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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Reproductive/Developmental toxicity screening test:


Data waiving: In accordance with column 2 of REACH Annex VIII, the screening for reproductive/ developmental toxicity study (required in section 8.7.1) does not need to be conducted as a pre-natal developmental toxicity study (Annex IX, 8.7.2) is available.


Supporting study. Read-across from analogue: Study with 12 male/ 12 female rats at doses of 100, 300, 1000 mg/kg/day, according to OECD Test Guideline 422. The result was as follows: fertility/reproductive toxicity NOAEL = 1000 mg/kg bw /day.


 


Two-generation reproductive toxicity study:


Data waving: In accordance with column 2 of REACH Annex IX, the extended one generation reproductive toxicity study (EOGRTS, required in section 8.7.3) does not need to be conducted as the 90-day study results do not indicate adverse effects on reproductive organs or tissues.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
1. HYPOTHESIS FOR THE ANALOGUE APPROACH: The analogue approach covers the substance Ultramarine Blue (EC nº 701-340-9) and the substance Ultramarine Violet (EC nº 701-186-2).The analogue Ultramarine Blue shares the same structure (Sodalite-type structure (SOD-Zeolite)) with the substance Ultramarine Violet. Therefore, they are expected to be toxicologically equivalent.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES) Ultramarine blue, C.I. Pigment blue 29, EC number: 701-340-9;
Ultramarine Violet, C.I. Pigment Violet 15, EC number: 701-186-2.
No relevant impurities.
3. ANALOGUE APPROACH JUSTIFICATION: The analogue approach covers the substance Ultramarine Blue (EC nº 701-340-9) and the substance Ultramarine Violet (EC nº 701-186-2).The analogue Ultramarine Blue shares the same structure (Sodalite-type structure (SOD-Zeolite)) with the substance Ultramarine Violet. Therefore, they are expected to be toxicologically equivalent.
4. DATA MATRIX (Please, see attached document "Reporting Format for the analogue approach")
Reason / purpose for cross-reference:
read-across source
Dose descriptor:
NOAEL
Remarks:
maternal toxicity
Effect level:
>= 300 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Remarks:
reproduction toxicity
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive function (oestrous cycle)
reproductive function (sperm measures)
reproductive performance
Key result
Dose descriptor:
NOAEL
Remarks:
offspring development
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Reproductive effects observed:
no

The analogue approach covers the substance Ultramarine Blue (EC nº 701-340-9) and the substance Ultramarine Violet (EC nº 701-186-2).


 


The analogue Ultramarine Blue shares the same structure (Sodalite-type structure (SOD-Zeolite)) with the substance Ultramarine Violet. Therefore, they are expected to be toxicologically equivalent.


 


Based on the experimental results obtained with the analogue, the read-across approach is applied and the NOAELs for the substance Ultramarine Violet are:


 


-NOAEL maternal toxicity >= 300 mg/kg bw /day


-NOAEL reproductive toxicity>= 1000 mg/kg bw/day


-NOAEL offspring development >= 1000 mg/kg bw/day

Conclusions:
Based on the experimental results obtained with the analogue, the read-across approach is applied and the NOAELs for the substance Ultramarine Violet are: -NOAEL maternal toxicity >= 300 mg/kg bw /day-NOAEL reproductive toxicity>= 1000 mg/kg bw/day-NOAEL offspring development >= 1000 mg/kg bw/day
Executive summary:

Based on the experimental results obtained with the analogue, the read-across approach is applied and the NOAELs for the substance Ultramarine Violet are:

-NOAEL maternal toxicity >= 300 mg/kg bw /day

-NOAEL reproductive toxicity>= 1000 mg/kg bw/day

-NOAEL offspring development >= 1000 mg/kg bw/day

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Species:
rat
Quality of whole database:
1 supporting study with analogue substance is available with a Klimisch score of 1.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Effects on developmental toxicity

Description of key information

Key Study: Toxicity to reproduction in rats. OECD 414, GLP study. The "no observed adverse effect level" (NOAEL) for reproduction and developmental toxicity is the highest dose used in this study of 1000mg/kg bw /day for maternal animals and pups in rats.


 


Supporting Study: Dose Range Finding Study in rats, OECD 414, non GLP. The oral administration of test item by gavage to presumed pregnant female Sprague Dawley Rats from Gestation Day 5 to 19 did not produce any indication of maternal and developmental toxicity at the dose levels of 100, 300 and 1000 mg/kg body weight/day under experimental conditions employed and the same are then considered appropriate for the definitive test.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
13 March 2020 to 08 May 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Reason / purpose for cross-reference:
reference to other study
Remarks:
main study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
DRF study, only 10 pregnant females.
GLP compliance:
no
Remarks:
Following the OECD Principles of Good Laboratory Practices [C (97) 186/Final], however GLP status will not be claimed for the study.
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: In-house bred animals
- Age at study initiation: 9 to 10 weeks
- Weight at study initiation: Males: 261.87 to 282.54 g, Females: 202.33 to 247.78 g
- Fasting period before study: no
- Housing: Acclimatization - Maximum of three animals of same sex per cage were housed in sterilized standard polypropylene cage (Size: L 430 × B 285 × H 150 mm).
- Housing: Cohabitation Period - During cohabitation, three animals (one male and two females) were housed in standard polypropylene cage (Size: L 430 × B 285 × H 150 mm).
- Housing: Post-mating - After confirming presence of sperm in the vaginal smear and / or vaginal plugs (Day 0 of pregnancy), the mated pairs were separated. Males were housed with their former cage mates while females were housed individually in polypropylene cage (Size: L 430 × B 285 × H 150 mm).
- Diet (e.g. ad libitum): ad libitum. Altromin maintenance diet for rats and mice (manufactured by Altromin Spezialfutter GmbH & Co. KG).
- Water (e.g. ad libitum): ad libitum. Deep bore-well water passed through reverse osmosis unit was provided in plastic water bottles with stainless steel sipper tubes.
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.1 to 23.6°C
- Humidity (%): 47 to 66%
- Air changes (per hr): 12 to 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light and 12 hours dark cycle

IN-LIFE DATES: From: 13 March 2020 To: 08 May 2020
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item formulations were freshly prepared before dose administration on each treatment day. The required quantity of test item was weighed and triturated well in a mortar with a small quantity of vehicle until a homogenous suspension was formed and thereafter the entire quantity of the formulation was transferred into measuring cylinder. A small quantity of vehicle was added to rinse the mortar and this was transferred into the measuring cylinder. The rinsing procedure of mortar and pestle was repeated (many times) to ensure the transfer of the contents to the measuring cylinder. Finally, the volume was made up to required quantity with vehicle to get desired concentration of 10, 30 and 100 mg/mL of test item for low, mid and high dose groups respectively. The test formulations were maintained under stirring conditions using magnetic stirrer to maintain homogeneity of the test item formulations.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test item was in-soluble in distilled water and uniformly suspended in 0.5% w/v Carboxy Methyl Cellulose at the concentration of 100 mg/mL (the highest dose concentration selected for the study considering the dose volume of 10 mL/kg body weight) as per in-house solubility/suspendibility test results. Hence, 0.5% w/v Carboxy Methyl Cellulose was used as vehicle for test item formulations and the details were recorded in the raw data and presented in the study report.
- Concentration in vehicle: 10 - 100mg/mL
- Amount of vehicle (if gavage): 10mL/kg bw
- Lot/batch no. (if required): BCBN1690V
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
The stability and homogeneity of the test item in dose formulations was not established under this dose range finding study. However, freshly prepared test item formulations were administered to the animals.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:2
- Length of cohabitation: 2 weeks or until confirmation of mating
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: not detailed
- Verification of same strain and source of both sexes: yes, in-house bred line
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
Gestation Day 5 to Day 19
Frequency of treatment:
once daily
Duration of test:
20 days
Dose / conc.:
0 mg/kg bw/day
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
No. of animals per sex per dose:
10 females in each group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: LD50 of test item Violet sodium polysulfide aluminosilicate with a sodalite (SOD-type) structure is >2000 mg/kg obtained from acute oral toxicity study in rats conducted as per OECD423 TG, The NOAEL of test item is 50 mg/kg when tested at 50, 500 and 5000 mg/kg obtained from a 90-day repeated oral toxicity study in rats conducted similar to ECD408 TG, The NOAEL for maternal toxicity is >=300 mg/kg, NOAEL for reproductive toxicity>=1000 mg/kg and NOAEL for offspring development >=1000 mg/kg when tested at 100, 300 and 1000 mg/kg body weight obtained from a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening study in rats conducted as per OECD422 TG, The NOAEL for maternal toxicity is >100 mg/kg and the NOAEL for fetal toxicity (teratogenicity) is >10000 mg/kg when tested at 10, 100, 1000, 10000 mg/kg obtained from a Prenatal Developmental Toxicity Study in rats conducted in lines with OECD414 TG. Based on the above available information, the doses of 100, 300 and 1000 mg/kg body weight for vehicle control, low dose, mid dose and high dose groups, respectively were selected.
- Rationale for animal assignment (if not random): The body weight of mated females on its GD 0 was recorded and arranged in the ascending order of their body weight until required number of mated females acquired for each group. These mated females were distributed to all the groups based on their body weights so as to maintain comparable mean body weight for all groups
- Fasting period before blood sampling for (rat) dam thyroid hormones: n/a
- Time of day for (rat) dam blood sampling: n/a
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily for clinical signs of toxicity and twice daily for mortality and morbidity


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily for clinical signs of toxicity and twice daily for mortality and morbidity

BODY WEIGHT: Yes
- Time schedule for examinations: GD 0, 3, 5, 8, 11, 14, 17, 19 and on 20

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): no feeding study


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): no drinking study


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: Ovaries, Uterus with cervix, Thyroid with Parathyroid glands

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:
- Plasma: No
- Serum: No
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: No
- Head examinations: No
- Anogenital distance of all live rodent pups: Yes
Statistics:
Parametric: One-way ANOVA with Dunnett’s post test: Gestation body weight (g), Percent change in gestation body weight (%), Corrected body weight (g), Percent change in corrected body weight (%), Gravid uterus weight (g), Feed consumption (g), Mean Fetal weight (g) per dam, Mean Fetal crown rump length (mm) per dam, Mean Fetal ano-genital distance (mm) and ratio per dam, Organ weight. Non-Parametric: Kruskal-Wallis: No. of corpora lutea per dam, No. of implantations per dam, Litter size per dam, No. of live/dead fetuses per dam, No. of early/late resorptions per dam, Percent of live/dead fetuses per dam, Percent of early/late resorptions per dam, Sex ratio (m/f) per dam, Pre/Post-implantation losses (%) per dam, Fetal external anomalies per dam. Frequencies Comparison: Cross Tabs - Chi-square test: Pregnancy rate, No. of dams with/without live fetuses, No. of dams with/without dead fetuses, No. of litters with/without resorptions
Indices:
- Corrected Body weight (g) = (Gestation day 20 body weight - Gestation day 5 body weight) -
Gravid uterus weigh
- Percent of Live Fetuses per dam = (Number of Live Fetuses / Litter Size) x 100
- Percent of Dead Fetuses per dam = (Number of Dead Fetuses/ Litter Size) x 100
- Percent of Early Resorptions (%) per dam = (Number of Early Resorptions/Number of Implantation sites) x 100
- Percent of Late Resorptions (%) per dam = (Number of Late Resorptions / Number of Implantation sites)x 100
- Pre-implantation Loss (%) per dam = [(Number of Corpora lutea - Number of Implantation sites)/ Number of Corpora lutea]x 100
- Post-implantation Loss (%) per Dam = [(Number of Implantation sites - Number of Viable fetuses )/ Number of Implantation sites]x 100
- Sex Ratio (m/f) = Number of live male fetuses / Number of live female fetuses
- Male/Female Fetuses (%) = (Number of live male/female fetuses/ Total number of live fetuses)x 100
- Ano-genital Distance Ratio = Cube root of Fetal weight (g) /Ano-genital distance measurement (mm)
- Fetal incidence (%) = (Number of Fetuses with particular observation per group/Total number of Fetuses examined per group)x 100
- Litter incidence (%) = (Number of Litters/dams with particular observation per group/Total number of Litters per group) x 100










Historical control data:
not available
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
A statistically significant reduction in the percent change in mean maternal body weight gain was noted during Gestation Day (GD) 8 to 11 at group G2 when compared with the vehicle control group. However, this noted change is considered as incidental and unrelated to treatment as there were no changes noted in feed consumption during this period and also no clinical signs were noted at any time point of treatment.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Endocrine findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
a statistically significant increase in the mean absolute thyroid and parathyroid weight was noted at group G3 when compared with the vehicle control group. However, this noted change is considered as incidental and unrelated to treatment, as the relative organ weight was unaffected at this dose level.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There were no effects noted in mean number of implantation sites at all the tested dose groups. The pre-and post-implantation losses per dam were unaffected at all the tested dose groups.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
There were no effects noted in mean number of resorptions.
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Description (incidence and severity):
There were no effects noted in mean live or dead fetuses at all the tested dose groups.
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
There were no effects noted in pregnancy rate at all the tested dose groups.
Other effects:
no effects observed
Description (incidence and severity):
There were no effects noted in mean gravid uterus weight at all the tested dose groups. There were no effects noted in mean number of corpora lutea at all the tested dose groups.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: No effects observed up to the highest dose tested
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
There were no changes noted in mean fetal weight at all the tested dose groups.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
effects observed, non-treatment-related
Description (incidence and severity):
A statistically significant decrease in mean male/female sex ratio was noted at group G2 when compared with vehicle control group. However, this noted change is considered as incidental and unrelated to treatment.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
There were no effects noted in mean litter size at all the tested dose groups.
Anogenital distance of all rodent fetuses:
no effects observed
Description (incidence and severity):
There were no changes noted in mean ano-genital distance measurement / ratio per dam at all the tested dose groups.
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
A total of 102 (9), 111 (10), 82 (8) and 106 (9) fetuses (litters) were available for gross external examination from G1, G2, G3 and G4 respectively. There were no test item- related external abnormalities noted from fetuses of all the tested dose group. However, subcutaneous haemorrhagic spots on skin were noted from tested dose group G3. This noted change is statistically significant when compared with vehicle control group. However, this noted occurrences are considered as incidental and unrelated to treatment, as these are alone incidences from group G3 only noted in three different fetuses belonging to three different dams and also such occasional findings are common in this species.
Skeletal malformations:
not examined
Visceral malformations:
no effects observed
Description (incidence and severity):
There were no test item-related changes noted in any of the group fetuses subjected to visceral (soft tissue) examination on the day of caesarean section.
Other effects:
no effects observed
Description (incidence and severity):
There were no changes noted in mean crown rump length at all the tested dose groups.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other:
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

TABLE 1 SUMMARY OF PREGNANCY STATUS, CLINICAL SIGNS OF TOXICITY AND MORTALITY RECORD                                                           



















































Group, Sex


& Dose


(mg/kg body weight/day)



Pregnancy Status



 



Clinical Signs and Mortality Record



No. of Presumed Pregnant Animals / Group



No. of Females confirmed with Pregnancy



Rate of Pregnancy (%)



Clinical Signs of Toxicity:


Observation (No. of Animals)



No. of Mortalities / Total No. of animals



G1, F & 0



10



9



90.0



N (10)



0/10



G2, F & 100



10



10



100.0



N (10)



0/10



G3, F & 300



10



8



80.0



N (10)



0/10



G4, F & 1000



10



9



90.0



N (10)



0/10



N: Normal


 


TABLE 2 SUMMARY OF GESTATION BODY WEIGHT (g) RECORD










































































































































































Group, Sex & Dose


(mg/kg body weight/day)


 

Body Weight (g) on Gestation Day (GD)



0



3



5



8



11



14



17



19



20



G1, F & 0



Mean



242.41



252.19



260.02



273.04



289.76



305.60



329.65



353.58



369.20



±SD



15.15



17.40



17.11



20.56



22.09



21.84



19.71



22.80



21.67



n



9



9



9



9



9



9



9



9



9



G2, F & 100



Mean



239.23



249.32



256.44



268.04



279.96



296.86



319.79



345.62



359.96



±SD



10.08



10.96



10.92



14.86



17.08



18.97



19.90



25.10



28.76



n



10



10



10



10



10



10



10



10



10



G3, F & 300



Mean



242.20



251.75



258.06



267.35



280.72



298.28



319.66



342.15



355.04



±SD



13.67



13.63



12.54



11.70



9.87



10.89



13.82



18.34



19.08



n



8



8



8



8



8



8



8



8



8



G4, F & 1000



Mean



242.71



251.42



259.28



268.03



283.67



298.68



324.57



344.43



362.16



±SD



6.00



5.27



6.59



7.10



7.92



10.68



15.90



18.41



19.99



n



9



9



9



9



9



9



9



9



9



F: Female; SD: Standard Deviation; n: Number of Animals


 


TABLE 3 SUMMARY OF PERCENT CHANGE IN GESTATION BODY WEIGHT (%) GAIN RECORD





























































































































































Group, Sex & Dose


(mg/kg body weight/day)


 

Percent Change in Body Weight (%) during Gestation Day (GD)



0 to 3



3 to 5



5 to 8



8 to 11



11 to 14



14 to 17



17 to 19



19 to 20



G1, F & 0



Mean



4.00



3.13



4.97



6.13



5.51



7.97



7.25



4.46



±SD



1.29



0.94



2.06



1.37



1.95



2.59



2.06



1.33



n



9



9



9



9



9



9



9



9



G2, F & 100



Mean



4.21



2.87



4.48



4.42*



6.03



7.77



8.03



4.11



±SD



0.72



1.39



1.93



0.96



1.71



2.96



2.65



1.67



n



10



10



10



10



10



10



10



10



G3, F & 300



Mean



3.96



2.53



3.63



5.05



6.26



7.17



7.01



3.77



±SD



0.75



0.62



1.84



2.17



1.82



2.62



2.26



1.09



n



8



8



8



8



8



8



8



8



G4, F & 1000



Mean



3.60



3.12



3.38



5.84



5.27



8.66



6.11



5.16



±SD



1.09



0.81



0.99



1.26



1.22



3.46



1.30



2.36



n



9



9



9



9



9



9



9



9



F: Female; SD: Standard Deviation; n: Number of Animals


*: Statistically significant (P<0.05) change than the vehicle control group


 


TABLE 4 SUMMARY OF GRAVID UTERUS WEIGHT (g) AND MATERNAL BODY WEIGHT CHANGE CORRECTED FOR GRAVID UTERINE WEIGHT (g)






































































































Group, Sex & Dose


(mg/kg body weight/day)



 



Body Weight Change (g)


GD 5 to 20



Gravid Uterus Weight (g)



Corrected Body weight (Gram)



Corrected Body weight (Percentage)



G1, F & 0



Mean



109.18



73.61



35.58



13.68



±SD



14.63



12.25



8.97



3.21



n



9



9



9



9



G2, F & 100



Mean



103.52



70.16



33.36



13.05



±SD



20.37



21.40



10.70



4.33



n



10



10



10



10



G3, F & 300



Mean



96.99



66.23



30.76



12.08



±SD



22.27



12.59



12.33



5.15



n



8



8



8



8



G4, F & 1000



Mean



102.87



70.75



32.13



12.35



±SD



14.51



7.99



9.06



3.34



n



9



9



9



9



F: Female; SD: Standard Deviation; n: No. of Animals


 


TABLE 5 SUMMARY OF AVERAGE GESTATION FEED CONSUMPTION (g/animal/day) RECORD





























































































































































Group, Sex & Dose


(mg/kg body weight/day)


 

Average Feed Consumption (g/animal/day) during Gestation Day (GD)



0 to 3



3 to 5



5 to 8



8 to 11



11 to 14



14 to 17



17 to 19



19 to 20



G1, F & 0



Mean



19.06



19.42



18.57



20.37



22.84



26.14



27.19



26.17



±SD



3.31



2.62



1.68



1.70



1.89



1.12



2.00



2.37



n



9



9



9



9



9



9



9



9



G2, F & 100



Mean



17.21



19.09



18.14



21.01



23.03



25.90



27.38



28.17



±SD



1.32



1.36



1.02



1.06



1.76



1.00



1.67



3.92



n



10



10



10



10



10



10



10



10



G3, F & 300



Mean



17.85



18.14



17.71



20.68



23.30



25.25



26.34



25.99



±SD



1.42



1.30



1.29



0.55



0.75



1.15



1.46



2.68



n



8



8



8



8



8



8



8



8



G4, F & 1000



Mean



17.35



19.72



18.19



20.75



22.83



25.68



26.70



26.83



±SD



1.05



1.70



1.33



1.66



0.84



2.29



2.29



4.42



n



9



9



9



9



9



9



9



9



F: Female; SD: Standard Deviation; n: Number of Animals


 


TABLE 6 SUMMARY OF UTERI OBSERVATIONS PER LITTER DURING CAESAREAN SECTION RECORD





























































































































































Group, Sex & Dose


(mg/kg body weight/day)


 

Average Feed Consumption (g/animal/day) during Gestation Day (GD)



0 to 3



3 to 5



5 to 8



8 to 11



11 to 14



14 to 17



17 to 19



19 to 20



G1, F & 0



Mean



19.06



19.42



18.57



20.37



22.84



26.14



27.19



26.17



±SD



3.31



2.62



1.68



1.70



1.89



1.12



2.00



2.37



n



9



9



9



9



9



9



9



9



G2, F & 100



Mean



17.21



19.09



18.14



21.01



23.03



25.90



27.38



28.17



±SD



1.32



1.36



1.02



1.06



1.76



1.00



1.67



3.92



n



10



10



10



10



10



10



10



10



G3, F & 300



Mean



17.85



18.14



17.71



20.68



23.30



25.25



26.34



25.99



±SD



1.42



1.30



1.29



0.55



0.75



1.15



1.46



2.68



n



8



8



8



8



8



8



8



8



G4, F & 1000



Mean



17.35



19.72



18.19



20.75



22.83



25.68



26.70



26.83



±SD



1.05



1.70



1.33



1.66



0.84



2.29



2.29



4.42



n



9



9



9



9



9



9



9



9



F: Female; SD: Standard Deviation; n: No. of Animals


 


TABLE 6 (Contd…). SUMMARY OF UTERI OBSERVATIONS PER LITTER DURING CAESAREAN SECTION RECORD






































































































Group, Sex & Dose


(mg/kg body weight/day)


 

No. of Female Live Fetuses



No. of Dead Fetuses



No. of Early Resorptions



No. of Late Resorptions



G1, F & 0



Mean



5.11



0.00



0.78



0.11



±SD



1.36



0.00



0.67



0.33



n



9



9



9



9



G2, F & 100



Mean



6.10



0.00



1.10



0.10



±SD



2.02



0.00



1.20



0.32



n



10



10



10



10



G3, F & 300



Mean



4.25



0.00



0.38



0.38



±SD



1.83



0.00



0.74



0.74



n



8



8



8



8



G4, F & 1000



Mean



4.56



0.00



0.78



0.11



±SD



1.51



0.00



0.97



0.33



n



9



9



9



9



F: Female; SD: Standard Deviation; n: No. of Animals


 


TABLE 7 SUMMARY OF MATERNAL DATA PER LITTER RECORD



















































































































Group, Sex & Dose


(mg/kg body weight/day)


 

Pre-Implantation Loss (%)



Post-Implantation Loss (%)



Percent of


Dead Fetus



Percent of


Early Resorptions



Percent of


Late Resorptions



G1, F & 0



Mean



2.50



7.61



0.00



6.81



0.79



±SD



3.76



6.44



0.00



5.93



2.38



n



9



9



9



9



9



G2, F & 100



Mean



3.06



12.65



0.00



11.74



0.91



±SD



5.60



17.97



0.00



18.39



2.87



n



10



10



10



10



10



G3, F & 300



Mean



2.98



6.35



0.00



3.46



2.88



±SD



4.12



9.78



0.00



7.20



5.72



n



8



8



8



8



8



G4, F & 1000



Mean



3.30



6.87



0.00



6.02



0.85



±SD



5.50



9.78



0.00



7.51



2.56



n



9



9



9



9



9



F: Female; SD: Standard Deviation; n: No. of Animals


 


TABLE 7 (Contd…). SUMMARY OF MATERNAL DATA PER LITTER RECORD






































































































Group, Sex & Dose


(mg/kg body weight/day)


 

Male / Female Sex ratio



Male Fetuses (%)



Female Fetuses (%)



Percent of Live Fetuses



G1, F & 0



Mean



1.26



54.99



45.01



100.00



±SD



0.29



5.99



5.99



0.00



n



9



9



9



9



G2, F & 100



Mean



0.80*



41.19*



58.81*



100.00



±SD



0.40



16.92



16.92



0.00



n



10



10



10



10



G3, F & 300



Mean



1.66



58.80



41.20



100.00



±SD



0.85



13.55



13.55



0.00



n



8



8



8



8



G4, F & 1000



Mean



1.84



62.02



37.98



100.00



±SD



0.90



9.91



9.91



0.00



n



9



9



9



9



F: Female; SD: Standard Deviation; n: No. of Animals


*: Statistically significant (P<0.05) change than the vehicle control group


 


TABLE 8 SUMMARY OF ABSOLUTE ORGAN WEIGHT (g) RECORD































































Group, Sex & Dose (mg/kg body weight/day)


 

Thyroid along with parathyroid #



G1, F & 0



Mean



0.0201



±SD



0.0011



n



9



G2, F & 100



Mean



0.0198



±SD



0.0013



n



10



G3, F & 300



Mean



0.0221*



±SD



0.0020



n



8



G4, F & 1000



Mean



0.0204



±SD



0.0017



n



9



F: Female; SD: Standard Deviation; n: No. of Animals; #: Weighed Post fixation


*: Statistically significant (P<0.05) change than the vehicle control group


 


TABLE 9 SUMMARY OF TERMINAL BODY WEIGHT (g) AND ORGAN WEIGHT (%) RELATIVE TO TERMINAL BODY WEIGHT RECORD












































































Group, Sex & Dose (mg/kg body weight/day)


 

Terminal Body Weight (g)



Thyroid along


with parathyroid



G1, F & 0



Mean



369.20



0.0055



±SD



21.67



0.0004



n



9



9



G2, F & 100



Mean



359.96



0.0055



±SD



28.76



0.0008



n



10



10



G3, F & 300



Mean



355.04



0.0063



±SD



19.08



0.0008



n



8



8



G4, F & 1000



Mean



362.16



0.0057



±SD



19.99



0.0007



n



9



9



F: Female; SD: Standard Deviation; n: No. of Animals


 


TABLE 10 SUMMARY OF MEAN FETAL WEIGHT (g) PER LITTER, MEAN FETAL CROWN RUMP LENGTH (mm) PER LITTER AND MEAN ANOGENITAL DISTANCE (AGD) RATIO PER LITTER RECORD



































































































































































Group, Sex & Dose (mg/kg body weight/day)


 

Mean Fetal Weight (g)


 

Mean Crown Rump Length (mm)


 

Ano-genital Distance Measurement (mm)


 

Mean Ano-genital Distance Ratio



Male



Female



Male



Female



Male



Female



Male



Female



G1, F & 0



Mean



4.12



3.86



37.94



36.20



3.55



2.51



2.21



1.60



±SD



0.18



0.21



0.34



0.49



0.13



0.13



0.08



0.09



n



9



9



9



9



9



9



9



9



G2, F & 100



Mean



4.21



3.79



38.19



36.24



3.56



2.51



2.21



1.61



±SD



0.26



0.23



0.61



0.81



0.16



0.12



0.08



0.06



n



9



10



9



10



9



10



9



10



G3, F & 300



Mean



4.01



3.77



37.93



36.00



3.51



2.46



2.21



1.58



±SD



0.14



0.13



0.37



0.43



0.17



0.10



0.10



0.07



n



8



8



8



8



8



8



8



8



G4, F & 1000



Mean



4.17



3.88



37.60



35.74



3.53



2.46



2.20



1.57



±SD



0.17



0.13



0.97



0.95



0.09



0.18



0.05



0.12



n



9



9



9



9



9



9



9



9



F: Female; SD: Standard Deviation; n: No. of Animals


 


TABLE 11 SUMMARY RECORD OF FETAL EXTERNAL EXAMINATION PER LITTER































































































































Parameters



Group



G1



G2



G3



G4



Dose
(mg/kg body weight/day)



0



100



300



1000



Number of Dams



9



10



8



9



Total Number of fetuses evaluated for
External Examination



102



111



82



106



 



 



 



 



 



No. of Fetuses (No. of Litters) noted with
No Abnormality Detected



102 (9)



108 (7)



82 (8)



106 (9)



Percentage of Fetuses (Percentage of Dams) with No Abnormality Detected



100.00 (100.00)



97.30
(70.00)



100.00 (100.00)



100.00 (100.00)



 



 



 



 



 



No. of Fetuses (No. of Dams) noted with Malformations



0 (0)



0 (0)



0 (0)



0 (0)



Percentage of Fetuses (Percentage of Dams) with Malformations



0.0 (0.0)



0.0 (0.0)



0.0 (0.0)



0.0 (0.0)



 



 



 



 



 



No. of Fetuses (No. of Dams) with Variations



0 (0)



3 (3)



0 (0)



0 (0)



Percentage of Fetuses (Percentage of Dams) with Variations



0.00 (0.00)



2.70 (30.00)



0.00 (0.00)



0.00 (0.00)



VARIATIONS



HAEMORRHAGIC SPOTS [EXTERNAL]



Haemorrhagic spot
(occurrence on different parts of the fetus)



Fetal Incidences



0



0.00



3



2.70



0



0.00



0



0.00



Litter Incidences



0



0.00



3*



30.00



0



0.00



0



0.00



*: Statistically significant (P<0.05) change than the vehicle control group


 


TABLE 12 SUMMARY OF FETAL VISCERAL EXAMINATION PER LITTER RECORD


































































































Parameters



Group



G1



G2



G3



G4



Dose


(mg/kg body weight/day)



0



100



500



1000



Number of Dams



9



10



8



9



Total Number of Fetuses evaluated for
Soft Tissue (Visceral) Examination



102



111



82



106



 



 



 



 



 



No. of Fetuses (No. of Litters) noted with
No Abnormality Detected



102 (9)



111 (10)



82 (8)



106 (9)



Percentage of Fetuses (Percentage of Dams) with
No Abnormality Detected



100.00 (100.00)



100.00 (100.00)



100.00 (100.00)



100.00 (100.00)



 



 



 



 



 



No. of Fetuses (No. of Dams) with Malformations



0 (0)



0 (0)



0 (0)



0 (0)



Percent of Fetuses (Percent of Dams) with Malformations



0.00 (0.00)



0.00 (0.00)



0.00 (0.00)



0.00 (0.00)



 



 



 



 



 



No. of Fetuses (No. of Dams) with Variations



0 (0)



0 (0)



0 (0)



0 (0)



Percentage of Fetuses (Percentage of Dams) with Variations



0.00 (0.00)



0.00 (0.00)



0.00 (0.00)



0.00 (0.00)



 


TABLE 13 SUMMARY OF GROSS PATHOLOGY FINDINGS RECORD





































































 


Parameters ↓



Sex



Female



Group & Dose (mg/kg body weight/day)



G1 & 0



G2 & 100



G3 & 300



G4 & 1000



No. of Animals
(both Pregnant and Non-pregnant)



10



10



10



10



No. of Animals found dead during treatment period



0



0



0



0



No. of Animals moribund sacrificed during treatment period



0



0



0



0



No. of Animals sacrificed terminally



10



10



10



10



Gross Pathology Findings



External



No. of animals with No Abnormality Detected



10



10



10



10



Internal



No. of animals with No Abnormality Detected



10



10



10



10



 


 

Conclusions:
The oral administration of test item by gavage to presumed pregnant female Sprague Dawley Rats from Gestation Day 5 to 19 did not produce any indication of maternal and developmental toxicity at the dose levels of 100, 300 and 1000 mg/kg body weight/day under experimental conditions employed.
Executive summary:

To establish doses for a definitive pre-natal developmental toxicity study a dose range finding study was performed similar to OECD 414, non GLP using Sprague Dawley Rats. Doses of 100mg/kg, 300mg/kg and 1000mg/kg were chosen in this study based on available toxicological data in the same or similar substance. 10 pregnant rats were allocated to each dose group and a concurrent vehicle control group of 10 animals was added. Carboxy methyl cellulose was chosen as vehicle and the test item was applied via oral (gavage) from gestation day 5 to 19 once daily. During the experiment, any clinical signs, mortality, body weight development, feed consumption was recorded. On GD20 the animals were euthanized and were subject to gross necropsy and relevant organs like ovaries, uterus with cervix and thyroid and parathyroid glands extracted. The sex, weight, crown-rump lengths, Ano-genital distance of the fetuses were determined and their soft tissue examined. Particular attention was paid to their reproductive tract and if external and internal sex matched. There were no signs of toxiticy in maternal animals for any endpoint. Similar, for pre-natal developmental toxicity no signs of toxicity could be noted besides some non test-item related alterations recorded during the external examination. The oral administration of test item by gavage to presumed pregnant female Sprague Dawley Rats from Gestation Day 5 to 19 did not produce any indication of maternal and developmental toxicity at the dose levels of 100, 300 and 1000 mg/kg body weight/day under experimental conditions employed and the same are then considered appropriate for the definitive test.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 August 2020 to 03 December 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to other study
Remarks:
DRF study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: In-house bred animals
- Age at study initiation: Minimum of 10 weeks
- Weight at study initiation: Males: 251.03 g to 285.09 g & Females: 200.23 g to 238.40 g
- Fasting period before study: No
- Housing: Pre-mating/Acclimatization - Maximum of three animals of same sex per cage were housed in sterilized standard polypropylene cage (Size: L 430 × B 285 × H 150 mm). Steam sterilized clean paddy husk was used as bedding material and was changed along with the cage at least twice a week
- Housing: Mating - During mating, three rats (one male and two females) were housed in standard polypropylene cages.
- Housing: Post mating - After confirming presence of sperm in the vaginal smear (Day 0 of pregnancy), the mated pairs were separated. Males were housed with their former cage mates while females were housed individually in polypropylene cages.
- Diet (e.g. ad libitum): ad libitum - Altromin maintenance diet for rats and mice (manufactured by Altromin Spezialfutter GmbH & Co. KG)
- Water (e.g. ad libitum): ad libitum - Deep bore-well water passed through reverse osmosis unit was provided in plastic water bottles with stainless steel sipper tubes.
- Acclimation period: for a minimum period of five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.1ºC to 23.2 ºC
- Humidity (%): relative humidity 50 to 61%
- Air changes (per hr): 12 to 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light and 12 hours dark

IN-LIFE DATES: From: To: 20 August 2020 to 13 October 2020
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The required quantity of test item was weighed and triturated well in a mortar with a small quantity of vehicle until a homogenous suspension was formed and thereafter the entire quantity of the formulation was transferred into measuring cylinder. A small quantity of vehicle was added to rinse the mortar and this was transferred into the measuring cylinder. The rinsing procedure of mortar and pestle was repeated many times to ensure the transfer of the contents to the measuring cylinder. Finally, the volume was made up to required quantity with vehicle to get desired concentration of 10, 30 and 100 mg/mL of test item for low, mid and high dose groups respectively.

VEHICLE
- Justification for use and choice of vehicle (if other than water): 0.5% w/v Carboxy Methyl Cellulose, test item was insoluble in distilled water
- Concentration in vehicle: 100 mg/mL (maximum)
- Amount of vehicle (if gavage): 10mL/kg
- Lot/batch no. (if required): BCBN1690V
- Purity: n/a
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Verification of concentration and homogonetiy of the test item was conduced by BIONEEDS INDIA PRIVATE LIMITED DEVARAHOSAHALLY, SOMPURA HOBLI, NELAMANGALA TALUK, BANGALORE RURAL DISTRICT, PIN - 562 111, KARNATAKA, INDIA which hold the Good Laboratory Practice Certificate (GLP). 0.1 mL of sample was pipetted out into a digestion tube, a volume of 6 mL of concentrated nitric acid was added and placed in microwave sample preparation system and digested by the selected method. After digestion, the digestion tubes were cooled to room temperature and the contents were transferred to suitable volumetric flasks and diluted with Milli-Q water.


TECHNIQUE AND TEST METHOD: ICP-MS


TEST PARAMETERS: Timings Sweeps : 30 Seconds, Reading : 1, Replicates : 3, Analyte : Aluminium, Mass : 26.9815, Sample Flush : 35 seconds, Read Delay : 15 Seconds, Wash : 45 seconds, Mode : KED, Flow (Helium) : 4.0 mL, Vehicle: 0.5% Carboxymethyl Cellulose


ACCEPTANCE CRITERIA: Homogeneity and dose formulation analysis of prepared test formulations for the dose concentration verification were performed during first and last week of the treatment and the obtained mean results were within the range of 85 to 115% of the nominal concentration and the relative standard deviation (% RSD) is ≤10%.
Details on mating procedure:
- Impregnation procedure: Cohoused
- If cohoused:
- M/F ratio per cage: 1:2 ratio (one male and two females)
- Length of cohabitation: until evidence of copulation was observed or for two weeks.
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility. yes
- Further matings after two unsuccessful attempts: no
- Verification of same strain and source of both sexes: In-house bred animals
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol: no
Duration of treatment / exposure:
Gestation days 5 to 19
Frequency of treatment:
once daily
Duration of test:
Acclimatization: 20 August 2020 to 25 August 2020
Cohabitation: 26 August 2020 to 23 September 2020
Treatment: 01 September 2020 to 12 October 2020
Necropsy: 16 September 2020 to 13 October 2020
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: dose range finding study, conducted with the doses of 100, 300 and 1000 mg/kg body weight based on the available literature. Oral administration of test item to pregnant female Sprague Dawley Rats from Gestation Day 5 to 19 did not produce any indication of maternal and prenatal-developmental toxicity at all these tested dose levels in the dose range finding study.
- Rationale for animal assignment (if not random): evenly distributed to all the groups based on their body weights so as to maintain comparable mean body weight for all groups
- Fasting period before blood sampling for (rat) dam thyroid hormones: 0
- Time of day for (rat) dam blood sampling: within 2 hours before necropsy.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily for clinical signs of toxicity and twice daily for mortality and morbidity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on gestation days (GD) 0, 3, 5, 8, 11, 14, 17, 19 and on 20 (day of caesarean section).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): not feeding study
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Recorded as g/animal/day during gestation days 0-3, 3-5, 5-8, 8-11, 11-14, 14-17, 17-19 and 19-20
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No applicable

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not drinking water study


POST-MORTEM EXAMINATIONS: Yes / No / No data
- Sacrifice on gestation day 20
- Organs examined: Ovaries, Uterus with cervix, Thyroid and Parathyroid glands
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: observation of uterine content.
Blood sampling:
- Plasma: No
- Serum: Yes
- Volume collected - n/a
- Other: blood samples were collected as follows:
- From all dams at termination for mandatory assessment of thyroid hormones T4, T3 and thyroid stimulating hormone (TSH) within 2 hours before necropsy.
- Blood samples from non-pregnant females were also collected but were not pooled with the pregnant dams.
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No
- Anogenital distance of all live rodent pups: Individual fetus was measured for its anogenital distance on the day of caesarean section
Statistics:
Parametric: One-way ANOVA with Dunnett’s post test: Maternal body weight, Percent change in maternal body weight , Corrected body weight for maternal increase, Gravid uterus weight, Maternal feed consumption, Mean fetal weight per dam, Mean fetal crown rump length per dam, Mean fetal anogenital distance per dam, Serum T3, T4 and TSH levels, Organ weight

Non-Parametric: Kruskal-Wallis: No. of corpora lutea per dam, No. of implantations per dam, Litter size per dam, No. of live/dead fetuses per dam, Percent of live/dead fetuses per dam, No. of early/late resorptions per dam, Percent of early/late resorptions per dam, Sex ratio (m/f) per dam, Pre/Post implantation losses (%) per dam, Fetal external / visceral / skeletal anomalies per dam

Frequencies Comparison: Cross Tabs -Chi-square test: No. of Pregnant / Non-pregnant females (Pregnancy status), No. of dams with / without live fetuses, No. of dams with / without dead fetuses, No. of dams with / without resorptions

Indices:
- Corrected Body weight (g) = (Gestation day 20 body weight - Gestation day 5 body weight) -
Gravid uterus weigh
- Percent of Live Fetuses per dam = (Number of Live Fetuses / Litter Size) x 100
- Percent of Dead Fetuses per dam = (Number of Dead Fetuses/ Litter Size) x 100
- Percent of Early Resorptions (%) per dam = (Number of Early Resorptions/Number of Implantation sites) x 100
- Percent of Late Resorptions (%) per dam = (Number of Late Resorptions / Number of Implantation sites)x 100
- Pre-implantation Loss (%) per dam = [(Number of Corpora lutea - Number of Implantation sites)/ Number of Corpora lutea]x 100
- Post-implantation Loss (%) per Dam = [(Number of Implantation sites - Number of Viable fetuses )/ Number of Implantation sites]x 100
- Sex Ratio (m/f) = Number of live male fetuses / Number of live female fetuses
- Male/Female Fetuses (%) = (Number of live male/female fetuses/ Total number of live fetuses)x 100
- Ano-genital Distance Ratio = Ano-genital distance (mm) / Cube root of Fetal weight (g)
- Fetal incidence (%) = (Number of Fetuses with particular observation per group/Total number of Fetuses examined per group)x 100
- Litter incidence (%) = (Number of Litters/dams with particular observation per group/Total number of Litters per group) x 100










Historical control data:
Historical control data not included in report. Historical control data from in-house of the same species and strain.
Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs of toxicity noted at all the tested dose groups and vehicle control group animals during the experimental period.
Mortality:
no mortality observed
Description (incidence):
There were no morbidity/mortality noted at all the tested dose groups and vehicle control group animals during the experimental period.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significant increase in mean gestation body weight on day 5 and statistically significant decrease in percent change in mean gestation body weight gain during day 5 to 8 was noted at group G4 when compared with the vehicle control group. These changes are considered as incidental and toxicologically insignificant as there were no changes noted in daily observations of animals and also no effects were noted in mean feed consumption during this period at this dose level.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significant increase in mean gestation (maternal) feed consumption during day 19 to 20 was noted at group G4 when compared with the vehicle control group. This change is considered as incidental and toxicologically insignificant as there were no changes noted in daily observations of animals and also no effects were noted in mean percent change in gestation body weight gain during this period at this dose level.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Endocrine findings:
effects observed, non-treatment-related
Description (incidence and severity):
Thyroid hormone T3: There were no test item-related changes noted for this parameter across the dose groups when compared to the vehicle control group. The noted statistically significant decrease in mean serum T3 levels (ng/mL) at group G4 is considered as incidental and un-related to treatment with test item due to lack of a dose-response relationship and also the obtained values were within lab historical control range of same species and strain.

Thyroid hormone T4: There were no test item-related changes noted for this parameter across the dose groups when compared to the vehicle control group. The noted statistically significant decrease in serum T4 levels (ng/mL) at group G4 is considered as incidental and unrelated to treatment with test item due to lack of a dose-response relationship and also the obtained values were within the lab historical control range of same species and strain.

Thyroid stimulating hormone THS: There were no test item-related changes noted for this parameter across the dose groups when compared to the vehicle control group. The noted statistically significant decrease in serum TSH levels (μIU/mL) at group G3 is considered as incidental and unrelated to treatment with test item due to lack of a dose-response relationship and also the obtained values were within the lab historical control range of same species and strain.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
The thyroid along with parathyroid was collected, preserved and weighed post fixation from all the dams of each dose group. There were no changes noted for mean absolute and relative weight for this organ in all the tested dose groups when compared to the vehicle control group.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no gross pathological changes noted in any of the animals from all the tested dose groups and vehicle control group during conduct of the necropsy.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related changes noted in thyroid along with the parathyroid subjected to histopathological examination at all the tested dose group animals. Single incidence of minimal, diffuse hypertrophy of follicular cells in groups G2 and G4, single incidence of minimal infiltration of inflammatory cells in group G3 and single incidence of ultimobranchial cyst/s in thyroid gland in each group (G1, G2, G3 and G4) were noted during microspic examination. However, all these changes were considered as incidental findings and/or were expected for laboratory rats of this age (Elizabeth McInnes, 2012).
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There were no statistically significant differences noted for percentage of pre- or post-implantation loss per litter at all the tested dose groups when compared to the vehicle control group.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Description (incidence and severity):
There were no statistically significant differences in the number and percentage of early or late resorptions per dam across dose groups when compared to the vehicle control group.
Dead fetuses:
no effects observed
Description (incidence and severity):
There were no statistically significant differences noted for number of dead fetuses at all the tested dose groups when compared to the vehicle control group.
Changes in pregnancy duration:
not specified
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Pregnancy with rates of 96%, 92%, 92% and 96% in groups G1, G2, G3 and G4 respectively.
Other effects:
no effects observed
Description (incidence and severity):
The mean number of corpora lutea per litter was 11.63, 12.26, 11.78 and 11.96 for groups G1, G2, G3 and G4 respectively. There were no changes or no statistically significant differences noted at all the tested dose groups when compared with vehicle control group for number of corpora lutea.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: Highest concentration tested, no effects observed
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
There were no changes noted in mean fetal weight of either sex across the dose groups when compared with the vehicle control group.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
There were no statistically significant differences noted for number of live fetuses at all the tested dose groups when compared to the vehicle control group.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There were no statistically significant differences noted for male/female sex ratio across the dose groups when compared to the control group.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
The mean litter sizes, assessed as the total number of fetuses in utero (live plus dead) per dam, was 10.33, 11.00, 10.26 and 10.67 for groups G1, G2, G3 and G4, respectively. There were no changes or no statistically significant differences noted at all the tested dose groups when compared with vehicle control group for litter size.
Anogenital distance of all rodent fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
The mean male fetal anogenital distance per litter was 4.44 mm, 4.47 mm, 4.47 mm and 4.37 mm and the mean female anogenital distance per litter was 2.43 mm, 2.50 mm, 2.50 mm and 2.43 mm for groups G1, G2, G3 and G4 respectively.
The mean male fetal anogenital distance ratio per litter was 2.69, 2.75, 2.76 and 2.69 and the mean female anogenital distance ratio per litter was 1.52, 1.56, 1.56 and 1.52 for groups G1, G2, G3 and G4 respectively.

Statistically significant increase in mean female fetal anogenital distance ratio per litter was noted at groups G2 and G3 when compared with the vehicle control group. These changes are considered as incidental and unrelated to treatment as the obtained range is within in-house historical control range.
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The general / developmental variations such as subcutaneous hemorrhagic spot/s beneath the skin on different regions of the body, pale skin colored fetuses, kinked tail and noted external malformations such as hyperextension of hindlimb – unilateral or short hindlimb - unilateral, noted across the tested dose group litters are considered incidental as these incidences are comparable with the vehicle control group and also these developmental alterations are common for this species and strain. Also, no remarkable differences or statistically significant changes were noted for these alterations in any of the tested dose groups when compared with vehicle control group.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related fetal skeletal malformations or skeletal developmental variations for any of the fetuses examined from all the tested dose group litters.
Occasional fetal incidences (litter incidences) of skeletal developmental variations were noted across the tested dose group litters: incomplete ossification of skull bones, incomplete ossification or unossification of sternabral bones, unilateral or bilateral wavy ribs, extra ossification site/s near to first lumbar vertebra i.e. immediately after 13th rib, dumbbell / semi-bipartite ossification of thoracic and lumbar verterbral centrum, incomplete ossification of thoracic verterbral centrum no. 10, unossification of metacarpal no. 5 of forelimb, incomplete ossification or unossification of proximal phalanges of forelimb - bilateral.
Occasional incidences of skeletal malformation were noted across the tested dose group litters:
-supplimentary rib - unilateral (14th rib) - two fetal incidences of different litters from group G3;
- supplimentary rib - bilateral (14th rib) - single fetal incidence from one litter from group G3;
- rudimentary rib - unilateral (14th rib) - two fetal incidences from one litter in group G3;
- thoracic vertebra centrum no. 9 - split - 1, 1, 0 and 0 fetal and litter incidences from group G1, G2, G3 and G4 respectively.
- thoracic vertebra centrum no. 10 - split 1, 1, 0 and 0 fetal and litter incidences from group G1, G2, G3 and G4 respectively.
- thoracic vertebra centrum no. 11 - split 1, 0, 0 and 1 fetal and litter incidences from group G1, G2, G3 and G4 respectively.
- thoracic vertebra centrum no. 12 - split 2, 1, 0 and 3 fetal and litter incidences from group G1, G2, G3 and G4 respectively.
- thoracic vertebra centrum no. 13 - split 1, 1, 1 and 0 fetal and litter incidences from group G1, G2, G3 and G4 respectively.

These skeletal developmental variations and skeletal malformations are considered as incidental and un-related to treatment as these findings occurred infrequently or at a frequency similar to the vehicle control group and did not occur in a dose-dependant manner. Also, the occurred mean litter/fetal proportions were within the lab historical control range of this species and strain.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related fetal visceral/soft tissue malformations or developmental variations for any of the fetuses examined from all the tested dose group litters. The developmental variations such as pale or discolored lung lobes, pale/discolored liver lobes, fused liver lobes or dilatation of renal pelvis noted across the tested dose group litters are considered incidental as these incidences are comparable with the vehicle control group and also these developmental variations are common for this species and strain. There were no remarkable differences or statistically significant changes noted for these variations in any of the tested dose groups when compared with vehicle control group.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
The mean male fetal crown rump length per litter was 37.38 mm, 38.10 mm, 38.10 mm and 38.17 mm and the female mean crown rump length per litter was 36.12 mm, 37.39 mm, 37.55 mm and 37.31 mm for groups G1, G2, G3 and G4 respectively. Statistically significant increase in mean male and female fetal crown rump length per litter was noted at all the tested dose groups when compared with the vehicle control group. These changes are considered as incidental and unrelated to treatment as the obtained range is within the lab historical control range.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Highest dose tested, no effects observed
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

TABLE 1. SUMMARY OF PREGNANCY STATUS, CLINICAL SIGNS OF TOXICITY AND MORTALITY RECORD                                                           



















































Group


& Dose


(mg/kg body weight/day)



Pregnancy Status



 



Clinical Signs and Mortality Record



No. of Presumed


Pregnant Animals / Group



No. of Females


confirmed with


Pregnancy



Rate of Pregnancy (%)



Clinical Signs of


Toxicity revealed


(No. of Animals)



No. of Mortalities /


Total No. of animals



G1 & 0



25



24



96.0



N (25)



0/25



G2 & 100



25



23



92.0



N (25)



0/25



G3 & 300



25



23



92.0



N (25)



0/25



G4 & 1000



25



24



96.0



N (25)



0/25



                                                       


TABLE 2. SUMMARY OF GESTATION BODY WEIGHT (g) RECORD










































































































































































Group & Dose


(mg/kg body weight/day)


 

Body Weight (g) on Gestation Day (GD)



0



3



5



8



11



14



17



19



20



G1 & 0



Mean



227.66



235.37



241.69



252.47



266.32



281.74



302.70



322.31



333.97



±SD



7.24



8.55



8.64



8.80



10.03



11.24



12.17



13.09



14.30



n



24



24



24



24



24



24



24



24



24



G2 & 100



Mean



229.32



237.70



243.76



254.17



268.02



284.04



306.43



327.34



339.75



±SD



7.43



8.57



8.41



9.83



9.59



10.45



12.93



14.14



15.10



n



23



23



23



23



23



23



23



23



23



G3 & 300



Mean



228.93



237.43



244.12



253.84



266.36



281.79



303.07



324.11



336.45



±SD



6.68



6.94



6.96



8.31



8.54



8.44



11.08



15.03



15.90



n



23



23



23



23



23



23



23



23



23



G4 & 1000



Mean



232.84



240.58



248.10*



257.18



270.47



285.42



306.45



327.75



339.95



±SD



10.50



9.99



10.09



10.07



9.32



9.38



10.98



10.51



11.44



n



24



24



24



24



24



24



24



24



24



*: Statistically significant (P<0.05) change than the control group


 


TABLE 3. SUMMARY OF PERCENT CHANGE IN GESTATION BODY WEIGHT (%) RECORD





























































































































































Group & Dose


(mg/kg body weight/day)


 

Percent Change in Body Weight Gain (%) during Gestation Day (GD)



0 to 3



3 to 5



5 to 8



8 to 11



11 to 14



14 to 17



17 to 19



19 to 20



G1 & 0



Mean



3.38



2.69



4.47



5.48



5.79



7.44



6.49



3.61



±SD



1.17



0.81



1.13



1.15



1.14



0.82



1.83



1.00



n



24



24



24



24



24



24



24



24



G2 & 100



Mean



3.66



2.57



4.26



5.47



5.98



7.87



6.83



3.79



±SD



1.97



1.68



1.24



1.40



1.11



1.71



1.69



0.79



n



23



23



23



23



23



23



23



23



G3 & 300



Mean



3.72



2.82



3.97



4.94



5.80



7.54



6.92



3.81



±SD



1.01



0.86



1.03



0.89



1.12



1.44



2.07



0.85



n



23



23



23



23



23



23



23



23



G4 & 1000



Mean



3.34



3.14



3.67*



5.19



5.54



7.37



6.97



3.72



±SD



1.18



1.11



0.94



1.21



0.87



1.66



1.51



0.93



n



24



24



24



24



24



24



24



24



*: Statistically significant (P<0.05) change than the control group


 


TABLE 4. SUMMARY OF GRAVID UTERUS WEIGHT (g) AND MATERNAL BODY WEIGHT CHANGE CORRECTED FOR GRAVID UTERINE WEIGHT (g)









































































































Group


& Dose


(mg/kg body weight/day)


 

Body Weight Change (g) from GD 5 to 20



Gravid Uterus Weight (g)



Corrected Body Weight



g



%



G1 & 0



Mean



92.29



64.90



27.38



11.37



±SD



10.08



8.44



6.38



2.84



n



24



24



24



24



G2 & 100



Mean



95.99



69.64



26.35



10.84



±SD



12.05



11.39



5.02



2.19



n



23



23



23



23



G3 & 300



Mean



92.33



65.24



27.09



11.13



±SD



14.08



12.54



6.28



2.72



n



23



23



23



23



G4 & 1000



Mean



91.85



66.79



25.06



10.15



±SD



10.71



8.17



5.81



2.52



n



24



24



24



24



 


TABLE 5. SUMMARY OF AVERAGE GESTATION FEED CONSUMPTION (g/animal/day) RECORD





























































































































































Group & Dose


(mg/kg body weight/day)



 



Feed Consumption (g/animal/day) during Gestation Day (GD)



0 to 3



3 to 5



5 to 8



8 to 11



11 to 14



14 to 17



17 to 19



19 to 20



G1 & 0



Mean



16.52



17.59



17.45



18.80



20.35



21.65



25.41



24.34



±SD



1.74



1.55



1.48



1.47



1.36



1.29



2.44



1.86



n



24



24



24



24



24



24



24



24



G2 & 100



Mean



16.81



18.47



17.55



19.15



20.56



21.98



26.49



25.47



±SD



1.51



1.29



1.49



1.40



1.28



1.25



1.80



1.74



n



23



23



23



23



23



23



23



23



G3 & 300



Mean



16.77



18.12



17.36



18.68



20.17



21.86



25.88



25.23



±SD



1.46



1.64



1.42



1.44



1.46



1.28



2.16



2.37



n



23



23



23



23



23



23



23



23



G4 & 1000



Mean



16.11



18.39



17.09



18.72



20.46



22.20



26.52



26.19*



±SD



1.59



2.07



1.28



1.27



1.20



0.92



1.88



1.96



n



24



24



24



24



24



24



24



24



*: Statistically significant (P<0.05) change than the control group


 


TABLE 6. SUMMARY OF UTERI OBSERVATIONS PER LITTER RECORD










































































































































































Group


& Dose


(mg/kg body weight/day)


 

No. of


Corpora lutea



No. of Implantations



Litter Size



No. of Live Fetuses



No. of
Dead Fetuses



No. of
Early Resorptions



No. of
Late Resorptions



Total



Male



Female



G1 & 0



Mean



11.63



11.08



10.33



10.33



5.38



4.96



0.00



0.75



0.00



±SD



2.04



2.00



1.83



1.83



1.74



1.78



0.00



0.79



0.00



n



24



24



24



24



24



24



24



24



24



G2 & 100



Mean



12.26



11.61



11.00



11.00



6.17



4.83



0.00



0.61



0.00



±SD



2.03



1.97



2.22



2.22



1.80



1.92



0.00



1.03



0.00



n



23



23



23



23



23



23



23



23



23



G3 & 300



Mean



11.78



10.83



10.26



10.26



5.35



4.91



0.00



0.57



0.00



±SD



2.07



2.08



1.96



1.96



1.99



1.35



0.00



0.73



0.00



n



23



23



23



23



23



23



23



23



23



G4 & 1000



Mean



11.96



11.29



10.67



10.63



5.63



5.00



0.04



0.58



0.04



±SD



1.88



1.57



1.71



1.69



1.56



1.32



0.20



0.72



0.20



n



24



24



24



24



24



24



24



24



24



 


TABLE 7. SUMMARY OF MATERNAL DATA PER LITTER RECORD


























































































































































































Group


& Dose


(mg/kg body weight/day)



 



Pre-Implantation Loss


(%)



Post-Implantation Loss


(%)



Percent of Dead Fetus (%)



Percent of Early Resorptions (%)



Percent of Late Resorptions (%)



Male/Female Sex Ratio



Male


Fetuses


(%)



Female Fetuses


(%)



Total
Live Fetuses (%)


 
 
 

G1 & 0



Mean



4.49



6.61



0.00



6.61



0.00



1.38



52.26



47.74



100.00


 

±SD



6.27



6.46



0.00



6.46



0.00



1.08



14.67



14.67



0.00


 

n



24



24



24



24



24



24



24



24



24


 

G2 & 100



Mean



5.18



5.27



0.00



5.27



0.00



1.56



56.68



43.32



100.00


 

±SD



7.16



9.08



0.00



9.08



0.00



0.88



13.54



13.54



0.00


 

n



23



23



23



23



23



23



23



23



23


 

G3 & 300



Mean



8.04



4.99



0.00



4.99



0.00



1.21



51.22



48.78



100.00


 

±SD



8.05



6.38



0.00



6.38



0.00



0.67



12.45



12.45



0.00


 

n



23



23



23



23



23



23



23



23



23


 

G4 & 1000



Mean



5.21



5.98



0.35



5.36



0.30



1.26



52.71



47.29



99.65


 

±SD



6.06



7.16



1.70



6.98



1.46



0.70



11.24



11.24



1.70


 

n



24



24



24



24



24



24



24



24



24


 

 


TABLE 8. SUMMARY OF ABSOLUTE ORGAN WEIGHT (g) RECORD































































Group & Dose


(mg/kg body weight/day)


 

Absolute Thyroid along with Parathyroid Weight (g)#



G1 & 0



Mean



0.0194



±SD



0.0011



n



24



G2 & 100



Mean



0.0205



±SD



0.0016



n



23



G3 & 300



Mean



0.0191



±SD



0.0025



n



23



G4 & 1000



Mean



0.0200



±SD



0.0012



n



24



 


TABLE 9. SUMMARY OF TERMINAL BODY WEIGHT (g) AND ORGAN WEIGHT (%) RELATIVE TO TERMINAL BODY WEIGHT RECORD












































































Group & Dose


(mg/kg body weight/day)


 

Terminal Body Weight (g)



Relative Thyroid along


with Parathyroid weight
(%)



G1 & 0



Mean



333.97



0.0058



±SD



14.30



0.0004



n



24



24



G2 & 100



Mean



339.75



0.0060



±SD



15.10



0.0004



n



23



23



G3 & 300



Mean



336.45



0.0057



±SD



15.90



0.0007



n



23



23



G4 & 1000



Mean



339.95



0.0059



±SD



11.44



0.0004



n



24



24



 


TABLE 10. SUMMARY OF SERUM TRIIODOTHYRONINE (T3) LEVELS (ng/mL) RECORD































































Group & Dose


(mg/kg body weight/day)


 

Serum T3 Levels (ng/mL)



G1 & 0



Mean



2.341



±SD



0.347



n



24



G2 & 100



Mean



2.219



±SD



0.117



n



23



G3 & 300



Mean



2.255



±SD



0.155



n



23



G4 & 1000



Mean



2.046*



±SD



0.190



n



24



* Statistically significant (P<0.05) change than the control group


 


TABLE 11. SUMMARY OF SERUM THYROXINE (T4) LEVELS (ng/mL) RECORD































































Group & Dose


(mg/kg body weight/day)


 

Serum T4 Levels (ng/mL)



G1 & 0



Mean



65.090



±SD



9.062



n



24



G2 & 100



Mean



62.287



±SD



5.247



n



23



G3 & 300



Mean



63.891



±SD



6.606



n



23



G4 & 1000



Mean



58.756*



±SD



6.290



n



24



* Statistically significant (P<0.05) change than the control group


 


TABLE 12. SUMMARY OF SERUM THYROID STIMULATING HORMONE (TSH) LEVELS (µIU/mL) RECORD































































Group & Dose


(mg/kg body weight/day)


 

Serum TSH Levels (µIU/mL)



G1 & 0



Mean



8.795



±SD



7.224



n



20



G2 & 100



Mean



7.262



±SD



5.641



n



19



G3 & 300



Mean



4.171*



±SD



4.266



n



21



G4 & 1000



Mean



9.573



±SD



6.067



n



17



* Statistically significant (P<0.05) change than the control group


 


TABLE 13. SUMMARY OF MEAN FETAL WEIGHT, MEAN FETAL CROWN RUMP LENGTH, MEAN FETAL ANOGENITAL DISTANCE AND RATIO RECORD



































































































































































Group & Dose
(mg/kg body weight/day)


 

Fetal Weight (g)


 

Crown Rump Length (mm)


 

Anogenital Distance Measurement (mm)


 

Anogenital Distance Ratio



Male



Female



Male



Female



Male



Female



Male



Female



G1 & 0



Mean



4.32



4.10



37.38



36.12



4.45



2.43



2.70



1.52



±SD



0.18



0.19



1.06



1.04



0.13



0.11



0.18



0.07



n



24



24



24



24



24



24



24



24



G2 & 100



Mean



4.29



4.12



38.10*



37.39*



4.47



2.50



2.75



1.56



±SD



0.12



0.11



0.66



0.65



0.13



0.10



0.06



0.05



n



23



23



23



23



23



23



23



23



G3 & 300



Mean



4.26



4.10



38.09*



37.55*



4.47



2.50



2.76



1.56



±SD



0.15



0.17



0.46



0.40



0.11



0.10



0.06



0.05



n



23



23



23



23



23



23



23



23



G4 & 1000



Mean



4.27



4.09



38.17*



37.31*



4.37



2.41



2.69



1.51



±SD



0.15



0.19



0.59



0.58



0.15



0.12



0.08



0.07



n



24



24



24



24



24



24



24



24



* Statistically significant (P<0.05) change than the control group


Note: The presented mean values are group mean of all litters from each group which was calculated from mean of individual fetal values obtained from each litter sex wise. 


 


TABLE 14. SUMMARY RECORD OF FETAL EXTERNAL EXAMINATION PER LITTER



































































































































Group



G1



G2



G3



G4



Dose (mg/kg body weight/day)



0



100



300



1000



 



Litters Evaluated for External Examination



No.



24



23



23



24



Fetuses Evaluated for External Examination



No.



248



253



236



255



Variations



Region/Tissue



Alteration


 

General



Subcutaneous hemorrhagic spot beneath the skin on different regions of the body



Litter Incidences



No. (%)



6



(25.0)



8



(34.8)



6



(26.1)



9



(37.5)



Fetal Incidences



No. (%)



9



(3.6)



10



(4.0)



8



(3.4)



10



(3.9)



General



Pale colored Fetus



Litter Incidences



No. (%)



2



(8.3)



4



(17.4)



1



(4.3)



3



(12.5)



Fetal Incidences



No. (%)



2



(0.8)



4



(1.6)



2



(0.8)



3



(1.2)



Tail



Kinked



Litter Incidences



No. (%)



0



(0.0)



1



(4.3)



0



(0.0)



0



(0.0)



Fetal Incidences



No. (%)



0



(0.0)



1



(0.4)



0



(0.0)



0



(0.0)



 







































































































Group



G1



G2



G3



G4



Dose (mg/kg body weight/day)



0



100



300



1000


 

Litters Evaluated for External Examination



No.



24



23



23



24



Fetuses Evaluated for External Examination



No.



248



253



236



255



Malformations



Region/Tissue



Alteration


 

Hindlimb



Hyperextension (unilateral)



Litter Incidences



No. (%)



1



(4.2)



0



(0.0)



0



(0.0)



0



(0.0)



Fetal Incidences



No. (%)



1



(0.4)



0



(0.0)



0



(0.0)



0



(0.0)



Hindlimb



Short (unilateral)



Litter Incidences



No. (%)



0



(0.0)



1



(4.3)



0



(0.0)



0



(0.0)



Fetal Incidences



No. (%)



0



(0.0)



1



(0.4)



0



(0.0)



0



(0.0)



 


TABLE 15. SUMMARY OF FETAL VISCERAL EXAMINATION PER LITTER RECORD





























































































































































































Group


 

G1



G2



G3



G4



Dose (mg/kg body weight/day)


 

0



100



300



1000


 

Litters Evaluated for External Examination



No.



24



23



23



24



Fetuses Evaluated for External Examination



No.



118



121



111



120



Variations



Region/Tissue↓



Alteration↓


 

Lungs



Pale/discolored



Litter Incidences



No. (%)



0



(0.0)



1



(4.3)



1



(4.3)



0



(0.0)



Fetal Incidences



No. (%)



0



(0.0)



1



(0.8)



1



(0.9)



0



(0.0)



Liver



Lobes Fused



Litter Incidences



No. (%)



0



(0.0)



0



(0.0)



0



(0.0)



1



(4.2)



Fetal Incidences



No. (%)



0



(0.0)



0



(0.0)



0



(0.0)



1



(0.8)



Liver



Pale/Discolored



Litter Incidences



No. (%)



2



(8.3)



1



(4.3)



0



(0.0)



1



(4.2)



Fetal Incidences



No. (%)



2



(1.7)



1



(0.8)



0



(0.0)



1



(0.8)



Kidneys



Renal Pelvis Dilatation (Bilateral)



Litter Incidences



No. (%)



5



(20.8)



2



(8.7)



4



(17.4)



3



(12.5)



Fetal Incidences



No. (%)



5



(4.2)



3



(2.5)



5



(4.5)



3



(2.5)



Ureters



Dilatation



Litter Incidences



No. (%)



4



(16.7)



4



(17.4)



4



(17.4)



3



(12.5)



Fetal Incidences



No. (%)



4



(3.4)



5



(4.1)



4



(3.6)



3



(2.5)



 


TABLE 16. SUMMARY OF SKELETAL EXAMINATION OF FETUSES PER LITTER RECORD














































































































































































































































































































Group



 



G1



G2



G3



G4



Dose (mg/kg body weight/day)



 



0



100



300



1000



 



Litters Evaluated for External Examination



No.



24



23



23



24



Fetuses Evaluated for External Examination



No.



130



132



125



135



 Variations



Region



Alteration



 



 



 



 



 



 



 



 



 



Skull



Parietal bones - Incomplete ossification



Litter Incidences



No. (%)



0



(0.0)



0



(0.0)



0



(0.0)



1



(4.2)



Fetal Incidences



No. (%)



0



(0.0)



0



(0.0)



0



(0.0)



1



(0.7)



Interparietal bones - Incomplete ossification 



Litter Incidences



No. (%)



0



(0.0)



1



(4.3)



0



(0.0)



1



(4.2)



Fetal Incidences



No. (%)



0



(0.0



3



(2.3)



0



(0.0)



1



(0.7)



Sternum


 



Sternebra No. 2 - Unossified 



Litter Incidences



No. (%)



0



(0.0)



0



(0.0)



0



(0.0)



1



(4.2)



Fetal Incidences



No. (%)



0



(0.0)



0



(0.0)



0



(0.0)



1



(0.7)



Sternebra No. 3 - Unossified 



Litter Incidences



No. (%)



0



(0.0)



0



(0.0)



0



(0.0)



1



(4.2)



Fetal Incidences



No. (%)



0



(0.0)



0



(0.0)



0



(0.0)



1



(0.7)



Sternebra No. 5 - Unossified 



Litter Incidences



No. (%)



8



(33.3)



2



(8.7)



3



(13.0)



7



(29.2)



Fetal Incidences



No. (%)



9



(6.9)



6



(4.5)



3



(2.4)



8



(5.9)



Sternebra No. 6 - Unossified 



Litter Incidences



No. (%)



2



(8.3)



2



(8.7)



2



(8.7)



2



(8.3)



Fetal Incidences



No. (%)



2



(1.5)



2



(1.5)



2



(1.6)



4



(3.0)



Sternebra No. 4 - Incomplete ossification



Litter Incidences



No. (%)



1



(4.2)



0



(0.0)



1



(4.3)



0



(0.0)



Fetal Incidences



No. (%)



1



(0.8)



0



(0.0)



1



(0.8)



0



(0.0)



Sternebra No. 5 - Incomplete ossification 



Litter Incidences



No. (%)



2



(8.3)



1



(4.3)



1



(4.3)



2



(8.3)



Fetal Incidences



No. (%)



3



(2.3)



1



(0.8)



1



(0.8)



2



(1.5)



Sternebra No. 6 - Incomplete ossification 



Litter Incidences



No. (%)



8



(33.3)



4



(17.4)



6



(26.1)



9



(37.5)



Fetal Incidences



No. (%)



13



(10.0)



12



(9.1)



14



(11.2)



18



(13.3)



 








































































































































































































































































































































































































































































































































































































































Group



 



G1



G2



G3



G4



Dose (mg/kg body weight/day)



 



0



100



300



1000



 



Litters Evaluated for External Examination



No.



24



23



23



24



Fetuses Evaluated for External Examination



No.



130



132



125



135



Variations 



Region



Alteration



 



 



 



 



 



 



 



 



 



Ribs


 



Rib No. 6 - Wavy (Unilateral) 



Litter Incidences



No. (%)



1



(4.2)



0



(0.0)



0



(0.0)



0



(0.0)



Fetal Incidences



No. (%)



1



(0.8)



0



(0.0)



0



(0.0)



0



(0.0)



Rib No. 7 - Wavy (Unilateral) 



Litter Incidences



No. (%)



1



(4.2)



0



(0.0)



0



(0.0)



0



(0.0)



Fetal Incidences



No. (%)



1



(0.8)



0



(0.0)



0



(0.0)



0



(0.0)



Rib No. 8 - Wavy (Unilateral) 



Litter Incidences



No. (%)



1



(4.2)



0



(0.0)



0



(0.0)



0



(0.0)



Fetal Incidences



No. (%)



1



(0.8)



0



(0.0)



0



(0.0)



0



(0.0)



Rib No. 9 - Wavy (Unilateral) 



Litter Incidences



No. (%)



1



(4.2)



0



(0.0)



0



(0.0)



0



(0.0)



Fetal Incidences



No. (%)



1



(0.8)



0



(0.0)



0



(0.0)



0



(0.0)



Rib No. 10 - Wavy (Unilateral) 



Litter Incidences



No. (%)



1



(4.2)



0



(0.0)



0



(0.0)



0



(0.0)



Fetal Incidences



No. (%)



1



(0.8)



0



(0.0)



0



(0.0)



0



(0.0)



Rib No. 11 - Wavy (Unilateral) 



Litter Incidences



No. (%)



1



(4.2)



0



(0.0)



0



(0.0)



0



(0.0)



Fetal Incidences



No. (%)



1



(0.8)



0



(0.0)



0



(0.0)



0



(0.0)



Rib No. 12 - Wavy (Unilateral)



 



 



 



 



 



 



 



 



 



Litter Incidences



No. (%)



1



(4.2)



0



(0.0)



0



(0.0)



0



(0.0)



Fetal Incidences



No. (%)



1



(0.8)



0



(0.0)



0



(0.0)



0



(0.0)



Rib No. 13 - Wavy (Unilateral) 



Litter Incidences



No. (%)



1



(4.2)



0



(0.0)



0



(0.0)



1



(4.2)



Fetal Incidences



No. (%)



1



(0.8)



0



(0.0)



0



(0.0)



1



(0.7)



Rib No. 4 - Wavy (Bilateral) 



Litter Incidences



No. (%)



0



(0.0)



2



(8.7)



0



(0.0)



0



(0.0)



Fetal Incidences



No. (%)



0



(0.0)



2



(1.5)



0



(0.0)



0



(0.0)



Rib No. 5 - Wavy (Bilateral) 



Litter Incidences



No. (%)



0



(0.0)



2



(8.7)



0



(0.0)



0



(0.0)



Fetal Incidences



No. (%)



0



(0.0)



2



(1.5)



0



(0.0)



0



(0.0)



Rib No. 6 - Wavy (Bilateral)



 



 



 



 



 



 



 



 



 



Litter Incidences



No. (%)



0



(0.0)



2



(8.7)



0



(0.0)



0



(0.0)



Fetal Incidences



No. (%)



0



(0.0)



2



(1.5)



0



(0.0)



0



(0.0)



Rib No. 7 - Wavy (Bilateral) 



Litter Incidences



No. (%)



0



(0.0)



2



(8.7)



0



(0.0)



0



(0.0)



Fetal Incidences



No. (%)



0



(0.0)



2



(1.5)



0



(0.0)



0



(0.0)



Rib No. 8 - Wavy (Bilateral) 



Litter Incidences



No. (%)



0



(0.0)



2



(8.7)



0



(0.0)



0



(0.0)



Fetal Incidences



No. (%)



0



(0.0)



2



(1.5)



0



(0.0)



0



(0.0)



Rib No. 9 - Wavy (Bilateral) 



Litter Incidences



No. (%)



0



(0.0)



2



(8.7)



0



(0.0)



0



(0.0)



Fetal Incidences



No. (%)



0



(0.0)



2



(1.5)



0



(0.0)



0



(0.0)



Rib No. 10 - Wavy (Bilateral) 



Litter Incidences



No. (%)



0



(0.0)



2



(8.7)



0



(0.0)



0



(0.0)



Fetal Incidences



No. (%)



0



(0.0)



2



(1.5)



0



(0.0)



0



(0.0)



Rib No. 11 - Wavy (Bilateral) 



Litter Incidences



No. (%)



0



(0.0)



2



(8.7)



0



(0.0)



0



(0.0)



Fetal Incidences



No. (%)



0



(0.0)



2



(1.5)



0



(0.0)



0



(0.0)



Rib No. 12 - Wavy (Bilateral) 



Litter Incidences



No. (%)



0



(0.0)



2



(8.7)



0



(0.0)



0



(0.0)



Fetal Incidences



No. (%)



0



(0.0)



2



(1.5)



0



(0.0)



0



(0.0)



Rib No. 13 - Wavy (Bilateral) 



Litter Incidences



No. (%)



0



(0.0)



2



(8.7)



0



(0.0)



0



(0.0)



Fetal Incidences



No. (%)



0



(0.0)



2



(1.5)



0



(0.0)



0



(0.0)



Ossification site at first lumbar vertebra (Unilateral) 



Litter Incidences



No. (%)



4



(16.7)



1



(4.3)



4



(17.4)



1



(4.2)



Fetal Incidences



No. (%)



5



(3.8)



1



(0.8)



5



(4.0)



2



(1.5)



Ossification site at first lumbar vertebra (Bilateral)  



Litter Incidences



No. (%)



1



(4.2)



1



(4.3)



2



(8.7)



1



(4.2)



Fetal Incidences



No. (%)



5



(3.8)



1



(0.8)



4



(3.2)



1



(0.7)



 













































































































































































































































































































Group


 

G1



G2



G3



G4



Dose (mg/kg body weight/day)


 

0



100



300



1000



Variations



Litters Evaluated for External Examination



No.



24



23



23



24



Fetuses Evaluated for External Examination



No.



130



132



125



135


 

Region



Alteration


         

Thoracic Vertebra



Centrum No. 5 - Dumbbell-shaped ossification



Litter Incidences



No. (%)



0



(0.0)



1



(4.3)



0



(0.0)



0



(0.0)



Fetal Incidences



No. (%)



0



(0.0)



1



(0.8)



0



(0.0)



0



(0.0)



Centrum No. 7 - Dumbbell-shaped ossification



Litter Incidences



No. (%)



0



(0.0)



1



(4.3)



0



(0.0)



0



(0.0)



Fetal Incidences



No. (%)



0



(0.0)



1



(0.8)



0



(0.0)



0



(0.0)



Centrum No. 8 - Dumbbell-shaped ossification



Litter Incidences



No. (%)



0



(0.0)



0



(0.0)



1



(4.3)



1



(4.2)



Fetal Incidences



No. (%)



0



(0.0)



0



(0.0)



1



(0.8)



1



(0.7)



Centrum No. 9 - Dumbbell-shaped ossification



Litter Incidences



No. (%)



1



(4.2)



4



(17.4)



2



(8.7)



1



(4.2)



Fetal Incidences



No. (%)



1



(0.8)



6



(4.5)



3



(2.4)



2



(1.5)



Centrum No. 10 - Dumbbell-shaped ossification



Litter Incidences



No. (%)



9



(37.5



8



(34.8)



7



(30.4)



7



(29.2)



Fetal Incidences



No. (%)



14



(10.8)



11



(8.3)



11



(8.8)



15



(11.1)



Centrum No. 11 - Dumbbell-shaped ossification



Litter Incidences



No. (%)



6



(25.0)



7



(30.4



7



(30.4)



7



(29.2)



Fetal Incidences



No. (%)



16



(12.3)



17



(12.9



17



(13.6)



16



(11.9)



Centrum No. 12 - Dumbbell-shaped ossification



Litter Incidences



No. (%)



8



(33.3)



8



(34.8)



8



(34.8)



11



(45.8)



Fetal Incidences



No. (%)



15



(11.5)



20



(15.2)



12



(9.6)



21



(15.6)



Centrum No. 13 - Dumbbell-shaped ossification



Litter Incidences



No. (%)



6



(25.0)



6



(26.1)



4



(17.4)



5



(20.8)



Fetal Incidences



No. (%)



8



(6.2)



8



(6.1)



6



(4.8)



6



(4.4)



Centrum No. 10 - Incomplete ossification



Litter Incidences



No. (%)



0



(0.0)



1



(4.3)



0



(0.0)



0



(0.0)



Fetal Incidences



No. (%)



0



(0.0)



1



(0.8)



0



(0.0)



0



(0.0)



 


































































































































































































Group


 

G1



G2



G3



G4



Dose (mg/kg body weight/day)


 

0



100



300



1000



Variations



Litters Evaluated for External Examination



No.



24



23



23



24



Fetuses Evaluated for External Examination



No.



130



132



125



135


 

Region



Alteration


         

Lumbar Vetebra



Centrum No. 1 - Dumbbell-shaped ossification



Litter Incidences



No. (%)



2



(8.3)



3



(13.0)



0



(0.0)



2



(8.3)



Fetal Incidences



No. (%)



2



(1.5)



3



(2.3)



0



(0.0)



2



(1.5)



Centrum No. 2 - Dumbbell-shaped ossification



Litter Incidences



No. (%)



1



(4.2)



0



(0.0)



0



(0.0)



0



(0.0)



Fetal Incidences



No. (%)



1



(0.8)



0



(0.0)



0



(0.0)



0



(0.0)



Forelimb



Proximal phalanges - Unossified (Bilateral)



Litter Incidences



No. (%)



8



(33.3)



9



(39.1)



11



(47.8)



9



(37.5)



Fetal Incidences



No. (%)



17



(13.1)



21



(15.9)



20



(16.0)



21



(15.6)



Proximal phalanges - Incompletely ossified (Bilateral)



Litter Incidences



No. (%)



1



(4.2)



1



(4.3)



2



(8.7)



2



(8.3)



Fetal Incidences



No. (%)



1



(0.8)



2



(1.5)



3



(2.4)



3



(2.2)



Metacarpel No. 5 - Unossified (Bilateral)



Litter Incidences



No. (%)



0



(0.0)



1



(4.3)



0



(0.0)



0



(0.0)



Fetal Incidences



No. (%)



0



(0.0)



5



(3.8)



0



(0.0)



0



(0.0)



 



























































































































































































































































































































































Group



 



G1



G2



G3



G4



Dose (mg/kg body weight/day)



 



0



100



300



1000



 



Litters Evaluated for External Examination



No.



24



23



23



24



Fetuses Evaluated for External Examination



No.



130



132



125



135



Malformations 



Region↓



Alteration↓



 



 



 



 



 



 



 



 



 



Ribs



Rib No. 14 - Supplementary (Unilateral)



 



 



 



 



 



 



 



 



 



Litter Incidences



No. (%)



0



(0.0)



0



(0.0)



2



(8.7)



0



(0.0)



Fetal Incidences



No. (%)



0



(0.0)



0



(0.0)



2



(1.6)



0



(0.0)



Rib No. 14 - Supplementary (Bilateral)



 



 



 



 



 



 



 



 



 



Litter Incidences



No. (%)



0



(0.0)



0



(0.0)



1



(4.3)



0



(0.0)



Fetal Incidences



No. (%)



0



(0.0)



0



(0.0)



1



(0.8)



0



(0.0)



Rib No. 14 - Rudimentary (Unilateral)



 



 



 



 



 



 



 



 



 



Litter Incidences



No. (%)



0



(0.0)



0



(0.0)



1



(4.3)



0



(0.0)



Fetal Incidences



No. (%)



0



(0.0)



0



(0.0)



2



(1.6)



0



(0.0)



Thoracic Vertebra



Centrum No. 9 - Split



 



 



 



 



 



 



 



 



 



Litter Incidences



No. (%)



1



(4.2)



1



(4.3)



0



(0.0)



0



(0.0)



Fetal Incidences



No. (%)



1



(0.8)



1



(0.8)



0



(0.0)



0



(0.0)



Centrum No. 10 - Split



 



 



 



 



 



 



 



 



 



Litter Incidences



No. (%)



1



(4.2)



1



(4.3)



0



(0.0)



0



(0.0)



Fetal Incidences



No. (%)



1



(0.8)



1



(0.8)



0



(0.0)



0



(0.0)



Centrum No. 11 - Split



 



 



 



 



 



 



 



 



 



Litter Incidences



No. (%)



1



(4.2)



0



(0.0)



0



(0.0)



1



(4.2)



Fetal Incidences



No. (%)



1



(0.8)



0



(0.0)



0



(0.0)



1



(0.7)



Centrum No. 12 - Split



 



 



 



 



 



 



 



 



 



Litter Incidences



No. (%)



2



(8.3)



1



(4.3)



0



(0.0)



3



(12.5)



Fetal Incidences



No. (%)



2



(1.5)



1



(0.8)



0



(0.0)



4



(3.0)



Centrum No. 13 - Split



 



 



 



 



 



 



 



 



 



Litter Incidences



No. (%)



1



(4.2)



1



(4.3)



1



(4.3)



0



(0.0)



Fetal Incidences



No. (%)



1



(0.8)



1



(0.8)



1



(0.8)



0



(0.0)



 

Conclusions:
In a pre-natal developmental toxicity study conducted in Sprague Dawley Rats, the NOAEL of the test substance for both maternal and fetal toxicity was determined to be 1000 mg/kg body weight/day under the experimental conditions employed.
Executive summary:

The developmental toxicity of the test item was determined using OECD Guideline 414 under GLP conditions. The study used spraque dawley rats from an in-house line to test the daily oral administration via gavage of the test item using Carboxymethyl cellulose as vehicle. Based on a preliminary dose range study doses of 100, 300 and 1000 mg/kg bw/day were set with a concurrent vehicle control. On day 5 until day 19 of gestation, the test item was administered orally once daily to 25 pregnant females per group. During this time weight, food consumption, clinical signs of toxicity, mortality were observed. On day 20 all animals were euthanized, and tissue, organs and fetuses extracted and examined. There were no clinical signs of toxicity, mortality, morbidity, changes in body weight, food consumption changes, gross pathological or visceral changes in any group of maternal animals. Pregnancy rate, mean gravid uterus weight, live fetuses, mean litter size, mean sex ratio, mean number of implantation sites, mean number of resorptions, mean number of corpora lutae were similarly not affected by the treatment. Also, the mean pre-and post-implantation losses per dam and the mean absolute and relative thyroid along with the parathyroid weight were unaffected. No test item-related changes were noted in mean thyroid hormonal levels (T3, T4 and TSH). No test item-related microscopic changes were noted in thyroid along with the parathyroid of all the dams. There were no test item-related changes noted in mean fetal weight, mean fetal crown rump length and mean fetal anogenital distance measurement/ratio per litter. No test item related effects on developmental or structural alterations could be observed. Some external, visceral and skeletal alterations are considered incidental and not related to the test item as they occur in all groups, including the control and are not dose dependent. During the study no test item related effects in maternal or offspring animals could be observed. Therefore the "no observed adverse effect level (NOAEL) for reproduction and developmental toxicity is the highest dose used in this study of 1000mg/kg bw /day for maternal animals and offspring in rats.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
1 key study is available with a Klimisch score of 1.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the available information, the substance is not classified for toxicity to reproduction in accordance with CLP Regulation (EC) no 1272/2008.

Additional information