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EC number: 265-449-9 | CAS number: 65113-55-5
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Developmental toxicity / teratogenicity
Administrative data
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- From 2016-08-04 to 2017-02-18
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 016
- Report date:
- 2017
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 414 (Prenatal Developmental Toxicity Study)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
Test material
- Reference substance name:
- [4-[p,p'-bis(dimethylamino)benzhydrylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium m-[[p-anilinophenyl]azo]benzenesulphonate
- EC Number:
- 265-449-9
- EC Name:
- [4-[p,p'-bis(dimethylamino)benzhydrylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium m-[[p-anilinophenyl]azo]benzenesulphonate
- Cas Number:
- 65113-55-5
- Molecular formula:
- C25H30N3.C18H14N3O3S
- IUPAC Name:
- [4-[p,p'-bis(dimethylamino)benzhydrylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium m-[[p-anilinophenyl]azo]benzenesulphonate
- Test material form:
- solid: particulate/powder
Constituent 1
Test animals
- Species:
- rat
- Strain:
- Wistar
- Remarks:
- females and males
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Vivo Bio Tech Ltd., Telangana, India. CPCSEA Registration No. 1117/PO/RcBiBt/S/07/CPCSEA.
- Age at study initiation: 15-16 weeks
- Weight at study initiation:
* Males : 289 g - 362 g
* Females : 201g - 239 g
- Fasting period before study: no
- Housing:
CAGES: Polycarbonate cages (size 31 cm (L) X 21 cm (W) X 20 cm (H)), cleaned and disinfected before the animals were brought in and cleaned at regular interval during the study.
* Acclimatation period: 1-3 rats /sex/cage
* During mating: 2 females and 1 male / cage
* Pregnant females, post-mating: 1 female/cage.
BEDDING MATERIAL: sterilized corn cob produced from pure corn, dried and free from dust, batch SPAR-33/2016, from Sparconn Life Sciences, Bangalore. Renewed as often as necessary to keep the animals dry and clean
STUDY ROOM: before initiation, the study room was cleaned and disinfected. During study: the floor of the study room and work tops were swept and mopped with disinfectant solution every day and when required.
- Diet (e.g. ad libitum): ad libitum: conventional laboratory pellet diet (batch 040716, 040816 and 040916) from Nutrivet Life Sciences, Pune.
- Water (e.g. ad libitum): ad libitum: aqua guard filtered drinking water in bottles. Samples of drinking water were subjected periodically to bacteriological tests and to chemical contaminant analysis (latests test results available)
- Acclimation period: 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-22.4°C
- Relative Humidity (%): 45.4 to 66.5%
- Air changes (per hr): 12 time / hr, and filtered adequately
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light
IN-LIFE DATES: From: To: 2016-08-05 to 2016-09-23
Administration / exposure
- Route of administration:
- oral: gavage
- Vehicle:
- corn oil
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
Dose formulations were prepared daily: the defined quantity of the test substance was transfered to the mortar and triturated using pestle. Vehicle is added in to mortar and mixed well.
The formulation was transfered to the measuring cylinder and make the volume up to desired quantity.
At the time of dosing, dose formulations were stirred continously on a magnetic stirrer for maitaining homogeneity of testing solutions.
VEHICLE
- Justification for use and choice of vehicle (if other than water): corn oil was used based on the solubility testing
- Concentration in vehicle: up to 20 mg test item / bw
- Amount of vehicle (if gavage): dose volume not exceeding 0.4 ml/100g bw - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- The testing solution samples at 0 mg/mL, 1.25 mg/mL, 2.5 mg/mL and 5 mg/L were analysed by HPLC. Two replicates of 2 ml samples from upper, middle and lower layer were sent to Analytical department for the determination of homogeneity and test substance content. The analysis was carried out 2 times during treatment at starting and end of the treatment period.
The analytical parameter has been validated with respect to the following parameters:
SPECIFICITY: evaluated by analysing the blank, standard, vehicle and sample.
LINEARITY: carried out by preparing and analysing standards solutions of 6 concentrations (covering the target analyte concentration). The coded calibration solutions were injected into the HPLC, a plot was drawn between the concentration and the peak area response. The correlation coefficient, slope and intercept of the linear regression were calculated.
ASSAY ACCURACY AND PRECISION: carried out by fortifying the standard in the vehicle at 2 levels (covering the target analyte concentration). Five preparations were carried out at each concentration level selected. One control at each concentration level was maintained. Mean, SD, % RSD were calculated.
The test item formulations were found to be homogeneous. - Details on mating procedure:
- - Impregnation procedure: cohoused
- M/F ratio per cage: 1/2
- Length of cohabitation: until pregancy occurs or 2 weeks elapsed.
- Verification of same strain and source of both sexes: Yes
- Proof of pregnancy: observation of sperm positive vaginal smear referred to as day 0 of gestation. - Duration of treatment / exposure:
- From gestation day 5 to gestation day 19 (i.e. 15 days).
- Frequency of treatment:
- Once daily
- Duration of test:
- 19 days from gestation day 0 (i.e. 20 days from gestation day 0).
Doses / concentrationsopen allclose all
- Dose / conc.:
- 0 mg/kg bw/day (nominal)
- Remarks:
- Basis: actual ingested
- Dose / conc.:
- 5 mg/kg bw/day (nominal)
- Remarks:
- Basis: actual ingested
- Dose / conc.:
- 10 mg/kg bw/day (nominal)
- Remarks:
- Basis: actual ingested
- Dose / conc.:
- 20 mg/kg bw/day (nominal)
- Remarks:
- Basis: actual ingested
- No. of animals per sex per dose:
- 25 females per dose:
G1: 0 mg/kg bw/day
G2: 5 mg/kg bw/day
G3: 10 mg/kg bw/day
G4: 20 mg/kg bw/day - Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: range finding study
- Rationale for animal assignment: based on body weight, all animals were assigned in descending order. Highest body weight animals were selected for mating. Randomization was done based on day 0 body weight. After confirmation of mating by vaginal smear examination, animals were assigned in unbaised manner to control and treated groups. The inseminated by same male were evenly distributed across the group.
Examinations
- Maternal examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily
BODY WEIGHT: Yes
- Time schedule : at arrival in the laboratory, after completion of the acclimatization period and before group allocation and D0, D3, D5, D8, D11, D14, D17 and D20 (day of scheduled killed) of pregnancy
FEED CONSUMPTION: Yes
- Time schedule : D0, D3, D5, D8, D11, D14, D17 and D20 (day of scheduled killed) of pregnancy
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20: Organs examined: gross observation of all maternal organes (for any structural abnormalities or pathological changes) with emphasis on uterus, number of live/viable foetuses, number of dead fetuses, number of corpora lutea, number of implantations sites, degree of resorption and weight of placenta recoded for the terminally sacrified females. The gravid uteri (including cervix) were weighed at necropsy - 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 resorptions: Yes
- Fetal examinations:
- Each fetuses were marked, sexed, weighted and crown rump length was measured
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: Yes: [half per litter] - Statistics:
- Raw data were processed using statistical software Sigma Plot 11.0: the mean and standard deviation were calculated using the excel and all data were summarized in tabular form.
Homogeneity checked for all continuous data (body weight, feed consumption, weight of uterus, relative weight of uterus, weight of ovaries, number of live fetuses, pre-implantation loss, number of implantation site, number of resorption, post-implantation loss, CL count, mean weight of fetus, mean weight of placenta, mean foetal CRL measurement) using Shapiro Wilk Test, followed by ANOVA and Kruskal Wallis one Way ANOVA
Data showing singificance in their variances were subjected to Holm-Sidak and Dunn's methods. - Indices:
- Pre-implantation loss, post-implantation loss, mean numbers of resorption, embryonic deaths and total foetuses
- Historical control data:
- No
Results and discussion
Results: maternal animals
General toxicity (maternal animals)
- Clinical signs:
- no effects observed
- Description (incidence and severity):
- No clinical signs were observed in any of the animals throughout the study period (see table 1).
- Dermal irritation (if dermal study):
- not examined
- Mortality:
- no mortality observed
- Description (incidence):
- No mortality and morbidity were oberved among any of the groups of animals throughout the study period (see Table 2)
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- There was no statistically significant difference in the body weight of dams of treated groups when compared to control group during study period.
There was no statistically significant difference in the percent body weight change of dams of treated groups when compared to control group during study period.
(see Tables 3 and 4). - Food consumption and compound intake (if feeding study):
- not examined
- Description (incidence and severity):
- Even if not a feeding study, food consumption was examined: there was no statistically significant diffrence in feed consumption of dams of trested groups when compared to control group during the study period.
- 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
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- not examined
- Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- External and internal gross examination of all dams in control and all treated groupds did not reveal any abnormality of pathological significance (see Table 5).
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- not examined
- Histopathological findings: neoplastic:
- not examined
- Other effects:
- no effects observed
- Description (incidence and severity):
- weight of uterus, weight of ovaries
Maternal developmental toxicity
- Number of abortions:
- not examined
- Pre- and post-implantation loss:
- no effects observed
- Description (incidence and severity):
- There was no statistically significant difference in pre- and post-implantation loss in any of the treated groups as compared to control group (see Table 7).
- Total litter losses by resorption:
- no effects observed
- Description (incidence and severity):
- no total litter loss by resorption was observed.
- Early or late resorptions:
- no effects observed
- Description (incidence and severity):
- There was no statistically significant difference in number resorptions in any of the treated groups as compared to control group (see Table 7).
- Dead fetuses:
- no effects observed
- Description (incidence and severity):
- There was no statistically significant difference in number of lives fetuses in any of the treated groups as compared to control group (see Table 7).
- Changes in pregnancy duration:
- no effects observed
- Description (incidence and severity):
- Intrauterine growth was unaffected by test item administration in G2, G3 and G4 compared to control group. All females were determined to be gravid, with the exception of 6 females in G1, 2 females in G2 and 4 females in G4.
(see Table 6). - Changes in number of pregnant:
- no effects observed
- Description (incidence and severity):
- Pregnancy rate of female in the group of control, and treated animals in G2, G3 and G4 was 76%, 92%, 100% and 84% respectively.
- Other effects:
- no effects observed
- Description (incidence and severity):
- There was no statistically significant difference in absolute and relative weights of uterus, weight of ovaries, corpus luteum, number of implemantation site in any of the treated groups as compared to control group (see Table 7).
Effect levels (maternal animals)
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- >= 20 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Basis for effect level:
- other: no test item related changes at the highest tested dose
Maternal abnormalities
- Key result
- Abnormalities:
- no effects observed
Results (fetuses)
- Fetal body weight changes:
- no effects observed
- Description (incidence and severity):
- There was no statistically significant difference in mean weight of fetus of both sexes of any of the treated group fetuses as compared to control group (see Table 8).
- Reduction in number of live offspring:
- no effects observed
- Description (incidence and severity):
- There was no statistically significant difference in number of lives fetuses in any of the treated groups as compared to control group.
- Changes in sex ratio:
- no effects observed
- Description (incidence and severity):
- Male/female sex ratio of fetuses in the group of control, and treated animals at in G2, G3 and G4 was 101/100, 135/139, 141/157 and 107/125 respectively (see Table 12).
- Changes in litter size and weights:
- not specified
- Changes in postnatal survival:
- not examined
- External malformations:
- effects observed, non-treatment-related
- Description (incidence and severity):
- The external gross examination of fetuses showed small fetuses:
- G1 : 2 fetuses
- G2 : 5 fetuses
- G3: 1 fetus
- G4: 2 fetuses
(see Table 9)
These observations were not dose dependent and inconsistent hence, not considered as treatment related, but considered as spontaneous in origin. - Skeletal malformations:
- effects observed, non-treatment-related
- Description (incidence and severity):
- There were no test item related fetal skeletal malformations noted at any of the dose levels tested.
(see Table 11) - Visceral malformations:
- no effects observed
- Description (incidence and severity):
- Visceral examination did not show any malformation in fetuses of treatment groups and control group (see Table 10).
- Other effects:
- no effects observed
- Description (incidence and severity):
- Head razor examination did not show any abnormality in treated group and control group (see Table 10)
Effect levels (fetuses)
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- >= 20 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: There was no test item induced adverse effects on structural development or growth in fetuses under the experimental conditions
Fetal abnormalities
- Key result
- Abnormalities:
- no effects observed
Overall developmental toxicity
- Key result
- Developmental effects observed:
- no
Any other information on results incl. tables
Table 1: Summary of General Clinical Signs Observation
Sex: Female |
|||||
Group |
Dose (mg/kg b. wt.) |
No. of Animals |
Animal Number |
Gestation Day |
Clinical Signs |
G1 |
0 |
25 |
01-25 |
0-20 |
Normal |
G2 |
5 |
25 |
26-50 |
0-20 |
Normal |
G3 |
10 |
25 |
51-75 |
0-20 |
Normal |
G4 |
20 |
25 |
76-100 |
0-20 |
Normal |
Keys:No. = Number, mg/kg b. wt. = milligram/ kilogram body weight
Table 2: Summary of Mortality and Morbidity
Sex: Female |
|||||
Group |
Dose (mg/kg b. wt.) |
No. of Animals |
Gestation Day |
Observation |
|
Morning |
Evening |
||||
G1 |
0 |
25 |
0-20 |
No mortality/ morbidity |
No mortality/ morbidity |
G2 |
5 |
25 |
0-20 |
No mortality/ morbidity |
No mortality/ morbidity |
G3 |
10 |
25 |
0-20 |
No mortality/ morbidity |
No mortality/ morbidity |
G4 |
20 |
25 |
0-20 |
No mortality/ morbidity |
No mortality/ morbidity |
Keys:No. = Number, mg/kg b. wt. = milligram/ kilogram body weight
Table 3: Summary of Body Weight (gram)
Sex: Female |
||||||||
Group (N) |
G1 (19) |
G2 (23) |
G3 (25) |
G4 (21) |
||||
Dose (mg/kg b. wt.) |
0 |
5 |
10 |
20 |
||||
Day |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
0 |
235.53 |
10.48 |
233.87 |
9.69 |
233.76 |
11.41 |
233.81 |
12.03 |
3 |
243.21 |
11.83 |
241.22 |
9.15 |
240.60 |
12.14 |
240.00 |
12.05 |
5 |
245.89 |
12.04 |
245.87 |
9.45 |
245.96 |
12.27 |
244.76 |
11.43 |
8 |
250.89 |
12.56 |
250.65 |
10.42 |
251.36 |
12.90 |
248.38 |
11.25 |
11 |
261.11 |
12.58 |
261.61 |
11.21 |
262.44 |
12.83 |
257.52 |
11.68 |
14 |
273.68 |
13.60 |
273.57 |
12.04 |
274.24 |
14.41 |
268.52 |
12.27 |
17 |
296.26 |
17.43 |
297.22 |
14.84 |
298.04 |
15.46 |
290.52 |
14.00 |
20 |
329.74 |
23.17 |
330.83 |
18.44 |
331.44 |
18.08 |
321.05 |
16.37 |
Keys:N= Number of Animals, SD= Standard Deviation, mg/kg b. wt. = milligram/ kilogram body weight
Table 4: Summary of Body Weight Change (%)
Sex: Female |
||||||||
Group (N) |
G1 (19) |
G2 (23) |
G3 (25) |
G4 (21) |
||||
Dose (mg/kg b. wt.) |
0 |
5 |
10 |
20 |
||||
Day |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
0-3 |
3.27 |
2.19 |
3.16 |
1.09 |
2.93 |
1.22 |
2.66 |
1.49 |
0-5 |
4.41 |
2.33 |
5.16 |
1.65 |
5.22 |
1.33 |
4.72 |
1.81 |
0-8 |
6.52 |
2.38 |
7.19 |
1.99 |
7.53 |
1.63 |
6.28 |
2.07 |
0-11 |
10.88 |
2.91 |
11.89 |
3.15 |
12.29 |
2.04 |
10.20 |
2.50 |
0-14 |
16.21 |
3.11 |
17.00 |
2.98 |
17.32 |
2.59 |
14.92 |
3.26 |
0-17 |
25.79 |
5.12 |
27.07 |
3.09 |
27.53 |
3.55 |
24.34 |
3.98 |
0-20 |
40.02 |
8.12 |
41.41 |
4.11 |
41.85 |
5.53 |
37.42 |
5.29 |
Table 5: Summary of Individual Gross observation
Sex: Female |
|||||
Group |
Dose (mg/kg b. wt.) |
No. of Animals |
Animal Number |
Macroscopic Observation |
|
External |
Internal |
||||
G1 |
0 |
25 |
01-25 |
NAD |
NAD |
G2 |
5 |
25 |
26-50 |
NAD |
NAD |
G3 |
10 |
25 |
51-75 |
NAD |
NAD |
G4 |
20 |
25 |
76-100 |
NAD |
NAD |
Keys:NAD = No Abnormality Detected, mg/kg b. wt. = milligram/ kilogram body weight
Table 6: Summary of Number of gravide uterus and pregnancy rate
Group (N) |
G1 (25) |
G2 (25) |
G3 (25) |
G4 (25) |
Dose (mg/kg b. wt.) |
0 |
5 |
10 |
20 |
No. of Gravid Uterus |
19 |
23 |
25 |
21 |
Pregnancy Rate (%) |
76 |
92 |
100 |
84 |
Keys:N= Number of animals,mg/kg b. wt. = milligram/ kilogram body weight, % = Percent
Table 7: Summary of Maternal Evaluation Data
Group (N) |
G1 (19) |
G2 (23) |
G3 (25) |
G4 (21) |
||||
Dose (mg/kg b. wt.) |
0 |
5 |
10 |
20 |
||||
Parameters |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Weight of Dam (g) |
329.737 |
23.1729 |
330.826 |
18.4407 |
331.44 |
18.081 |
321.048 |
16.3661 |
Weight of Uterus (g) |
55.77 |
13.24 |
62.66 |
8.79 |
64.12 |
9.39 |
58.19 |
9.83 |
Relative Uterus Weight (%) |
16.76 |
3.28 |
18.90 |
2.11 |
19.09 |
2.60 |
18.1017 |
2.8028 |
Weight of Ovaries (g) |
0.16 |
0.04 |
0.16 |
0.02 |
0.15 |
0.02 |
0.14 |
0.03 |
CL Count |
12.16 |
1.74 |
12.91 |
1.41 |
12.80 |
1.47 |
13.14 |
1.59 |
Number of Live Fetus |
10.47 |
2.63 |
11.91 |
1.56 |
11.88 |
1.67 |
11.05 |
2.09 |
Number of Dead Fetus |
0.05 |
0.23 |
0.00 |
0.00 |
0.04 |
0.20 |
0.00 |
0.00 |
Number of Implantation Site |
11.79 |
2.02 |
12.61 |
1.75 |
12.72 |
1.40 |
12.00 |
2.24 |
Number of Resorption |
1.21 |
1.32 |
0.65 |
0.78 |
0.76 |
0.97 |
0.95 |
0.97 |
Pre-Implantation Loss (%) |
3.17 |
7.43 |
2.53 |
6.88 |
0.19 |
9.73 |
8.32 |
14.69 |
Post-Implantation Loss (%) |
12.05 |
13.15 |
5.21 |
6.17 |
6.69 |
7.45 |
7.53 |
7.97 |
Keys:N = Number of Dams, n = Number of Dams including Dams with Complete Resorption, g = Gram, CL = Corpus Luteum, SD = Standard Deviation,% = Percent,mg/ kg b. wt. = milligram/ kilogram body weight
Table 8: Summary of Fetal weight (g), Placenta weight (g) and CRL Measurment (cm)
Group (N) |
G1 (19) |
G2 (23) |
G3 (25) |
G4 (21) |
|||||
Dose (mg/kg b. wt.) |
0 |
5 |
10 |
20 |
|||||
Sex |
Parameters |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Males |
Fetus Weight (g) |
3.4058 |
0.3107 |
3.4789 |
0.2757 |
3.5694 |
0.2939 |
3.5248 |
0.2209 |
Placenta Weight (g) |
0.4700 |
0.0536 |
0.4494 |
0.0456 |
0.4412 |
0.0385 |
0.4359 |
0.0356 |
|
CRL (cm) |
3.4288 |
0.1919 |
3.4309 |
0.1224 |
3.4588 |
0.1384 |
3.4644 |
0.1780 |
|
Females |
Fetus Weight (g) |
3.2420 |
0.2542 |
3.3270 |
0.2318 |
3.4079 |
0.2326 |
3.3480 |
0.2943 |
Placenta Weight (g) |
0.4688 |
0.0948 |
0.4405 |
0.0464 |
0.4331 |
0.0460 |
0.4271 |
0.0493 |
|
CRL (cm) |
3.3418 |
0.1759 |
3.3712 |
0.0975 |
3.4447 |
0.1538 |
3.3383 |
0.1725 |
|
Male and Female |
Fetus Weight (g) |
3.3323 |
0.2504 |
3.4010 |
0.2269 |
3.4747 |
0.2487 |
3.4331 |
0.2525 |
Placenta Weight (g) |
0.4734 |
0.0802 |
0.4437 |
0.0439 |
0.4371 |
0.0390 |
0.4329 |
0.0409 |
|
CRL (cm) |
3.3829 |
0.1762 |
3.4049 |
0.1032 |
3.4518 |
0.1443 |
3.3951 |
0.1690 |
Keys:N= Number of animals, SD= Standard Deviation, g = Gram, cm = Centimeter, CRL = Crown to Rump Length,mg/kg b. wt. = milligram/ kilogram body weight.
Note:Fused placenta was observed in fetus number 09 and 10 of animal number 08. Hence placenta weight was not considered in mean weight of Placenta in male and female separately but considered in combined male and female.
Table 9: Summary of External, Visceral and Head Razor Evaluation Data
Group |
Fetus |
Litters |
||||||
G1 |
G2 |
G3 |
G4 |
G1 |
G2 |
G3 |
G4 |
|
Dose (mg/kg b. wt.) |
0 |
5 |
10 |
20 |
0 |
5 |
10 |
20 |
Number Examined Externally |
201 |
274 |
298 |
232 |
19 |
23 |
25 |
21 |
Dead Fetus |
1 |
0 |
1 |
0 |
1 |
0 |
1 |
0 |
Small Fetus |
2 |
5 |
1 |
2 |
1 |
3 |
1 |
2 |
NAD |
198 |
269 |
296 |
230 |
19 |
23 |
25 |
21 |
Number Examined Viscerally |
104 |
142 |
156 |
123 |
19 |
23 |
25 |
21 |
NAD |
104 |
142 |
156 |
123 |
19 |
23 |
25 |
21 |
Number Examined by Head Razor |
104 |
142 |
156 |
123 |
19 |
23 |
25 |
21 |
NAD |
104 |
142 |
156 |
123 |
19 |
23 |
25 |
21 |
Keys:NAD = No Abnormality Detected, mg/kg b. wt. = milligram/ kilogram body weight
Table 10: Summary of Percent (%) External, Visceral and Head Razor Evaluation Data
Group |
% Fetus |
% Litters |
||||||
G1 |
G2 |
G3 |
G4 |
G1 |
G2 |
G3 |
G4 |
|
Dose (mg/kg b. wt.) |
0 |
5 |
10 |
20 |
0 |
5 |
10 |
20 |
Number Examined Externally |
201 |
274 |
298 |
232 |
19 |
23 |
25 |
21 |
% Dead Fetus |
0.50 |
0.00 |
0.34 |
0.00 |
5.26 |
0.00 |
4.00 |
0.00 |
% Small Fetus |
1.00 |
1.82 |
0.34 |
0.86 |
5.26 |
13.04 |
4.00 |
9.52 |
% NAD |
100 |
100 |
100 |
100 |
100 |
100 |
100 |
100 |
Number Examined Viscerally |
105 |
142 |
156 |
123 |
19 |
23 |
25 |
21 |
% NAD |
100 |
100 |
100 |
100 |
100 |
100 |
100 |
100 |
Number Examined by Head Razor |
105 |
142 |
156 |
123 |
19 |
23 |
25 |
21 |
% NAD |
100 |
100 |
100 |
100 |
100 |
100 |
100 |
100 |
Keys:NAD = No Abnormality Detected, mg/kg b. wt. = milligram/ kilogram body weight,% = Percent
Table 11: Summary of Number and Percent (%) Skeletal Evaluation Data
Skeletal Malformations / Variations |
Group: No. of Fetus (% foetus) |
Group: No. of Litters (% litters) |
||||||||
G1 (96) |
G2 (132) |
G3 (137) |
G4 (109) |
G1 (19) |
G2 (23) |
G3 (25) |
G4 (21) |
|||
Name of Bone |
Malformation/ Variation |
|||||||||
NAD |
45 (46.88%) |
61 (46.21%) |
67 (48.91%) |
73 (66.97%) |
1 (5.26%) |
2 (8.70%) |
3 (12.00%) |
7 (33.33%) |
||
Hyoid |
Unossified / Incomplete Ossification |
5 (5.21%) |
3 (2.27%) |
3 (2.19%) |
4 (3.67%) |
4 (21.05%) |
1 (4.35%) |
2 (8.00%) |
4 (19.05%) |
|
Frontal/ Parietal/ Interparietal/ Occipital |
Unossified/Incomplete Ossification |
8 (8.33%) |
15 (11.36%) |
13 (9.49%) |
3 (2.75%) |
4 (21.05%) |
5 (21.74%) |
7 (28.00%) |
3 (14.29%) |
|
Zygomatic |
Incomplete Ossification |
1 (1.04%) |
5 (3.79%) |
7 (5.11%) |
6 (5.50%) |
1 (5.26%) |
1 (4.35%) |
4 (16.00%) |
3 (14.29%) |
|
Fused |
0 (0.00%) |
2 (1.52%) |
2 (1.46%) |
0 (0.00%) |
0 (0.00%) |
2 (8.70%) |
1 (4.00%) |
0 (0.00%) |
||
Rib |
Supernumerary |
19 (19.79%) |
20 (15.15%) |
28 (20.44%) |
17 (15.60%) |
9 (47.37%) |
10 (43.48%) |
12 (48.00%) |
6 (28.57%) |
|
Wavy |
0 (0.00%) |
2 (1.52%) |
4 (2.92%) |
0 (0.00%) |
0 (0.00%) |
1 (4.35%) |
1 (4.00%) |
0 (0.00%) |
||
Incomplete Ossification |
0 (0.00%) |
1 (0.76%) |
2 (1.46%) |
0 (0.00%) |
0 (0.00%) |
1 (4.35%) |
1 (4.00%) |
0 (0.00%) |
||
Sternebra |
Unossified/Incomplete Ossification |
32 (33.33%) |
41 (31.06%) |
42 (30.66%) |
21 (19.27%) |
15 (78.95%) |
18 (78.26%) |
18 (72.00%) |
10 (47.62 |
|
Sternoschisis |
4 (4.71%) |
2 (1.52%) |
3 (2.19%) |
3 (2.75%) |
3 (15.79%) |
2 (8.70%) |
3 (12.00%) |
2 (9.52%) |
||
Bipartite Ossification |
0 (0.00%) |
2 (1.52%) |
1 (0.73%) |
0 (0.00%) |
0 (0.00%) |
2 (8.70%) |
1 (4.00%) |
0 (0.00%) |
||
Misaligned |
0 (0.00%) |
0 (0.00%) |
1 (0.73%) |
0 (0.00%) |
0 (0.00%) |
0 (0.00%) |
1 (4.00%) |
0 (0.00%) |
||
Thoracic centrum |
Bipartite Ossification |
0 (0.00%) |
3 (2.27%) |
2 (3.65%) |
0 (0.00%) |
0 (0.00%) |
3 (13.04%) |
2 (8.00%) |
0 (0.00%) |
|
Dumbell Ossification |
0 (0.00%) |
3 (2.27%) |
5 (3.65%) |
0 (0.00%) |
0 (0.00%) |
3 (13.04%) |
3 (12.00%) |
0 (0.00%) |
||
Lumbar Vertebra |
Unossified / Incomplete Ossification |
0 (0.00%) |
1 (0.76%) |
0 (0.00%) |
0 (0.00%) |
0 (0.00%) |
1 (4.35%) |
0 (0.00%) |
0 (0.00%) |
|
Thoracic Vertebra |
Incomplete Ossification |
0 (0.00%) |
1 (0.76%) |
0 (0.00%) |
0 (0.00%) |
0 (0.00%) |
1 (4.35%) |
0 (0.00%) |
0 (0.00%) |
|
Ischium |
Unossified / Incomplete Ossification |
0 (0.00%) |
2 (1.52%) |
1 (0.73%) |
0 (0.00%) |
0 (0.00%) |
1 (4.35%) |
1 (4.00%) |
0 (0.00%) |
|
Pubis |
Unossified/Incomplete Ossification |
0 (0.00%) |
0 (0.00%) |
1 (0.73%) |
0 (0.00%) |
0 (0.00%) |
0 (0.00%) |
1 (4.00%) |
0 (0.00%) |
|
Squamosal |
Incomplete Ossification |
0 (0.00%) |
3 (2.27%) |
4 (2.92%) |
0 (0.00%) |
0 (0.00%) |
1 (4.35%) |
1 (4.00%) |
0 (0.00%) |
|
Metacarpals and Metatarsals |
Incomplete Ossification |
0 (0.00%) (0.00%) |
1 (0.76%) |
1 (0.73%) |
0 (0.00%) |
0 (0.00%) |
1 (4.35%) |
1 (4.00%) |
0 (0.00%) |
Keys:NAD = No Abnormality Detected,No. = Number, % = Percent.
Table 12: Summary of Fetal Sex Ratio
Group (N) |
G1 (201) |
G2 (274) |
G3 (298) |
G4 (232) |
Dose (mg/kg b. wt.) |
0 |
5 |
10 |
20 |
Sex Ratio (Male/Female) |
101/100 |
135/139 |
141/157 |
107/125 |
Key:N= Number of Fetus,mg/kg b. wt. = milligram/ kilogram body weight
Applicant's summary and conclusion
- Conclusions:
- Based on the findings of prenatal developmental oral toxicity study of the test item in Wistar rat at dose level 0, 5, 10, 20 mg/kgbw, no test item related changes were observed in any of the doses tested hence, the maternal No Observed Adverse Effect Level (NOAEL) of the test item is considered to be 20 mg/kg bw in Wistar rat. Since, there was no test item induced adverse effects on structural development or growth in the fetuses, the developmental No Observed Adverse Effect Level (NOAEL) of the test item in fetuses is considered to be 20 mg/kg bw under the experimental conditions.
- Executive summary:
The objective of this study was to provide evaluations of prenatal developmental toxicity of the test item in Wistar rats.
The results of the study provides general information concerning the effects of prenatal exposure on the pregnant animal and fetus, also include assessment of maternal effects as well as death, structural abnormalities, or altered growth in the fetus and determination of the maternal and developmental No Observed Adverse Effect Level (NOAEL).
The animals were randomly allocated to the four groups (25 Females/group). The doses selected for groups were; 0, 5, 10 and 20 mg/kg body weight.
Control group animals received vehicle (Corn oil) aloneand treatment groups were administered with test item daily by oral route, throughout the study period (Gestation day5-19).
Test item formulation was found to be homogeneous. The mean active ingredient content of AY36 at 1.25, 2.5 and 5 mg/ml concentrationwas 1.197,2.377, 4.701 mg/ml and 1.186, 2.356, 4.730 mg/mlat first week (initialsample)and last week (finalsample)of treatment, respectively.The mean active ingredient content of BV3 at 1.25, 2.5 and 5 mg/ml concentrationwas1.205, 2.391, 4.759 mg/ml and 1.194, 2.393, 4.747mg/mlat first week (initialsample)and last week (finalsample)of treatment, respectively.
No mortality and morbidity were observed among any of the groups of animals throughout the study period.
No clinical signs were observed in any of the animals throughout the study period.
There was no statistically significant difference in the body weight of dams of treated groups when compared to control group G1 during study period.
There was no statistically significant difference in the percent body weight change of dams of treated groups when compared to control group G1 during study period.
There was no statistically significant difference in feed consumption of dams of treated groups when compared to control group during study period.
External and internal gross examination of all dams in control and all treated groups did not reveal any abnormality of pathological significance.All females were determined to be gravid, with the exception of 6, 2, 4 females in G1 (0 mg/kg body weight), G2 (5 mg/kg body weight) and G4 (20 mg/kg body weight), respectively.
There was no statistically significant difference in absolute and relative weights of uterus, weight of ovaries, number of live fetuses, pre-implantation loss, number of implantation site, number of resorptions, post-implantation loss, CL count in any of the treated groups as compared to control group. Intrauterine growth and survival of fetuses was unaffected by test item administration at 5 (G2), 10 (G3) and 20 (G4) mg/kg body weight. Pregnancy rate of female in the group of G1, G2, G3 and G4 was 76%, 92%, 100% and 84%, respectively.
There was no statistically significant difference in mean weight of fetus, mean weight of placenta and mean foetal CRL measurement in male, female and both sexes of any of the treated group fetuses as compared to control group.
The external gross examination of fetuses showed small fetuses 2 (G1), 5 (G2), 1 (G3), 2 (G4) and dead fetuses 1(G1), 1(G3). These Observations were not dose dependent and inconsistent hence, not considered as treatment related, but considered as spontaneous in origin. Male/ Female sex ratio of fetuses in the group G1, G2, G3 and G4 was 101/100, 135/139, 141/157 and 107/125, respectively.
Visceral examination did not show any malformation in the fetuses of treatment groups and control group.
Head razor examination did not show any abnormality in control and treatment groups.
There were no test item related fetal skeletal malformation noted at any of the dose levels tested. Skeletal malformations like Hyoid: Unossified/ Incomplete Ossification were noted for 5 (4), 3 (1), 3 (2), 4 (4) fetus (litter) in G1, G2, G3 and G4, respectively. Frontal/ Parietal/ Interparietal/Occipital: Unossified/Incomplete Ossification were noted for 8 (4), 15 (5), 13 (7), 3 (3) fetus (litter) in G1, G2, G3 and G4, respectively. Zygomatic: incomplete ossification were noted for 1 (1), 5 (1), 7 (4), 6 (3) fetus (litter) in G1, G2, G3 and G4, respectively and Zygomatic: fused were noted for 2 (2) and 2 (1) fetus (litter) in G2 and G3, respectively. Supernumerary rib were noted for 19 (9), 20 (10), 28 (12), 17 (6) fetus (litter) in G1, G2, G3 and G4, respectively. Wavy ribs were noted for 2 (1), 4 (1), fetus (litter) in G2 and G3 respectively. Incomplete ossification of ribs were noted for 1 (1), 2 (1), fetus (litter) in G2 and G3 respectively.
Sternebra: unossified/ Incomplete ossification were noted for 32 (15), 41 (18), 42 (18), 21 (10) fetus (litter) in G1, G2, G3 and G4, respectively. Sternebra: sternoschisis were noted for 4 (3), 2 (2), 3 (3), 3 (2) fetus (litter) in G1, G2, G3 and G4, respectively. Sternebra: bipartite ossification were noted for 2 (2), 1 (1) fetus (litter) in G2 and G3, respectively. Sternebra: misaligned was noted for 1 (1) fetus (litter) in G3. Thoracic centrum: Bipartite ossification were notedfor 3 (3), 2 (2), fetus (litter) in G2 and G3, respectively.
Thoracic centrum: dumbell ossification were noted for 3 (3), 5 (3) fetus (litter) in G2 and G3, respectively. Lumbar vertebra: Unossified/ Incomplete ossification was noted for 1 (1) fetus (litter) in G2. Thoracic vertebra: Incomplete ossification was noted for 1 (1) fetus (litter) in G2. Ischium: Unossified/ Incomplete ossification were noted for 2 (1), 1 (1) fetus (litter) in G2 and G3, respectively. Pubis: Unossified/ Incomplete ossification was noted for 1 (1) fetus (litter) in G3. Squamosal: Incomplete ossification were noted for 3 (1), 4 (1) fetus (litter) in G2 and G3, respectively. Metacarpals and Metatarsals: Incomplete ossification were noted for 1 (1), 1 (1) fetus (litter) in G2 and G3, respectively.
There were no test item related fetal skeletal developmental variations noted at any of the dose levels tested. These findings occured infrequently and were noted similarly in the concurrent control group, and /or were noted in a manner thatwas not considered to be treatment related. As these observations were not dose dependent therefore, these skeletal developmental variations were not considered to be test item related.
Conclusion
Based on the findings ofprenatal developmentaloral toxicity study of the item in Wistar rat at dose level 0, 5, 10 and 20 mg/kg body weight, no test item related changes were observed in any of the doses tested hence, the maternal No Observed Adverse Effect Level (NOAEL) of the test item is considered to be 20 mg/ kg body weight in Wistar Rat. Since, there was no test item induced adverse effects onstructural development or growth in the foetuses, the developmental No Observed Adverse Effect Level (NOAEL) of test item in fetuses is considered to be 20 mg/ kg body weight under the experimental conditions.
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