Phenol, 4-[[4-(dimethylamino)phenyl]azo]-, reaction products with sodium sulfide (Na2(Sx)), leuco derivatives

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

25 January 2018 - 23 March 2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

14 days exposure instead of 28 days

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

2008

Deviations:

yes

Remarks:

only 14 days exposure

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Version / remarks:

2008

Deviations:

yes

Remarks:

only 14 days exposure

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3050 Repeated Dose 28–Day Oral Toxicity Study in Rodents, EPA Health Effects Test Guidelines

Version / remarks:

2000

Deviations:

yes

Remarks:

only 14 days exposure

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reaction product of 4-​aminophenol with 2-​ethyl-​6-​methylbenzenamine and sodium polysulfide

EC Number:

600-519-8

Cas Number:

1040873-93-5

Molecular formula:

not applicable

IUPAC Name:

Reaction product of 4-​aminophenol with 2-​ethyl-​6-​methylbenzenamine and sodium polysulfide

Test material form:

solid: particulate/powder

Details on test material:

Test item: Blue Sema
Appearance: Black to brownish black, solid
CAS No: 1040873-93-5
EC No: 600-519-8

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The rat is a commonly used species for toxicological studies in accordance with international recommendations. The Wistar rat was the system of choice because it has been the preferred and most commonly used species for oral toxicity tests and is a well-known laboratory model with sufficient historical data.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Toxi-Coop Zrt. 1103 Budapest, Cserkesz u. 90., Hungary
- Females nulliparous and non-pregnant: yes
- Age at study initiation: Male animals: 48 – 53 days, Female animals: 63 – 68 days
- Weight at study initiation: 202 – 223 g for male animals, 145 – 176 g for female animals
- Housing: 2-3 animals of the same sex/ cage (Type II polypropylene/ polycarbonate)
- Diet: ssniff® SM R/M-Z+H complete diet for rats and mice produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany, ad libitum
- Water: tap water ad libitum
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): > 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
- Rate of preparation of diet: Formulations were prepared in the formulation laboratory of the Test Facility beforehand not longer than for three days and stored at 5 +/- 3 °C until use.

VEHICLE
- Concentration in vehicle: 20, 60 and 200 mg/mL by the active ingredient content (corrected concentrations; respectively to uncorrected concentrations of 23, 69 and 230 mg/mL)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical dose verification of the formulations was performed once during the study. Five aliquots of 5 mL of each formulation to be administered to the animals (20, 60 and 200 mg/mL) and five aliquots of 5 mL control substance (vehicle) were taken. The suitability of the chosen vehicle (recovery and stability) for the test item at the intended concentrations was analytically verified up front. The recovery of the test item from the vehicle was within the acceptance criteria (relative to nominal concentrations: 95 % at ca. 1 mg/mL and 103 % at ca. 200 mg/mL). The test substance proved to be stable in distilled water at the intended concentrations at room temperature for 24 hours and at 5+/-3 °C for three days.

Duration of treatment / exposure:

14 days

Frequency of treatment:

7 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

corresponding to 0 mg dye/kg bw/d

Doses / concentrations 2

Dose / conc.:

115 mg/kg bw/day (actual dose received)

Remarks:

corresponding to 100 mg dye/kg bw/d

Doses / concentrations 3

Dose / conc.:

345 mg/kg bw/day (actual dose received)

Remarks:

corresponding to 300 mg dye/kg bw/d

Doses / concentrations 4

Dose / conc.:

1 150 mg/kg bw/day (actual dose received)

Remarks:

corresponding to 1000 mg dye/kg bw/d

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose setting with 0, 100, 300 and 1000 mg/kg bw/day was based on the literary data of chemically similar compounds. Doses were selected with the aim of inducing toxic effects but no mortality or suffering at the highest dose and a NOAEL at the lowest dose.

Positive control:

no

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a day
- Parameters checked in table No.1 were examined.

BODY WEIGHT: Yes
- Time schedule for examinations: Day 0 (prior to study start) and twice weekly (on Days 0, 4, 7, 10 and 13)

FOOD CONSUMPTION: Yes
- Time schedule: determined once weekly

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: before the terminal necropsy
- Anaesthetic used for blood collection: Yes, Isofluran
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table No. 2 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: before the terminal necropsy
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table No. 3 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes.
The organs listed in table 4 were removed and preserved.

HISTOPATHOLOGY: No
The organs listed in table 4 were removed and preserved. Histopathological examinations were not performed because no adverse effects were observed up to the top dose applied.

Other examinations:

Organ Weight
The following organ weights were determined and recorded: paired organs were weighed together: liver, kidneys, testes, epididymides, uterus, thymus, spleen, brain and heart, prostate and seminal vesicles with coagulating glands, as a whole, adrenal glands.

Statistics:

Statistical analysis was done with SPSS PC+ software for the following data: body weight, food consumption, hematology, blood coagulation, clinical chemistry and organ weight.
The heterogeneity of variance between groups was checked by Bartlett's homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance was carried out. If the obtained result was positive, Duncan's Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorov-Smirnov test. In case of a none-normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was used. If there was a positive result, the inter-group comparisons were performed using the Mann-Whitney U-test.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

The test item did not cause clinical signs at 100, 300 or 1000 mg/kg bw/day in surviving male or female animals during the course of 14-day administration. The behavior and physical condition of all animals (male and female) were normal during the 14-day observation period. Clinical signs of dead female animal (see below) were not considered to be related to the test item due its singular occurrence. Transiently diarrhea was noted for one male animal at 300 mg/kg bw/day (1/5) on Day 2 but there were no clinical signs during the following days.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female animal (1/5, no. 42) at 1000 mg/kg bw/day was found dead on Day 10. Preceding clinical signs – decreased activity, dyspnea and noisy breathing – and decrease in the body weight were noted for this animal from Day 4 up to and including Day 9. The organs and tissues were significantly autolyzed therefore detection of necropsy findings was not feasible. There was no mortality in the control, 100 or 300 mg/kg bw/day groups during the 14-day treatment period (male and female).

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The body weight and body weight gain of the male and female animals were unaffected in all test item treated groups (100, 300 or 1000 mg/kg bw/day) throughout the entire observation period. The mean body weight and mean body weight gain was similar in male animals in the control and test item treated groups (100, 300 and 1000 mg/kg bw/day) during the entire observation period. The body weight of the dead female animal decreased between Days 4 and 7. A slight, but statistically significant difference with respect to the control was detected at the slightly higher mean bodyweight gain of female animals at 100 mg/kg bw/day between Days 4 and 7. This slight change was considered to be indicative of biological variation and not related to the test item.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no test item related changes in the mean daily food consumption at 100, 300 or 1000 mg/kg bw/day. The mean daily food consumption was similar in the control and test item treated groups (male and female) during the 14-day observation period.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item related alterations in the examined hematological parameters in male or female animals at 100, 300 or 1000 mg/kg bw/day compared to their controls.
Slight but statistically significant differences compared to the control group were noted in some of the white blood cell parameters in male animals as follows: elevated mean white blood cell count (WBC) and lower mean percentage of neutrophil granulocytes (NEU) at 100 and 1000 mg/kg bw/day; lower mean percentage of basophil granulocytes (BASO) at 300 mg/kg bw/day; slightly higher mean percentage of lymphocytes (LYM) and lower mean percentages of monocytes (MONO) at 1000 mg/kg bw/day. In the female animals, statistical significance was detected at the slightly lower mean percentage of reticulocytes (RET) at 100 and 300 mg/kg bw/day as well as at the slightly longer mean prothrombin time (PT) at 100 mg/kg bw/day when compared to the concurrent control. These slight but statistically significant differences with respect to control (WBC, NEU, BASO, LYM and MONO in male animals and RET, PT in female animals) were considered to be toxicologically not relevant as these were at a low magnitude, were not related to doses or were only present in the low or mid dose groups.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical chemistry evaluation did not reveal test item related changes in the investigated parameters at 100, 300 or 1000 mg/kg bw/day (male and female). The examined clinical chemistry parameters were similar in male animals in the control and test item treated groups (100, 300 and 1000 mg/kg bw/day). Slight but statistically significant differences with respect to the control were detected at the lower mean concentration of albumin (ALB) and albumin: globulin ratio (A/G) at 100 mg/kg bw/day and at the higher mean glucose concentration at 300 and 1000 mg/kg bw/day. These findings in the clinical chemistry parameters were judged to be indicative of biological variation and of no biological significance.

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):

Test item related organ weight changes were not observed in male and female animals at 100, 300 or 1000 mg/kg bw/day. In male animals, statistical significances were noted for the slightly lower heart weights (absolute and relative to body and brain weights) at 100 and 300 mg/kg bw/day and at the higher mean weights of seminal vesicle-coagulating gland-prostate complex (absolute and relative to body and brain weights) at 100 mg/kg bw/day when compared to the relevant control. In the female animals, there were no statistically or biologically significant differences between the control and treated groups (100, 300 or 1000 mg/kg bw/day) in the weights of examined organs. Changes in the weights of the above mentioned organs were of low degree and not related to doses. Therefore these findings were judged to be toxicologically not relevant.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item related macroscopic findings were observed in male or female animals (100, 300 or 1000 mg/kg bw/day). In the female animal found dead, autolysis of visceral organs was seen therefore macroscopic observation of tissues was not possible. There were no macroscopic changes in the organs or tissues of male animals in the control 100, 300 or 1000 mg/kg bw/day groups at the necropsy. Slight or moderate hydrometra was observed in single female animals in control (1/5) group and at 300 mg/kg bw/day (1/5). Hydrometra, related to the female sexual cycle, is a frequent observation in experimental rats. In the lack of related inflammatory or other pathological signs it was judged to be toxicologically not relevant.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: absence of test item related adverse effects

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

Under the conditions of the present study, the test item did not cause adverse effects in male or female Hsd.Han: Wistar rats after the consecutive 14-day oral (by gavage) administration of 100, 300 or 1000 mg dye/kg bw/d.

Executive summary:

A study was conducted to obtain first information on the toxic potential of the test item in rats at three dose levels to allow a dose-setting for a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (main study according to TG OECD 422). Doses of 0 (vehicle only), 100, 300 and 1000 mg dye/kg bw/d (corrected doses, corresponding to 0, 115, 345 and 1150 mg test item/kg bw) were orally administered (gavage) to four groups of Hsd.Han:Wistar rats consisting of five animals per group and sex at a dosing volume of 5 mL/kg. Blue Sema was administered in concentrations of 20, 60 and 200 mg/mL by the active ingredient content (corrected concentrations; respectively to uncorrected concentrations of 23, 69 and 230 mg/mL). A group of vehicle (distilled water) treated animals (n= 5/sex) served as a control. Detailed clinical observations were performed daily after the treatment and before the necropsy. Body weights were recorded twice weekly. The food consumption was determined weekly to coincide with body weight measurements during the study. Clinical pathology (hematology, blood coagulation and clinical chemistry) and gross pathology examinations were conducted one day after the last treatment (on Day 14). Selected organs were weighed. One male animal at 1000 mg/kg bw/day was found dead on study Day 10. Dyspnea, noisy breathing and decreased activity were observed on the preceding day (from Days 4 to 9).The test item did not induce clinical signs at 100, 300 or 1000 mg/kg bw/day in surviving male or female animals during the two weeks treatment period. No test item-related body weight or body weight gain changes were observed at 100, 300 or 1000 mg/kg bw/day in male or female animals. The mean daily food consumption was comparable in the control and test item treated groups – 100, 300 or 1000 mg/kg bw/day, male and female. Hematological evaluation did not reveal test item related changes in the examined parameters at 100, 300 or 1000 mg/kg bw/day (male and female). There were no test item related alterations in the examined clinical chemistry parameters (100, 300 or 1000 mg/kg bw/day, male or female). Specific macroscopic findings were not detected in the organs or tissues of male or female animals at 100, 300 or 1000 mg/kg bw/day. The weights of examined organs were not affected by the treatment with 100, 300 or 1000 mg/kg bw/day dose of the test item. Therefore, under the conditions of the present study, the test item did not cause adverse effects in male or female Hsd.Han: Wistar rats after the consecutive 14-day oral (by gavage) administration of 100, 300 or 1000 mg/kg bw/d. A NOAEL of 1000 mg/kg bw/d was determined.