α,α-bis[4-(dimethylamino)phenyl]-4-(phenylamino)naphthalene-1-methanol

Toxicity to reproduction

Administrative data

Endpoint:

reproductive toxicity, other

Remarks:

organ toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data from study report

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 407

Principles of method if other than guideline:

Effects on male and female reproductive organs were studies in the 28 day repeated oral toxicity study

GLP compliance:

yes

Limit test:

no

Justification for study design:

No data available

Test material

Test material information

Constituent 1

Reference substance name:

α,α-bis[4-(dimethylamino)phenyl]-4-(phenylamino)naphthalene-1-methanol (Solvent Blue 4) (containing less than 0.1% Michler’s Ketone)

IUPAC Name:

α,α-bis[4-(dimethylamino)phenyl]-4-(phenylamino)naphthalene-1-methanol (Solvent Blue 4) (containing less than 0.1% Michler’s Ketone)

Test material form:

solid

Details on test material:

- Name of test material: 1-Naphthalenemethanol, .alpha.,.alpha.-bis[4-(dimethylamino)phenyl]-4-(phenylamino)-
- Molecular formula: C33H33N3O
- Molecular weight: 487.644 g/mol
- Smiles notation: N(c1ccc(cc1)C(c1ccc(N(C)C)cc1)(c1c2c(c(Nc3ccccc3)cc1)cccc2)O)(C)C
- InChl: 1S/C33H33N3O/c1-35(2)27-18-14-24(15-19-27)33(37,25-16-20-28(21-17-25)36(3)4)31-22-23-32(30-13-9-8-12-29(30)31)34-26-10-6-5-7-11-26/h5-23,34,37H,1-4H3
- Substance type: Organic
- Physical state: solid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

No data available

Sex:

male/female

Details on test animals or test system and environmental conditions:

Source: Central Animal Facility (CAF), NIPER, Sector-67, S.A.S. Nagar, 160 062, Punjab, India.
- Age at study initiation: (P) x wks; (F1) x wks:
( P) 7 to 8 weeks old
- Weight at study initiation: (P) Males: x-x g; Females: x-x g: Male 196.26 - 241.83 g,
Female 182.80-213.04 g
(F1) Males: x-x g; Females: x-x g: No data available
- Fasting period before study: A fast of 2h before chemical administered.
- Housing: Animals were housed four rats per sex per cage in sterilized solid bottom polypropylene cages with stainless steel grill tops with facilities for food, water bottles and bedding of clean paddy husk. The cages were suspended on stainless steel racks. Identified by assigned a unique identification (ID) number written on the tail, also specified on individual cage tag.
- Diet (e.g. ad libitum): Standard laboratory sterile extruded pelleted rodent feed, ad libitum.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30-70 %
- Air changes (per hr): 25 ± 5 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark

- Water (e.g. ad libitum): Potable tap water filtered through Reviva Reverse Osmosis
System (water filter cum purifier) of Eureka Forbes was provided in polypropylene bottles with stainless steel sipper tubes, ad libitum.
- Acclimation period: 5 days

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Dose of the test item was freshly prepared prior to dosing on each day. The test chemical was administered to each rat at the dose levels of 50, 150 or 450 mg/kg in the dose volume of 10 ml/kg body weight. The test item was weighed on a weighing balance. Then, it was transferred to calibrated falcon tube. Some quantity of the corn oil was added initially and vortexed. The sufficient quantity of vehicle was added to make up the required volume of dose.
Justification for use and choice of vehicle (if other than water): Corn oil was used as a vehicle for this study as the test item was not soluble in water and to deliver the desired dose levels because it is also recommended in the toxicological evaluation guidelines.
- Concentration in vehicle: 0, 50, 150 or 450 mg/kg body weight /day
Amount of vehicle (if gavage): 10 ml/kg body weight
- Lot/batch no. (if required): No data available

Details on mating procedure:

No data available

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

28 days

Frequency of treatment:

Daily

Details on study schedule:

No data available

Doses / concentrations

Remarks:

0, 50, 150 or 450 mg/kg/body weight/day

No. of animals per sex per dose:

Total : 56
0 mg/kg/body weight/day: 7 male, 7 female
50 mg/kg/body weight/day: 7 male, 7 female
150 mg/kg/body weight/day: 7 male, 7 female
450 mg/kg/body weight/day: 7 male, 7 female

Control animals:

yes, concurrent vehicle

Details on study design:

No data available

Positive control:

No data available

Examinations

Parental animals: Observations and examinations:

Mortality and changes in body weight of the male and female reproductive organs like the testis, epidydimydes, uterus and ovary respectively were noted. In addition, food consumption, water consumption and locomotor activity effects were also recorded.
In male rats, relative organ weight of brain was significantly increased while no changes were observed in female organ weight when treated with 50 mg/kg/body weight/day as compare to control.

Oestrous cyclicity (parental animals):

No data available

Sperm parameters (parental animals):

No data available

Litter observations:

No data available

Postmortem examinations (parental animals):

Testes, ovaries and uterus were pathologically examined after termination. Examinations also included weight of brain, epididymides, and ovaries (incl. paired ovaries and uterus, including cervix).

Postmortem examinations (offspring):

No data available

Statistics:

Statistical analysis was carried out by using Microsoft Excel and IBM SPSS statistics version- 20.0. All analyses and comparisons were evaluated at the 5 % level, statistically significant differences (p ≤ 0.05) indicated by appropriate notation. The focus was to examine the mean differences and their significance in control vs low-dose group only as there was total mortality observed in mid and high-dose groups in both the sexes. The statistical decision was taken by preparing the univariant GLM MODEL to check the significance between above mentioned groups. For multiple comparisons Turkey’s HSD test was applied.

Reproductive indices:

No data available

Offspring viability indices:

No data available

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Some abnormalities like nasal discharge, red crust around nostrils and soft feces were common to all the groups in both the sexes and hence can be considered as background notations. Vocalization on handling was observed in low and mid-dose groups of both sexes. Decreased locomotion was confined to few mid and high-dose groups. Shallow/abdominal breathing was observed in both mid and high-dose groups. There was one case of dehydration and thin body condition in mid-dose male animals. The staining of fore paw was confined to high–dose group of male and female animals.

Dermal irritation (if dermal study):

not specified

Mortality:

mortality observed, treatment-related

Description (incidence):

In high-dose group of male rats the mortality was observed on the day 3, 4, 5, whereas in the mid-dose male rats the mortality was observed in the day 4, 5, 7, 8, and 11. In high-dose females the mortality was seen on day 3, 4 and 5 and in the mid-dose female animals of the mortality was found on day 5, 6, 7, 8, and 10. Only the animals of control and low-dose group survived throughout the treatment period of 28 days.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean body weights of all dose groups of surviving animals were calculated and compared with the control animal data. In male animals, a significant decrease has been noticed in the body weight of low-dose group as compared to control animals on days 15, 22, 28 and also on terminal day. A significant decrease was also noticed in the body weight of female animal treated with low-dose taken on days 22, 28 and terminal day.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

In low-dose group animals the average food consumption (g/rat/day) was calculated and compared with the control. No difference was observed among low-dose group of male as well as female animals. The data of mid-dose and high-dose group could not be used for statistics as there was mortality observed in both the sexes in the first week itself.

Food efficiency:

not specified

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

no effects observed

Description (incidence and severity):

Average water consumption (ml/rat/day) was calculated and compared with the control. No difference was observed among low-dose group of male as well as female animals. The data of mid-dose and high-dose group could not be used for statistics as there was mortality observed in
both the sexes in the first week itself.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No abnormalities were found in the ophthalmological examination (control vs. low-dose treated group).

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

In male rats, RBC count and percentage of monocytes increased significantly (p≤0.05) in lowdose treated group as compared to control animals. However, in female rats, no significant changes were observed in any of the hematological parameters.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

In male rats, the levels of potassium and total proteins were elevated significantly (p≤0.05) in low-dose group as compared to control animals. In female rats, low-dose treatment resulted in significant decrease in the level of total bile acid (p≤0.01) as compared to the control animals

Urinalysis findings:

not specified

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Locomotor activity was decreased significantly in the female animals of low-dose group as compared to control animals.

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

No significant changes were observed in control as well as in low-dose group animals. Few microscopic findings were observed in control as well as in low-dose treated animals which include excess of lymphocytes in bronchi and alveoli of lungs, in ileum and colon. These types of findings may be considered to be within the range of normal background lesions which may be seen in rats of this strain and age of this study type and were considered incidental in nature with carbon dioxide inhalation and terminal changes at sacrifice, reflecting the usual individual variability.

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

not specified

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

water consumption and compound intake

ophthalmological examination

haematology

clinical biochemistry

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

Remarks on result:

other: No effects on reproductive organ weight

Target system / organ toxicity (P0)

Critical effects observed:

not specified

System:

other: not specified

Organ:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

not specified

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

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

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:

other: not specified

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

Overall reproductive toxicity

Reproductive effects observed:

not specified

Treatment related:

not specified

Any other information on results incl. tables

Summary of Reproductive Organ Weights (Male Rats)

Group/Dose		Testes	Epididymides
1/0 mg/kg bw	Mean	0.9930	0.4157
	SD	0.1199	0.0675
	SEM	0.0453	0.0255
2/50 mg/kg bw	Mean	1.0826	0.4780
	SD	0.1111	0.0530
	SEM	0.0420	0.0200

No significance attributed to the body weight changes in comparison to control groups

Summary of Reproductive Organ Weights (Female Rats)

Group/Dose		Uterus	Ovaries
5/0 mg/kg bw	Mean	0.2915	0.0441
	SD	0.0519	0.0188
	SEM	0.0196	0.0071
6/50 mg/kg bw	Mean	0.2346	0.0412
	SD	0.0701	0.0155
	SEM	0.0265	0.0059

No significance attributed to the body weight changes in comparison to control groups

 

Applicant's summary and conclusion

Conclusions:

Male and female rats dosed with 50 mg/kg bw of Solvent blue 4 did not show appreciable difference in the weight of testes & epidydimides and uterus and ovaries respectively, as compared to the control. No significance attributed to the reproductive organ body weight changes in comparison to control groups. Hence the no observed adverse effect level (NOAEL) for the parents has been concluded to be 50 mg/kg bw

Executive summary:

In a 28 days repeated dose toxicity study, the effect of test chemical (containing less than 0.1% Michler's Ketone) was evaluated in male and female Sprague Dawley rats .TheSprague Dawley (SD) rats of 7-8 weeks old were divided into four groups of each sex and each group comprised of either 7 males or 7 females. Test chemical was administered at the dose levels of 50, 150 and 450 mg/kg/day for 28 consecutive days by oral route. Male animals were divided into four groups, group 1 served as control and group 2, 3 and 4 served as low, mid and high-dose group respectively. Female animals were divided into four groups, group 5 served as control and group 6, 7 and 8 served as low, mid and high-dose group respectively. Corn oil was used as vehicle for the present study.

In 450 mg/kg/day dose treatment group in male, the mortality was observed on the day 3, 4, 5, whereas in the 150 mg/kg/day dose treatment group in male rats the mortality was observed in the day 4, 5, 7, 8, and 11. In 450 mg/kg/day dose treatment group in females the mortality was seen on day 3, 4 and 5 and in the 150 mg/kg/day –dose treatment group in female animals’ mortality was found on day 5, 6, 7, 8, and 10 after the administration of test chemical.At the end of the 28 days study only male and female animals of control and 50 mg/kg/day dose groupsurvived up to the completion of the treatment period. During the treatment period, detailed clinical signs, body weight, food and water consumption were recorded weekly. In the groups survived after 28 days of treatment, no abnormalities were found in the ophthalmological examination (control vs. low-dose treated group). However, a significant decrease was observed in the motor activity of treated female animals as compared to the control animals.After the completion of dosing period, blood was collected from all the surviving animals forhematology and clinical biochemistry analysis. All survived animals were sacrificed on the day of

study termination and gross lesions as well as the weight of different organs were recorded. Allorgans were fixed in 10 % formal saline, except the reproductive organs, which were fixed inBouin’s fixative for short duration and thereafter shifted to 10 % formal saline.

In the terminally sacrificed and the animals died during treatment, the necropsy findings that werecommon to all the dosed treated groups include bluish stained perineum and alimentary canal whichinclude stomach and intestine.The finding of stomach blotted with gastric content was common to all the treated group maleanimals and in one female animal of 450 mg/kg/day dose treated group. Mild blue tinged patches were observed in 50 mg/kg/day dose treated male rats. The enlargement of atria in heart, lung congestion and marked thinning of fore stomach wall was confined to 150 mg/kg/day and 450 mg/kg/day dose groups of both the sexes as observed after mortality. White / pale patches on liver / liver congestion / dark reddish liver were confined to 450 mg/kg/day dose group of male and female animals during the time of gross necropsy after mortality. In male rats, the relative weight of brain was significantly increased in 50 mg/kg/day -dose group as compared to control group.Hematology of male animals showed a significant elevation in the red blood cell count as well asthe percentage of monocytes in the 50 mg/kg/day dose group as compared to control animals.

In male rats, significant elevation in the levels of potassium and total proteins was noticed with 50 mg/kg/day dose treatment as compared to control animals.In female rat, significant decrease in the level of total bile acid was noticed in the 50 mg/kg/day dose treated animals as compared to control animals.In the microscopic histopathological study there was no significant change observed in 50 mg/kg/day dose group animals as compared to control animals. Few microscopic findings were observed in control

as well as in 50 mg/kg/day dose treated animals, which include excess of lymphocytes in bronchi and alveoli of lungs, ileum and colon. These types of findings may be considered within the range of normalbackground lesions, which are seen in rats with the age groups used in the present study. Further,these types of changes were considered incidental in nature with carbon dioxide inhalation. Male and female rats dosed with 50 mg/kg bw of Solvent blue 4 did not show appreciable difference in the weight of testes & epidydimides and uterus and ovaries respectively, as compared to the control. No significance attributed to the reproductive organ body weight changes in comparison to control groups. Hence the no observed adverse effect level (NOAEL) for the parents has been concluded to be 50 mg/kg bw