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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:
From 11 May 2021 to 19 July 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021

Materials and methods

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

Test material

Constituent 1
Chemical structure
Reference substance name:
Sodium chlorite
EC Number:
231-836-6
EC Name:
Sodium chlorite
Cas Number:
7758-19-2
Molecular formula:
ClHO2.Na
IUPAC Name:
sodium chlorite
Test material form:
liquid

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Age at study initiation: 11-13 weeks old
- Weight at study initiation: before initiation of pairing: males: 322 - 383 g; females: 185 - 258 g
- Fasting period before study: No
- Housing: The animals were kept individually in IVC cages (type III H, polysulphone cages) on Altromin saw fibre bedding.
- Diet: ad libitum access to Altromin 1324 maintenance diet
- Water: ad libitum access to drinking tap water
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 55 ± 10%
- Air changes (per hr): 10x / hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
aqua ad injectionem
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
A correction factor of 3.15 was applied to consider the amount of active components in the test item. The test item was weighed into a tared vial on a suitable precision balance and the vehicle was added to give the appropriate final concentration of the test item. The formulation was vortexed and/or stirred until visual homogeneity was achieved. The prepared formulation was stored protected from light and at room temperature.

VEHICLE
- Concentration in vehicle: 0.5, 1.25 and 2.5 mg/mL
- Amount of vehicle: 10 mL/kg bw/day
- Lot/batch: 2005066
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Before beginning of the treatment period, formulation samples of 0.5, 1, 12.5 and 25 mg/mL were prepared and analysed in order to obtain knowledge about stability and homogeneity of the test item in the selected vehicle. The formulations were stable for 6 h and for 10 days at room temperature and for 10 days at 2 to 8 and -15 to -35 °C. The test item formulation was shown to be homogenous. Samples (duplicates) were taken for substance concentration determination by a previously validadted analytical method. in the first and in the last week of the study for all doses (8 samples in total).
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1:2
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy.
Duration of treatment / exposure:
7 days per week from GD 5 to GD 19.
Frequency of treatment:
Once a day
Duration of test:
From 11 May 2021 to XXXX
Doses / concentrationsopen allclose all
Dose / conc.:
5 mg/kg bw/day (nominal)
Remarks:
Low dose
Dose / conc.:
12.5 mg/kg bw/day (nominal)
Remarks:
Mid dose
Dose / conc.:
25 mg/kg bw/day (nominal)
Remarks:
High dose
No. of animals per sex per dose:
23
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: Based on a dose range finding study (see endpoint "Developmental toxicity / teratoginicity DRF"), the highest dose of 25 mg/kg bw/day was chosen with the aim of inducing toxic effects, but not death or severe suffering.
- Fasting period before blood sampling for (rat) dam thyroid hormones: No
- Time of day for (rat) dam blood sampling: Haematological parameters from all dams were examined at the end of the treatment period prior to or as part of the sacrifice of the animals

Examinations

Maternal examinations:
CLINICAL OBSERVATIONS: Yes
- Time schedule: General clinical observations were made at least once a day. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.
- Cage side observations included: spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size. Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed once before initiation of pairing. The sperm-positive females were weighed on gestation days 0, 5, 8, 11, 14, 17 and 20.

FOOD CONSUMPTION: Yes. Food consumption of the pregnant females was controlled on days of body weight determination.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Observations: Each dam was examined macroscopically for any structural abnormalities or pathological changes. Any macroscopic findings were preserved in 4% neutral-buffered formaldehyde. Organs: all gross lesions, kidneys, liver, larynx, lungs, nasal cavities, oesophagus, spleen, stomach, trachea, ovaries, thymus.
- Histopathology: Thyroid/parathyroid glands from all dams were preserved in 4% neutral-buffered formaldehyde. The weight of thyroid/parathyroid glands was measured after 24 hour fixation.
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
Blood sampling:
- Blood: Yes (for haematology parameters)
- Serum: Yes (for thyroid hormones)
- Volume: 500 µL
- Haematology parameters: haematocrit value (HCT); haemoglobin content (HGB); red blood cell count (RBC); mean corpuscular volume (MCV); mean corpuscular haemoglobin (MCH); mean corpuscular haemoglobin concentration (MCHC)
reticulocytes (RET); platelet count (PLT); white blood cells (WBC); neutrophils (Neut); lymphocytes (Lym); monocytes (Mono); eosinophils (Eos); basophils (Baso); large unstained cells (Luc).
- Thyroid hormones: T3, T4, TSH.


Fetal examinations:
- External examinations: Yes: all per litter
- Reproductive tract: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
- Anogenital distance of all live rodent pups: Yes: all per litter
Statistics:
A statistical assessment of the results of the body weight and food consumption was performed by comparing values of dosed animals with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Results of absolute and relative organ weights, thyroid hormones and foetal evaluation parameters like external, visceral, craniofacial and skeletal parameters were statistically analysed by comparing values of dosed with control animals using either a parametric one-way ANOVA and a post-hoc Dunnett Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests. The statistics were performed with GraphPad Prism V.6.01 software or Ascentos 1.3.4 software (p<0.05 is considered as statistically significant).

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
None of the females showed signs of abortion or premature delivery prior to the scheduled sacrifice.
Clinical signs observed which includes skin and fur, hairless area in one animal of the low dose group on right forepaw and one animal of the high dose group had hairless at right forepaw and abdomen. These clinical signs are considered to be incidental findings. No clinical signs were observed in mid dose group.
On the high dose group an animal showed slight spontaneous reduced activity, moving the bedding, slight wasp waist, nasal discharge, red urine and slight piloerection at post dose observation on days 16-18. One animal showed slight, spontaneous reduced activity. Another animal showed slight piloerection. Clinical signs observed in these animal are considered to be test item related during the treatment period. No clinical signs of toxicity or test item related were observed in low and mid dose groups.
Mortality:
mortality observed, treatment-related
Description (incidence):
In the high dose group two animals were euthanized (before the date of scheduled sacrifice) due to animal welfare reasons and clinical signs of toxicity. One of the animals showed vocalization, moderate spontaneous reduced activity, moving the bedding, moderate wasp waist, moderate piloerection, slight increased salivations and red urine at post dose observation day 10. The other animal showed moderate spontaneous reduced activity, moderate piloerection and abnormal breathing at post dose observation day 14. These findings are considered to be test item related findings. Both rats were found to be pregnant at necropsy observation. No mortality occurred in other treated group and control during the treatment period. All other females survived until the date of scheduled sacrifice.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no test item related changes in mean body weight and mean body weight gain observed when compared to control on GDs 5-20.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The mean food consumption observed during GDs 5-20 comparable to control. No statistical significance was observed in mean food consumptions. No test item-related effect on mean food consumption was observed in the treated groups.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
At haematological evaluation, slight but statistically significant lower mean RBC count was observed in high dose group (9.43% below control) when compared to control. Haemoglobin was slightly lowered in high dose groups (8.97% below control) but without statistical significance. Statistically significant higher percent reticulocytes were observed in high dose group (181.11% above control) when compared to control. Statistically significant higher mean reticulocytes count was observed in high dose group (98.36% above control) when compared to control. The statistical significant higher reticulocytes were observed due to two individual animal abnormal values of reticulocytes in two animals at the highest dose. However, there were slight trend in decrease in mean RBC count, mean haemoglobin values and increase in mean reticulocytes count/percentage were observed in high dose groups and It is considered to be test item related findings.
Increase or decrease mean WBC count and/or percent eosinophils, basophils and leucocytes were observed in treated groups without dose dependency or statistical significance, hence they are not considered to be test item related.
Endocrine findings:
no effects observed
Description (incidence and severity):
In all terminally sacrificed females, no statistically significant or toxicologically relevant effects were observed on group mean T3, T4 and TSH hormone levels and values were comparable to the control.
Statistical analysis of post-fixed thyroid/parathyroid weights from all dams revealed, no statistically significant differences in the absolute and relative (to body weight) thyroid/parathyroid weights of the treated groups when compared to the control.
At the histopathology evaluation, there were no histology changes in the thyroid and parathyroid glands that could be related to the treatment with the test item
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
On the control group, one animal showed dark red lungs and liver, and thymus with spotted was found at necropsy.
At the low dose group, in seven animals, findings in heart (adhesion), lungs (dark red), oesophagus (black coloured), liver (dark red), kidneys (dark colored), spleen (enlarged), thymus (small, spotted or red) and/or head (abnormal red color) were observed at necropsy.
At the mid dose group, in eight animals, findings in stomach (spotted and abnormal coloured), colon (gas filled), lungs (supernumerary lobe), kidneys (right pelvis dilatation), ovaries (cyst), adrenal glands (dark coloured and enlarged), spleen (enlarged), thymus (small size or spotted red) or parotid lymph node (swollen) were observed at necropsy.
At the high dose group, in ten animals, findings in spinal cord(black coloured), thoracic cavity (clear fluid filled and pale, small or enlarged thymus), heart (adhesion), lungs (dark red or adhesion), stomach (gas filled), duodenum and ileum (dark red), peyer's patches (enlarged), cecum (yellow fluid), pancreas (pale), kidneys (black), urinary bladder (dark red fluid filled), adrenals (enlarged), spleen (black or enlarged), vagina (dark red fluid), lumbar node (enlarged) and head (atrophie) were observed at necropsy.
These findins cannot be excluded to be test substance related, since these findings were also evidenced in the dose range finding study as well.
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related histopathological findings were observed in thyroid glands and parathyroid of all females.

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
None of the females showed signs of abortion or premature delivery prior to the scheduled sacrifice.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
No test item-related effects of toxicological relevance were noted for any prenatal parameters including implantation sites.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
No test item-related effects of toxicological relevance were noted for any prenatal parameters including losses by resorption.
Early or late resorptions:
no effects observed
Description (incidence and severity):
No test item-related effects of toxicological relevance were noted for any prenatal parameters including late resorptions.
Dead fetuses:
no effects observed
Description (incidence and severity):
No test item-related effects of toxicological relevance were noted for any prenatal parameters including number of live foetuses. No dead foetuses were noted in any of the groups.
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
No test item-related effects of toxicological relevance were noted for any prenatal parameters including terminal body weight, adjusted maternal weight (carcass weight), uterine weight, number of corpora lutea.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
< 5 mg/kg bw/day (nominal)
Based on:
act. ingr.
Basis for effect level:
gross pathology

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
No test item-related and statistically significant effects in mean foetal weight, male and female foetal weight on a per litter basis (group mean of individual litter mean) were observed in any of the treated groups when compared to the control group.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
No test item-related effects of toxicological relevance were noted for any prenatal parameters including number of male foetuses and sex ratios in treatment groups when compared to the control group.
Changes in litter size and weights:
no effects observed
Anogenital distance of all rodent fetuses:
no effects observed
Description (incidence and severity):
In males and female foetuses, the absolute and relative anogenital distance (AGD) in treatment groups remained unaffected when compared to the control group.
All male foetuses were checked for indications of incomplete testicular descent/ cryptorchidism and evaluation revealed the completion of testicular descent (abdominal) in all male foetuses from all groups.
External malformations:
no effects observed
Description (incidence and severity):
No test item-related external abnormalities were observed in any of the treated groups.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Wavy ribs are typically classified as transient and reversible post-natally and may thus be considered as variations but not malformations. Observed ossification-related findings were observed at lower or higher incidences and without dose-dependency and thus were not considered as toxicologically relevant. There were also slightly higher or lower litter incidences of skeletal findings in the LD and MD groups, but they did not show any statistical significance or dose dependency.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Visceral findings observed in the treated groups were at frequencies generally comparable to or, in some cases, slightly higher or lower in frequency compared to the control. As the observed findings were either minor variations and/or due to a lack of dose dependency and consistency, no toxicological significance can be attributed to these findings and they were considered to be spontaneous in nature. In addition, all these values were within the historical control range for this strain.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Craniofacial examination findings were considered to be spontaneous in nature and not related to the treatment with the test item. Statistical analysis of the data revealed no statistical significance for any of these findings. In addition, all values were within the historical control range for this strain.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
> 25 mg/kg bw/day
Based on:
act. ingr.
Remarks on result:
not determinable due to absence of adverse toxic effects

Fetal abnormalities

Key result
Abnormalities:
effects observed, non-treatment-related

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

Dose formulation analysis:


Nominal concentrations were confirmed for all dose groups, as measured concentrations were within acceptance criterion of 10%.


Summary of macroscopic findings (dams):


 



































































































































































































































































































































Tissue/OrgansMacroscopic ObservationsGroups
Control(0 mg/kg bw/day)LD(5 mg/kg bw/day)MD(12.5 mg/kg bw/day)HD(25 mg/kg bw/day)
No. of animals23232323
Spinal cordAbnormal colour, black0001 (78+)
Thoracic cavityFluid filled clear0001 (84+)
HeartAdhesion01 (24)02 (71, 84+)
LungDark, dark read1 (8)002 (78+, 84+)
Adhesion1 (22)001 (71)
StomachAbnormal colour, spotted001 (57)0
Gas filled0001 (78+)
Fluid filled, yellow0001 (77)
DuodenumAbnormal colour, dark red0001 (78+)
JejunumAbnormal colour, dark red0001 (78+)
IleumAbnormal colour, dark red0001 (78+)
Peyer’s patchesEnlarged0001 (78+)
CecumFluid filled, yellow0001 (78+)
ColonGas filled001 (56)0
LiverAbnormal colour dark red1 (8)1 (29)00
Supernumerary lobe001 (65)0
PancreasAbnormal colour, pale0001 (78+)
KidneyAbnormal colour, black, dark01 (30)03 (78+, 84+, 77)
Dilatation001 (67)0
Urinary bladderFluid filled, dark red0001 (78+)
OvariesCyst002 (56, 63)0
VaginaFluid filled, dark red0001 (84+)
Thyroid glandAbnormal colour, pale0001 (77)
Parathyroid glandsAbnormal colour, pale0001 (77)
Adrenal glandsAbnormal colour, dark, reddish001 (58)1 (84+)
Enlarged002 (58, 54)2 (84+, 78+)
SpleenAbnormal colour, black0001 (78+)
Enlarged01 (33)4 (49, 51, 56, 61)8 (71, 77, 80, 81, 83, 84+, 85, 90)
ThymusAbnormal color, pale, red, spotted2 (7, 8)2 (40, 31)01 (84+)
Adhesion0001 (71)
Enlarged0001 (78+)
Small01 (32)1 (58)1 (77)
Attached to heart01 (24)00
Mesentric LymphnodeEnlarged0001 (78+)
Parotid lymph nodeSwelling001 (51)0
Lumbar lymph nodeEnlarged0002 (83, 90)
Abdominal cavityAdhesion0001 (78+)
HeadAbnormal colour, red01 (30)00
Atrophie0001 (76)

(animal) (+ = intercurrent death)
All the above macroscopic findings observed in the LD, MD and HD groups cannot be excluded to be test item related, since these findings were also evidenced in the dose range finding study.

Applicant's summary and conclusion

Conclusions:
The NOAEL for maternal toxicity could not be established due to macroscopic lesions in all the treated groups. The NOAEL for fetal toxicity could be established at 25 mg/kg bw/day (highest dose tested) since no test item-related and toxicologically relevant external, visceral, craniofacial and skeletal findings were observed in the foetuses of the treatment groups.
Executive summary:

A prenatal developmental test in rats was performed according to OECD Guideline 414 (GLP test). Sodicum chlorite was administered daily by gavage from gestation day (GD) 5 to 19 to 23 pregnant rats per group at 5 (LD), 12.5 (MD) and 25 (HD) mg/kg bw/day. During the period of administration, the animals were observed precisely each day for signs of toxicity and mortality. Body weight and food consumption were measured on GD 0, 5, 8, 11, 14, 17 and 20. Animals that died during the study were examined macroscopically. All surviving female animals were sacrificed on the respective GD 20. Following the gross necropsy, the uteri and ovaries were removed, weighed and examined for number of implantations, resorptions (early and late), live and dead foetuses. Foetuses were identified using numbered plates, sexed and weighed. All foetuses were observed for external abnormalities, half of the foetuses for visceral and craniofacial abnormalities and the remaining half of the litter was observed for skeletal abnormalities. The uteri of the non-pregnant females were processed with 10% ammonium sulphide solution and checked for the early embryonic deaths. Thyroid/parathyroid glands from all dams were preserved. The weight of thyroid/parathyroid glands was measured after fixation. Blood samples were assessed for serum levels for thyroid hormones. A histopathological evaluation of the preserved thyroid/parathyroid glands was performed. Two HD group animals were euthanized due to animal welfare reason and clinical signs of toxicity. Based on clinical signs and gross lesion at necropsy observations, this mortality is considered to be test item related. No mortality occurred in other treated group and control during the treatment period. All other females survived until the date of scheduled sacrifice. Test item-related clinical signs were observed in three animals of HD groups; includes spontaneous reduced activity, moving the bedding, slight wasp waist, nasal discharge, red urine, piloerection, vocalization, increased salivations and abnormal breathing. No clinical signs of test item related were observed in LD and MD groups. No test item-related effects on mean body weight gain and food consumption were observed in any of the treated animals during the study. No test item-related effects were observed on prenatal parameters including terminal body weight, adjusted maternal weight (carcass weight), uterine weight, number of corpora lutea, implantation sites, early and late resorptions, percent pre- and post-implantation loss, number of live foetuses, anogenital distance (AGD), foetal weight, number of male and female foetuses, sex ratio and testicular descent in treatment groups when compared to the control. No test item or statistically significant or toxicologically relevant effect was observed on group mean T3, T4 and TSH hormone levels and no test item related changes were observed in thyroid/parathyroid weights from all dams. No gross or histopathological lesions were observed in thyroid/parathyroid glands. The macroscopic findings observed in dams in heart (adhesion), lungs (abnormal color, fluid filled, adhesion), stomach/intestine (abnormal color, enlarged), liver (abnormal color, enlarged), kidneys (abnormal color), thymus (abnormal color, adhesion and small), spleen (enlarged) and adrenals (enlarged) in the treated groups cannot be excluded to be test item related effects. Furthermore, no test item-related and toxicologically relevant external, visceral, craniofacial and skeletal findings were observed in the foetuses of treatment groups. Therefore, the NOAEL for maternal toxicity could not be established due to macroscopic lesions in all the treated groups. The NOAEL for fetal toxicity could be established at 25 mg/kg bw/day (highest dose tested).