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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:
2018-11-05 to 2018-12-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2020

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:
solid: crystalline

Test animals

Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: S & K-LAP Kft., Császár út 135, 2173 Kartal, Hungary
- Age at study initiation: young adult
- Weight at study initiation: 3498-4202 g
- Fasting period before study: no
- Housing: individually in metal cages
- Diet: ad libitum, S&K LAP separating rabbit diet (Cargill Takarmány Zrt., 5300 Karcag, Madarasi út 0399, Hungary)
- Water: ad libitum, tap water
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15-21
- Humidity (%): 32-70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the vehicle (water, Aqua purificata) in concentrations of 50 mg/mL, 15 mg/mL and 5 mg/mL. Formulations were prepared in the formulation laboratory of the Test Facility with a frequency and stored according to the results of stability measurements in the frame of the analytical method validation (552-100-3004).

VEHICLE
- Concentration in vehicle: 50 mg/mL, 15 mg/mL and 5 mg/mL
- Amount of vehicle (if gavage): 2 mL dose preparation/kg body weight
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test item was formulated in the vehicle (water, Aqua purificata) in concentrations of 50 mg/mL, 15 mg/mL and 5 mg/mL. Formulations were prepared in the formulation laboratory of the Test Facility with a frequency and stored according to the results of stability measurements in the frame of the analytical method validation (552-100-3004). The sample preparation frequency ranged from daily to every three days and the sample was stored at room temperature or in the refrigerator. The suitability of the chosen vehicle for the test item was analytically proven. Recovery was between 101-102% of nominal concentrations at 0.1 mg/mL and 200 mg/mL in water (Aqua purificata), respectively. Diammonium peroxodisulphate (APS) was proved to be stable in the formulations for three days at room temperature and in the refrigerator (5 ± 3°C). A separate analytical report provided these results (Toxi-Coop study no. 552-100-3004). Analytical control of dosing solutions (control of test item concentration and homogeneity) was performed in the Analytical Laboratory of Test Facility two times during the study.
Five samples from different places were taken from each concentration for analysis of concentration and homogeneity on two occasions. Similarly, five samples were taken from the vehicle (Control, Group 1) and analyzed. Results of analysis are added to the Study Report.
Details on mating procedure:
- Impregnation procedure: purchased timed pregnant
Duration of treatment / exposure:
gestational 6 to 27
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
10 mg/kg bw/day
Remarks:
5 mg/mL
Dose / conc.:
30 mg/kg bw/day
Remarks:
15 mg/mL
Dose / conc.:
100 mg/kg bw/day
Remarks:
50 mg/mL
No. of animals per sex per dose:
25-26
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose setting was based on findings obtained in the preliminary non-GLP “Dose Range Finding Prenatal Developmental Toxicity Study with Diammonium peroxodisulphate (APS) in the Rabbit by Oral (Gavage) Administration” Study number: 552-410-4018, by Toxi-Coop Zrt.
According to this dose range finding study the dose level of 200 mg/kg bw/day caused extremely low food consumption, marked weight loss, expressly underfeed condition of the animals and abortion or early delivery of 3 of 6 females.
The 100 mg/kg bw/day dose caused lower food consumption and weight loss of the maternal animals. There was a clear dose response seen in the 100 and 200 mg/kg doses in the lower fetal weight and length as well as the increased incidence of retarded fetuses versus control values.
In this main study 100 mg/kg bw/day was chosen accordingly with the aim to induce some developmental and maternal toxicity but not death or severe suffering. The low dose was selected to induce no toxicity. The intermediate dose level of 30 mg/kg bw/day was interpolated geometrically.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily (morbidity and mortality were checked twice daily)
- Cage side observations included check of behavior and general condition, duration and severity of the clinical signs and observations for signs of morbidity and mortality.

BODY WEIGHT: Yes
- Time schedule for examinations: on gestation days 0, 3, 6, 9, 12, 15, 18, 21, 24, 27 and 28

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 28
- Organs examined: Uterus with cervix and ovaries
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
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [all per litter]
- Skeletal examinations: Yes: [all per litter]
- Head examinations: Yes: [half per litter]
Statistics:
The statistical evaluation of data will be performed with the program package SPSS PC+4.0.
The homogeneity of variance between groups will be checked by Bartlett’s homogeneity of variance test.
Where no significant heterogeneity is detected a one-way analysis of variance (ANOVA) is carried out. If the obtained result is significant Duncan’s Multiple Range test will be used to assess the significance of inter-group differences. If significance is the result of the Bartlett’s test, the Kruskal-Wallis analysis of variance will be used and the inter-group comparisons will be performed using Mann-Whitney U-test.
Does or litters were excluded from the data evaluation in cases of:

- A disease or death of the doe unrelated to the treatment (total exclusion)
- Non pregnant females i.e. females with no implantation and no corpora lutea (total exclusion)
- Females with total pre-implantation loss (only the intra-uterine parameters were evaluated total exclusion)
- Circumstances unrelated to the test item which are considered to be reason for exclusion, at the discretion of the Study Director
- Females and litters with early delivery (total exclusion)

Although these animals were excluded from the data evaluation the study report contains all data. A male/female fetus was considered as retarded in body weight/crown-rump length, when its weight/length was below the average minus twofold standard deviation of the control male/female fetuses. This was noted in the raw data only and not used for data evaluation.
Historical control data:
The results were compared to the laboratory's historical control data.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no effects of treatment with the test item at any dose level on the clinical condition of the rabbits.
With the exception of two control rabbits, clinical symptoms were observed only in those animals excluded from the study for reasons of premature delivery, misdosing or ill health and were therefore incidental to treatment with the test item.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There was a clear effect of treatment with 100 mg/kg bw/day on maternal body weight gain (please refer to the attached result tables below).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
There was a clear effect of treatment with 100 mg/kg bw/day on maternal food consumption (please refer to the attached result tables below).
Significantly reduced food consumption was observed from start of the treatment (from GD 6) up to GD 24 in the 100 mg/kg bw/day dose group (p<0.01 from g.d. 6 to 21 and p<0.05 from g.d. 21 to 24). Between GD 24 and 28 the mean food consumption was similar to the control in this group.
There was some variation in maternal food consumption at 30 mg/kg bw/day but differences from control were small, inconsistent and not statistically significant. No adverse effect of treatment with the test item at 30 mg/kg bw/day on maternal food consumption was indicated.
There was no adverse effect of treatment with 10 mg/kg bw/day on maternal food consumption.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no effects of treatment with the test item at any dose level on the incidence of macroscopic findings at necropsy.
The most common observations were pinhead-sized haemorrhages in the lungs of animals from all groups without a dose response including controls.
One rabbit in the 30 mg/kg bw/day group was found to have a congenital abnormality; the right uterine horn was interrupted and the right kidney was absent. The data for this animal were therefore excluded from the study.

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
There was an effect of 100 mg/kg bw/day on the survival of the fetuses in utero manifest as increased post-implantation loss with a consequential reduction in the number of viable fetuses (please refer to the attached result tables below). Increase of early embryonic death and post-implantation loss (p<0.01) was observed for the 100 mg/kg bw/day dose group. In addition, total intrauterine mortality was significantly increased at 100 mg/kg bw/day. Accordingly, a slightly lower mean number of viable fetuses (without a statistical significance) was observed in the 100 mg/kg bw/day dose group. No treatment related adverse effect was indicated in the number of implantations.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There was an effect of 100 mg/kg bw/day on fetal body weight and on placental weight (please refer to attached result tables).
Significantly lower fetal weight (p<0.01 for each calculation except p<0.05 if the weight of male and female fetuses was evaluated together) and crown rump length were observed at the 100 mg/kg bw/day dose level. The placental weight was slightly lower (p<0.05 if the males and females evaluated together and for the female fetuses). The relative placental weight was slightly higher (p<0.05 if the male and female fetuses were evaluated together). The findings were considered a consequence of the maternal toxicity induced by the test item.
There was no effect of treatment at 10 or 30 mg/kg bw/day on fetal body weight or placental weight.
Changes in sex ratio:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Details on embryotoxic / teratogenic effects:
There were 5/22, 3/23, 6/21 and 9/20 litters with malformed fetuses. The slightly higher incidence of litters with malformed fetuses in the 100 mg/kg bw/day dose group was not statistically significant different from controls.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
Oral (gavage) treatment of pregnant New Zealand White rabbits from gestation day 6 up to day 27 (the day before Caesarean section) with Diammonium peroxodisulphate (APS) at the dose level of 100 mg/kg bw/day induced maternal toxicity manifest as initial weight loss and subsequently reduced body weight gain together with reduced food consumption. The NOAEL for maternal toxicity is 30 mg/kg bw/day.
The severity of the maternal toxicity at 100 mg/kg bw/day was considered to impact fetal viability and growth and to slightly delay ossification. This dose of Diammonium peroxodisulphate (APS) did not induce fetal malformations. The NOAEL for developmental toxicity is 30 mg/kg bw/day.
Executive summary:

Diammonium peroxodisulphate (APS) was examined for its possible prenatal developmental toxicity. Groups of 25 (low and mid dose) and 26 (high dose) inseminated New Zealand White rabbits were treated with Diammonium peroxodisulphate (APS) by oral (gavage) administration daily at three dose levels of 10, 30 and 100 mg/kg bw/day respectively from day 6 up to and including day 27 post insemination. A control group of 25 inseminated females was included and the animals were given the vehicle water (aqua purificata). The treatment volume was 2 mL/kg bw.

Diammonium peroxodisulphate (APS) was proved to be stable in water formulations at 0.1 mg/mL and 200 mg/mL concentration levels for three days at room temperature and in the refrigerator (5 ± 3°C) according to the analytical method validation (Study no.: (Toxi-Coop study no. 552-100-3004)) at Toxi-Coop Zrt. The suitability of the chosen vehicle for the test item was analytically proven. Analytical control of dosing solutions was performed during the first and last week of treatment. The mean of the test item concentrations of the test item in the dosing formulations varied in the acceptable range between 96 and 99 % of nominal concentrations at both analytical occasions confirming proper dosing.

During the study, mortality was checked and clinical observations were performed. Body weight and food consumption of the does were recorded. The day of insemination was regarded as day 0 of gestation. Caesarean section and gross pathology were performed on gestational day 28. The number of implantations, early and late resorptions, live and dead fetuses in each uterine horn and the number of corpora lutea were recorded. Each fetus was weighed, measured and examined for gross external abnormalities. The placentas were weighed and examined externally.

Fresh visceral examination of each fetus was performed including the determination of the gender.

Head of about 50 % of each litter was removed, fixed in modified Sanomiya solution and washed in 90 % isopropanol. Examination of the heads was done by Wilson's free-hand razor blade method. All skeletons were examined by means of a dissecting microscope after cartilage-bone staining.

 

Results

 

Mortality, moribund animals, abortion

There was no test item related mortality, moribund state or abortion observed.

 

Implantation sites, litters

In total, on gestation day 28 there were 22, 23, 21 and 20 evaluated litters in the control, 10, 30 and 100 mg/kg bw/day group respectively.

 

Clinical signs, necropsy findings

There were no test item related clinical signs and pathological macroscopic findings observed.

 

Food consumption, body weight

Treatment with the test item at 100 mg/kg bw/day induced maternal toxicity manifest as an initial weight loss and subsequent reduction in body weight gain (77% between GD 6-28). Corrected body weight and corrected body weight gain clearly reflected the effect at 100 mg/kg bw/day. The reduction in body weight correlated with a reduction in food consumption, observed from the start of the treatment.

Treatment with the test item at 30 mg/kg bw/day did not induce maternal toxicity. Variations in weight gain were not statistically significant during the study. Variations in the food consumption were not statistically significant at 30 mg/kg bw/day except for GD 18-21. This did not result in statistically lower body weight gain.

There was no effect of 10 mg/kg bw/day on maternal body weight or food consumption.

 

Intrauterine mortality

There was evidence of an increase in early embryonic death/post-implantation loss/total intrauterine mortality and a slightly lower mean number of viable fetuses (without a statistical significance) in the 100 mg/kg bw/day dose group. This outcome was considered to be related to the severity of the maternal toxicity induced.

There was no effect of treatment at 10 or 30 mg/kg bw/day on intrauterine mortality.

 

Fetal examinations

Significantly lower fetal weight and crown-rump length were observed in the 100 mg/kg bw/day dose group. These smaller fetuses showed evidence of delayed ossification (e.g. larger or slightly larger anterior fontanelle, reduced or asymmetric ossification of the bones of the digits (including pollex) or small hole in xiphoid cartilage. These effects were considered to be a consequence of the maternal toxicity induced. There was no evidence of treatment-related malformation at 100 mg/kg bw/day.

 

There was no effect of treatment at 10 or 30 mg/kg bw/day on fetal growth or development.

Conclusion

Oral (gavage) treatment of pregnant New Zealand White rabbits from gestation day 6 up to day 27 (the day before Caesarean section) with Diammonium peroxodisulphate (APS) at the dose level of 100 mg/kg bw/day induced maternal toxicity manifest as initial weight loss and subsequently reduced body weight gain together with reduced food consumption. The NOAEL for maternal toxicity is 30 mg/kg bw/day.

The severity of the maternal toxicity at 100 mg/kg bw/day was considered to impact fetal viability and growth and to slightly delay ossification. This dose of Diammonium peroxodisulphate (APS) did not induce fetal malformation.

The NOAEL for developmental toxicity is 30 mg/kg bw/day.