Disodium; 4-Amino-3-{4-[4-(2,4-diamino-phenylazo)-benzenesulfonylamino]-phenylazo}-5-hydroxy-6-phenylazo-naphthalene-2,7-disulfonate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

disodium 4-amino-3-[2-(4-{4-[2-(2,4-diaminophenyl)diazen-1-yl]benzenesulfonamido}phenyl)diazen-1-yl]-5-hydroxy-6-(2-phenyldiazen-1-yl)naphthalene-2,7-disulfonate

EC Number:

605-104-5

Cas Number:

157577-99-6

Molecular formula:

C34H26N10Na2O9S3

IUPAC Name:

disodium 4-amino-3-[2-(4-{4-[2-(2,4-diaminophenyl)diazen-1-yl]benzenesulfonamido}phenyl)diazen-1-yl]-5-hydroxy-6-(2-phenyldiazen-1-yl)naphthalene-2,7-disulfonate

Details on test material:

- Name of test material (as cited in study report): Acid Black 234

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Supplier: SPF breeding, VELAZ s.r.o., Lysolajské údolí 15/53, 165 00 Prague 6, Czech Republic, RČH CZ 11760500

Sex:

male/female

Details on test animals or test system and environmental conditions:

Age of animals: 10 weeks on arrival
Acclimatization: 5 days
Number of animals: 6 males and 6 females per group
Housing conditions: SPF conditions according to internal SOP No.12, transparent polysulfonate cages (VELAZ).
Animal per cage: 2 rats of the same sex in one cage in pre-mating period, during mating period – one male and one female in one cage, pregnant females – individually
Food: complete pelleted diet for rats and mice in SPF breeding
Water: drinking water ad libitum, Regulation No. 252/2004 Czech
Coll. of Law, Ministry of Health
Light cycle: 12 hour light/12 hour dark
Microclimate: 22  3 °C, relative humidity 30 - 70 %
Bedding: sterilized soft wood fibers Lignocel (producer: JRS GMBH+Co.KG, Rosenberg, Germany)
Selection of animals: random selection according to the internal SOP No.42 – at the beginning of the study the weight variation of animals in groups of each sex should not exceed + 20% of the group mean weight
Identification of animals: the animals were identified by the colour marks on their fur; each cage was marked with the number of animals, sex, number of cage, name and dose level of the test item.

Administration / exposure

Route of administration:

oral: unspecified

Details on route of administration:

The application form of the test item was administered to the stomach by gavage. The test item concentration at single dose level was adjusted so that the administered volume was constant at all dose levels – 1 ml/100 g body weight based on the most recent body weight determination. The administration form of the test item was prepared daily before and during administration. It was mixed by the magnetic stirrer for 10 minutes.

Vehicle:

water

Details on oral exposure:

The application form (test item in aqua pro iniectione) was administered to the stomach by gavage. The vehicle control group was administered by aqua pro iniectione in the same volume. The test item concentration at single dose level was adjusted so that the administered volume was constant at all dose levels – 1 ml/100 g body weight.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

The animals were treated 7 days per week at the same time (8.00 – 10.00 am).
The test item was administered in graduated dose levels to males and females 7 day before mating and during mating period. Then males were administered after mating up to a total of 3 weeks. Pregnant females were administered during pregnancy (0 – 19th day of pregnancy ).

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

Dose / conc.:

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

No. of animals per sex per dose:

6 females and 6 males

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: Dose levels were determined in accordance with the requirements of the Sponsor for comparing with the similar substance. The test item was administered daily in graduated three doses to groups of experimental males for 42 days and females a for max. 40 days.
The dose levels for the dose-range finding experiment have been chosen with respect to the information about acute toxicity the test item (LD50 ˃ 2000 mg/kg bw) and according to the demands of OECD Test Guideline No. 422

- Rationale for animal assignment (if not random): random selection according to the internal SOP No.42 – at the beginning of the study the weight variation of animals in groups of each sex should not exceed + 20% of the group mean weight
Experimental Data Collection

Mating Procedure: Animals were mated from the 8th to 21st day of study. Mating 1:1 (one male to one female) was in this study. Each morning the females were examined for presence of spermatozoa in vaginal smears. Day 0 of pregnancy was defined as the day the sperms were found


Vaginal smears: daily in mating period
Body weight: males: before first application and then weekly
females: before first application and then weekly
during pregnancy: on the 0, 3rd, 6th, 9th, 12th, 15th and 20th day
Food consumption: weekly and in the same days as body weight (except the mating period)
Mortality control: daily
Health condition control: daily
Clinical observations: twice a day (except weekend, when clinical observation was performed once a day)
Observation od sperm: motility – during necropsy
morphology – after necropsy
Haematological examination: males: after the end of application
females: 20th day of pregnancy
Pathological examination: males: after the end of application
females and foetuses: 20th day of pregnancy
Histopathological examination: males and females: after the end of necropsy

-

Positive control:

no

Examinations

Observations and examinations performed and frequency:

Health Condition Control
The health condition of animals was controlled daily. Pre-experimental observation of all rats was performed to ensure that only the animals exhibiting normal behavioural activity would be entered into the study. In administration period this observation was performed before, during and immediately after application.

Clinical Observation
All animals were observed daily after administration. This observation was made in order to record possible clinical effects after application and all changes in behaviour of animals. So, it was done after application at the same time every day – at the time of expectation of maximal effect of the test item. Animals were observed in natural conditions in their cages. The condition of the animals was recorded including mortality, moribundity, pertinent behavioural changes, and all signs of overt toxicity.

Body Weight
The body weight of animals was recorded on automatic balances with group average computing module. Males were weighting before first application and then weekly. Females were weighting before first application and then weekly. During pregnancy were weighted 0, 3st, 6th, 9th, 12th, 15th and 20th day of pregnancy (before necropsy). Weight increment was computed as an average per group (in grams).

Food Consumption
Food consumption were determined in the same days as body weight (except the mating period), when were weighting animals.
In a specified day the remainder of pellets were weighed in each cage, the new food was weighed out and the food consumption for the previous week was computed.
In males mean values was calculated for each week of the study (except the mating period). Food consumption for animal/day was calculated from mean values of each group. The same way of calculation of mean food consumption was used for females in pre-mating period. In pregnancy mean individual values (grams/animal/day) were calculated for each week of the study. Mean food consumption for each group was calculated from individual values. Non-pregnant females were not included in calculation of mean food consumption in pregnancy period.

Examination of Vaginal Smears
Each morning in the mating period vaginal smears were prepared from all the mated females. These smears were examined for presence of spermatozoa. Vaginal smears were prepared and examined according to internal SOP No. M/74

Haematological Examination
Pregnant females and males were collected blood under the light ether narcosis by glass micropipette under the light ether narcosis into the PVC test tubes (containing anticoagulation system), which were prepared for individual examinations.
In the dose-range finding experiment the following parameters were determined: total erythrocyte count, total leucocyte count, total platelet count, haematocrit, haemoglobin concentration and mean corpuscular volume by haematology analyzer URIT-5160Vet

Observation of Sperm
In all males were examined the following sperm parameters: sperm motility and sperm morphology. Observation of sperm were prepared and examined according to internal SOP No. M/82

Pathological Examination Males and Females
Males were done necropsy after the end of application (43rd day of study) and during necropsy were done macroscopic examination and observations of sperm (motility, morphology). The following organs were collected from all males at necropsy for further histopathology evaluation: spleen (fixed in buffered 4% formaldehyde solution (v/v)) for and testicles (fixed in Davidson’s solution).
Females were killed one day prior to the expected day (20th day of pregnancy) of delivery. As soon as possible after euthanasia gravid uterus (incl. the cervix) was removed and weighed and pregnancy status determined. In uterus number of viable foetuses, number of dead foetuses, number of early resorptions (implantations without recognisable embryo/foetus) and number of late resorptions (dead embryo or foetus with external degenerative changes) were recorded. Number of corpora lutea on ovaries was found out.
Uteri of non-pregnant females was examined to confirm the non-pregnant status. Then was performed macroscopic examinations in all females

Pathological Examination of Foetuses
Number, sex and body weight of all live foetuses, and number and sex of dead foetuses were recorded. Each foetus was examined for external alteration and pathological changes, which were recorded

Histopathological Fxamination
For histopathological processing the routine histological paraffin technique with synoptic haematoxylin-eosin staining was used. Other special staining was used in indicated cases.
In males, histopathology investigation was performed at the organs as spleen and testicles collected during the necropsy. Due to macroscopic finding on spleen in females at the intermediate dose level, so histopathological examination was also performed.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes

Statistics:

For statistical evaluation the software Statgraphic® Centurion (version XV, USA) was used. The data from control group were compared with data from treated groups.

The parametric test was used for statistical evaluation of:
• Haematological examination of males and females

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Change health condition and clinical symptom of intoxication as piloerection were recorded in males and females at the dose 1000 mg/kg/day. At the dose levels 450 and 1000 mg/kg/day coloured faeces were observed in males and females. This change of colour of faeces is associated with the colour of the test item (black).

Mortality:

mortality observed, treatment-related

Description (incidence):

Females: Control group (0 mg/kg/day): no mortality occurred.
150 mg/kg/day: no mortality occurred.
450 mg/kg/day: no mortality occurred.
1000 mg/kg/day: All six females gradually died during the 1st week of application. First female was found dead the 3rd day of study. Other females were found dead the 4th, 6th and 7th day of study.
Males:Control group (0 mg/kg/day): no mortality occurred.
150 mg/kg/day: no mortality occurred except one male who died 29th day of study, due to incorrect application of the test item (not related with the test item toxicity).
450 mg/kg/day: no mortality occurred.
1000 mg/kg/day: All six males gradually died during the 1st week of application. First male was found dead the 3rd day of the study. Other males were found dead the 6th and 7th day of the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Males: Body weight and total weight increment of males at all treated groups were quite balanced.
Females: During premating period, the total weight increments of females at dose levels 150 and 450 mg/kg were slightly decreased compared with group of control females. In pregnancy period, mean weight increments of treated groups of females were comparable with group of control females. Corrected body weights of all treated females were similar to the control females.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Females: In pregnancy period, only females which were found to be pregnant 20th day of pregnancy (females with live foetuses) were used for calculation of mean food consumption.
The food consumption of females at the dose level 450 mg/kg/day in premating period was slightly decreased compared with group of control females. The food consumption of treated groups of females during the pregnancy was similar in comparison with the control group of females.
During mating period, the food consumption was not observed
Males: The food consumption of males was quite balanced at all treated groups. During mating period, the food consumption was not observed.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Males: All measured values of treated males were compared with group of control males. Haematological examination showed differences among the dose levels and the control group of males. The statistical evaluation was performed. Statistically significant differences at the dose levels 150 and 450 mg/kg/day were detected. It revealed decreased total erythrocyte count, haemoglobin, haematocrit and increased mean corpuscular volume.
All other measured parameters were quite balanced to group of control males
Females: All measured values of treated groups of females were compared with group of control females. Haematological examination showed minor differences among the dose levels and control females. The statistical evaluation was performed. Statistically significant differences at the dose level 450 mg/kg/day were detected. It revealed the increase of total leucocyte count and mean corpuscular volume. All other measured parameters were comparable to group of control females

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:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Pathological examination of males at the dose level 450 mg/kg/day revealed dark enlarged spleen and in females at this dose level dark spleen only were found.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathological examination of testicles and spleen in all males and macroscopically changes organs – spleen in group of females at the dose level 450 mg/kg/day was performed.
In males at the dose levels 150 and 450 mg/kg/day, microscopic examination of spleen revealed mild to marked siderosis, minimal to mild extramedullary hemopoiesis and mild to marked venostasis. No pathological findings of testicles in all dose levels, except for atrophy of solitary seminiferous tubules in one male (spontaneous origin).
In females at the dose level 450 mg/kg/day, microscopic examination of spleen revealed mild to marked siderosis and marked extramedullary hemopoiesis.

Effect levels

Key result

Dose descriptor:

LOAEL

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

haematology

histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

gastrointestinal tract

Organ:

spleen

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Any other information on results incl. tables

For females (per dose)	Dose (mg/kg/day)				Notes
	Control	150	450	1000*  Females No. 131-136 – died during 1st week of application.
Mortality	0/6	0/6	0/6	6/6	-
Body Weight (g) - corrected (group mean ± SD)	326.81±19.09	332.91±26.59	329.05±25.06	-	-
Clinical signs: description, severity, time of onset and duration	none	none	Faeces coloured (throughout the application of the test item)	Faeces coloured and piloerection (during 1^st week the application of the test item)	-
Pathological examination	none	none	Dark spleens	Autolysis organs*	* Females were found dead
Haematological examination – statistically significant	-	-	↑ WBC, MCV	-	-
Histopathological examination	none	none	Siderosis (minimal-mild) Extramedullary hemopoiesis (marked)	-	-

For males (per dose)	Dose (mg/kg/day)				Notes
	Control	150*	450	1000**
Mortality	0/6	1/6	0/6	6/6	-
Total weight increment (g) - group mean	74.84	69.54	76.58	-	-
Clinical signs: description, severity, time of onset and duration	none	none	Faeces coloured (throughout the application of the test item)	Faeces coloured and piloerection (during 1^st week the application of the test item)	-
Pathological examination	none	none	Dark and enlarged spleens	Autolysis organs*	* Males were found dead
Haematological examination – statistically significant	-	↓ RBC, Hgb, Hct ↑ MCV	↓ RBC, Hgb, Hct ↑ MCV	-	-
Histopathological examination	none	none	Siderosis (minimal-mild), Extramedullary hemopoiesis (marked), Venostasis (mild-marked)	-	-

* Male No. 11 – died the 29th after application.
** Males No. 31-36 – died during 1st week of application.

Applicant's summary and conclusion

Conclusions:

Deaths occurred during the Dose-range finding experiment with the test item Acid Black 234. All males and females at the dose level 1000 mg/kg/day died during the 1st week of study. One male died at the dose level 150 mg/kg/day, but its death was accidental (intubation error), not related with the test item administration. No death was recorded in males and females at the dose level 450 mg/kg/day and control group.

Change health condition and clinical symptom of intoxication as piloerection were recorded in males and females at the dose 1000 mg/kg/day. At the dose levels 450 and 1000 mg/kg/day coloured faeces were observed in males and females. This change of colour of faeces is associated with the colour of the test item (black).

Lower body weight increment was recorded in females (during premating period) at the dose levels 150 and 450 mg/kg/day. The negative effect of the test item administration on the body weight of males and pregnant females was not observed at any dose levels.

The negative effect of the test item administration on the food consumption was not observed at any dose levels.

Haematological examination showed some statistically significant differences among the dose level 450 mg/kg/day and other dose levels in females. The administration of the test item caused in females increased total leucocyte count and mean corpuscular volume. In males at the dose levels 150 and 450 mg/kg/day were observed statistically significant decrease of total erythrocyte count, which was associated with the decline decrease of haemoglobin, haematocrit and increase of mean corpuscular volume.

Pathological examination of males at the dose level 450 mg/kg/day revealed dark enlarged spleen and in females at this dose level dark spleen only were found. No macroscopic changes were found during the pathological examination of foetuses.

The evaluation of reproduction parameters showed the following: In females at the dose levels 150 and 450 mg/kg/day were slightly increased resorptions. Total number of foetuses were the lowest at the dose level 450 mg/kg/day. No death foetuses were found in any dose levels. The body weight of foetuses was slightly decreased at the dose level 150 mg/kg/day.
No significant changes in sperm motility and morphology of treated group of males were found during the observation of sperms.

Histopathological examination of testicles and spleen in all males and macroscopically changes organs – spleen in group of females at the dose level 450 mg/kg/day was performed.
In males at the dose levels 150 and 450 mg/kg/day, microscopic examination of spleen revealed mild to marked siderosis, minimal to mild extramedullary hemopoiesis and mild to marked venostasis. No pathological findings of testicles in all dose levels, except for atrophy of solitary seminiferous tubules in one male (spontaneous origin).
In females at the dose level 450 mg/kg/day, microscopic examination of spleen revealed mild to marked siderosis and marked extramedullary hemopoiesis.

Due to mortality of males and females at the dose level 1000 mg/kg/day, macroscopic and histopathological findings on the spleens of males and females at the dose level 450 mg/kg/day, the following level doses have been proposed.

On the basis of the results given above the following dose levels – 75, 150 and 300 mg/kg/day were proposed for the future main Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test.

Executive summary:

The test item Acid Black 234, was tested for Dose-range finding experiment for Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test using the OECD Test Guideline No. 422 Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, Adopted by the Council on the 29th July, 2016.The dose-range finding experiment was performed with 3 groups of treated males and females and control group of males and females treated by vehicle. Females were mated with males in ratio 1:1.The Dose levels were: 0 (control), 150, 450 and 1000 mg/kg/day.After acclimatization the application form (test item in aqua pro iniectione) was administered to animals to the stomach by gavage in three doses (low, intermediate and high). The control group received only vehicle (aqua pro iniectione). Each dose group consisted of 6 males and 6 females. The test item concentration at single dose level was adjusted so that the administered volume was constant at all dose levels – 1 ml/100 g body weight based on the most recent body weight determination. Males were dosed before, during mating period and three weeks after mating up to and including the day before scheduled kill. Females were dosed throughout the study. This included one week prior to mating, during the mating period and the duration of pregnancy.Control of fertilization was made by the help of the vaginal smears. Vaginal smears were carried out after 24 hours of the first removing to male and then daily at the same time and the presence of sperms were examined. Day 0 of pregnancy was the day on which sperms in vaginal smear were found out. Males, after the end of application were collected blood for haematological examination under the light ether narcosis by glass micropipette and then was performed necropsy. During necropsy macroscopic examination and observations of sperm (motility, morphology) were performed. The following organs were collected from all males at necropsy for further histopathology evaluation: spleen and testicles.
On day 20 of pregnancy, blood was collected from females under the light ether narcosis for haematological examination. Then the females were necropsied, macroscopic abnormalities were recorded and the examination of reproductive parameters (implantations, resorptions and number of corpora lutea) and macroscopic examinations of all foetuses (sex, number, body weight, pathological changes and external alteration) were performed.

Results showed that there was an unplanned death of all females and males during the first week of study at the dose level 1000 mg/kg/day (cause unknown), and one male at the dose level 150 mg/kg/day (intubation error). Changes of health condition and clinical symptoms of intoxication as piloerection was found in females and males at the dose level 1000 mg/kg/day.
During control of body weight increments of males at all the dose levels were not detected toxicologically significant treatment-related effects. In females (during premating period) at the dose levels 150 and 450 mg/kg/day lower body weight increment was recorded.
Food consumption in males and females at all dose levels and control groups, toxicologically significant treatment-related effects were not detected.
Minor statistically significant differences were showed in haematological examination. In males, at the dose levels 150 and 450 mg/kg/day was observed decrease of total erythrocyte count, which was associated with the decline decreased of haemoglobin, haematocrit and increased of mean corpuscular volume. In females were detected decrease of total erythrocyte count and mean corpuscular volume at the dose level 450 mg/kg/day .
The evaluation of reproduction parameters revealed slightly increased resorptions at the dose level 150 and 450 mg/kg/day and slightly decreased the body weight of foetuses at the dose level 150 mg/kg/day. Sperm motility and morphology not showed significant changes.
Macroscopical structure of organs of males, females and foetuses – in all males at the dose level 450 mg/kg/day were found dark enlarged spleen. In all females at the same dose level were found dark spleen. No macroscopic changes were found during the pathological examination of foetuses.
Subsequent histopathological examination of spleen in males at the dose levels 150 and 450 mg/kg/day and in females at the dose level 450 mg/kg/day revealed siderosis, extramedullary hemopoiesis and venostasis. Testicles in all dose levels did not show any pathological findings.