3,6-bis(diethylamino)-9-[2-(methoxycarbonyl)phenyl]xanthylium tetrachlorozincate

Toxicity to reproduction

Administrative data

Endpoint:

reproductive toxicity, other

Remarks:

Subchronic reproductive study

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Justification for type of information:

Data from peer reviewed journal

Data source

Reference

Reference Type:

publication

Title:

Subchronic reproductive study of test material was conducted on female rats for 36 days

Author:

Maryanti et al

Year:

2014

Bibliographic source:

Cukurova Medical Journal 2014

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

other: As mentioned below

Principles of method if other than guideline:

Subchronic reproductive study of test material was conducted on female rats for 36 days

GLP compliance:

not specified

Limit test:

no

Justification for study design:

No data available

Test material

Test material information

Constituent 1

Reference substance name:

9-(2-carboxyphenyl)-3,6-bis(diethylamino)xanthylium chloride

EC Number:

201-383-9

EC Name:

9-(2-carboxyphenyl)-3,6-bis(diethylamino)xanthylium chloride

Cas Number:

81-88-9

Molecular formula:

C28H31N2O3.Cl

IUPAC Name:

9-(2-carboxyphenyl)-3,6-bis(diethylamino)xanthenium chloride

Details on test material:

- Name of test material (as cited in study report): Rhodamine B
- Molecular formula : C28H31N2O3Cl
- Molecular weight : 479.017 g/mole
- Substance type: Organic
- Physical state: Solid

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

No data available

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Laboratory Pharmacology Brawijaya University.
- Weight at study initiation: (P) -150-200 g
- Diet : ad libitum
- Water: ad libitum
- Temperature (°C):22’C
- Photoperiod (hrs dark / hrs light): 12-h light/dark

Administration / exposure

Route of administration:

other: orally using probe.

Vehicle:

other: double distilled water

Details on exposure:

Test material was dissolved with double distilled water and administered orally using probe.

Details on mating procedure:

No data available

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

36 days

Frequency of treatment:

No details

Details on study schedule:

No data available

Doses / concentrations

Remarks:

4.5, 9 ,and 18 mg/200gm body weight=22.5,45, 90 mg/kg

No. of animals per sex per dose:

28 rats

Control animals:

yes, concurrent vehicle

Details on study design:

No data available

Positive control:

No data available

Examinations

Parental animals: Observations and examinations:

OTHER: Cycle determination was started at the end of
the treatment. The cycle of each female rat was determined by observation of vaginal smears, which were taken using a plastic tip.

Oestrous cyclicity (parental animals):

Estrous cyclicity was observed

Sperm parameters (parental animals):

No data available

Litter observations:

No data available

Postmortem examinations (parental animals):

No data available

Postmortem examinations (offspring):

No data available

Statistics:

Data are presented as mean ± SD and differences between groups were analyzed using 1-way ANOVA with SPSS 19.0 statistical package. Post Hoc test was used if the ANOVA was significant. p < 0.05 was considered statistically significant.

Reproductive indices:

No data available

Offspring viability indices:

No data available

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

not specified

Dermal irritation (if dermal study):

not specified

Mortality:

not specified

Body weight and weight changes:

not specified

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The test material reduces thickness of endometrium significantly compared to the control (P < 0.05). The 22.5,45 mg/kg doses did not generate significant difference in the thickness of endometrium (P> 0.05)

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, treatment-related

Description (incidence and severity):

In the 90mg/kg dose group increase ovarian MDA levels significantly than the control (P < 0.05). There was no significant difference in the
In 22.5 and 45 mg/kg dose group compared to the third dose (P > 0.05).Administration of test material in 45 and 90mg/kg dose group in female rats can reduce thenumber of primary, secondary, and De Graaf follicles significantly compared to the control (P < 0.05). For primary follicles, there were no significant differences among the three doses group(P> 0.05) Administration of test material in 45 and 90mg/kg dose group in female rats can reduce 17β-estradiol level significantly compared to the control (P < 0.05).

Reproductive function: sperm measures:

not specified

Reproductive performance:

not specified

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

22.5 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

histopathology: non-neoplastic

reproductive function (oestrous cycle)

Remarks on result:

other: overall no effects on estrous cycle

Target system / organ toxicity (P0)

Critical effects observed:

not specified

System:

other: not specified

Organ:

not specified

Treatment related:

not specified

Dose response relationship:

not specified

Relevant for humans:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality / viability:

not examined

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

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

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Other effects:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Dose descriptor:

other: not specified

Generation:

F1

Based on:

not specified

Sex:

not specified

Basis for effect level:

other: not specified

Remarks on result:

not measured/tested

Target system / organ toxicity (F1)

Critical effects observed:

not specified

System:

other: not specified

Organ:

not specified

Treatment related:

not specified

Dose response relationship:

not specified

Relevant for humans:

not specified

Overall reproductive toxicity

Reproductive effects observed:

not specified

Treatment related:

not specified

Relation to other toxic effects:

not specified

Dose response relationship:

not specified

Relevant for humans:

not specified

Applicant's summary and conclusion

Conclusions:

NOAEL was considered to be 22.5 mg/kg base on ovarian toxicity through oxidative stress, a decrease in the number of follicles, and decreased level of 17β-estradiol which ultimately lowered the thickness of endometrium, When female rats were treated with test chemical.

Executive summary:

The subchronic reproductive study conducted on Wister female rats for 36 days. The test material wasdissolved with double distilled water and administeredat concentrations of 22.5 ,45 and 90mg/kg bw orally using probe.28Female rats were randomly divided into the following four groups: the control group and three treatment group Malondialdehyde level in ovary was measured thiobarbituric acid method using TBARS Assay Kit (R&D system, Catalog Series KGE013, USA). The numbers of primary, secondary, tertiary and de Graaf follicles were calculated from the right ovary cut transversely and then preparation was made and stained histologically with HE and the follicles were calculated using Dotslide Olympus Camera XC 10. The entire cross-section was analyzed and further identified with magnification of 100 X. The cycle of each female rat was determined by observation of vaginal smears, which were taken using a plastic tip. Saline was placed on the vaginal opening, aspirated, and then placed on a microscopic slide.After the sample had dried, it was stained with hematoxylin-eosin. When the dye was removed, the slide was washed in de-ionized water and examined under a binocular microscope. Animals in the diestrus phase were used. The remainder of the animals continued to have their estrous cycles checked daily, being sacrificed always when in diestrus.

 

The test material reduces thickness of endometrium significantly compared to the control (P < 0.05). The22.5,45 mg/kgdoses did not generate significant difference in the thickness of endometrium (P> 0.05).In the 90mg/kg dose group increase ovarian MDA levels significantly than the control (P < 0.05). There was no significant difference in the 22.5 and 45 mg/kg dose group compared to the 90mg/kg dose group(P > 0.05).Administration of test material in 45 and 90mg/kg dose group in female rats can reduce thenumber of primary, secondary, and De Graaf follicles significantly compared to the control (P < 0.05). For primary follicles, there were no significant differences among the three doses group(P> 0.05) Administration of test material in 45 and 90mg/kg dose group in female rats can reduce 17β-estradiol level significantly compared to the control (P < 0.05).Hence NOAEL was considered to be 22.5 mg/kg based onovarian toxicity through oxidative stress, a decrease in the number of follicles, and decreased level of 17β-estradiol which ultimately lowered the thickness of endometrium, When female rats were treated with test material orally.