 1000 mg/kg/day had any adverse effect on litter data, as assessed by the mean numbers of implantations, resorptions, live young and pre- and post-implantation losses, at any of the doses investigated. Sex ratio, as assessed by the percentage of males per litter, was in line with expectations and unaffected by maternal treatment.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
There was no evidence that maternal treatment with Sodium Xylene Sulphonate at doses up to and including 1000 mg/kg/day had any adverse effect on litter data, as assessed by the mean numbers of implantations, resorptions, live young and pre- and post-implantation losses, at any of the doses investigated. Sex ratio, as assessed by the percentage of males per litter, was in line with expectations and unaffected by maternal treatment.
Other effects:
no effects observed

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
ca. 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
food efficiency
gross pathology
maternal abnormalities
number of abortions
pre and post implantation loss
total litter losses by resorption
effects on pregnancy duration
early or late resorptions
dead fetuses
changes in pregnancy duration
changes in number of pregnant
necropsy findings

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
There was no effect of maternal treatment on mean placental, litter or fetal weights at any dose level investigated.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): There was no effect of maternal treatment on mean placental, litter or fetal weights at any dose level investigated.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Sex ratio, as assessed by the percentage of males per litter, was in line with expectations and unaffected by maternal treatment.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
There was no effect of maternal treatment on mean placental, litter or fetal weights at any dose level investigated.
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Description (incidence and severity):
The incidence of major and minor fetal abnormalities and skeletal variants showed no relationship to maternal treatment with Sodium Xylene Sulphonate.
Skeletal malformations:
no effects observed
Description (incidence and severity):
The incidence of major and minor fetal abnormalities and skeletal variants showed no relationship to maternal treatment with Sodium Xylene Sulphonate.
Visceral malformations:
no effects observed
Description (incidence and severity):
The incidence of major and minor fetal abnormalities and skeletal variants showed no relationship to maternal treatment with Sodium Xylene Sulphonate.
Other effects:
no effects observed

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
ca. 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
reduction in number of live offspring
changes in sex ratio
fetal/pup body weight changes
changes in litter size and weights
changes in postnatal survival
external malformations
skeletal malformations
visceral malformations

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
Based on the results obtained in this study of embryo-fetal development, it was concluded that the dose level of 1000 mg/kg/day represented the No Observed Adverse Effect Level (NOAEL) for maternal toxicity and for embryo-fetal survival, growth and development.
Executive summary:

Based on the results obtained in this study of embryo-fetal development, it was concluded that the dose level of 1000 mg/kg/day represented the No Observed Adverse Effect Level (NOAEL) for maternal toxicity and for embryo-fetal survival, growth and development.