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

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

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

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animal species: laboratory rat
Strain: Wistar CRL (SPF quality - guaranteed)
Supplier: SPF breeding, VELAZ s.r.o., Lysolajské údolí 15/53, 165 00 Prague 6, Czech Republic, RČH CZ 11760500
Selection of animal species: laboratory rat has been chosen because our testing laboratory has long experience with this species
Sex: males and females (nulliparous and non-pregnant)
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: gavage

Vehicle:

water

Details on exposure:

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
Aqua pro iniectione
Batch No.: 2004280281 expiration: 04/2022
2005110305 expiration: 05/2022
Manufacturer: Ardeapharma Ševětín, Czech Republic

Details on 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.

Details on study schedule:

Dose Levels and Administration
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.

1. Control – vehicle 0 mg/kg/day 6 females and 6 adult males
2. Low dose 150 mg/kg/day 6 females and 6 adult males
3. Intermediate dose 450 mg/kg/day 6 females and 6 adult males
4. High dose 1000 mg/kg/day 6 females and 6 adult males

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. 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 (nominal)

Doses / concentrations 2

Dose / conc.:

450 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

150 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

0 mg/kg bw/day (nominal)

No. of animals per sex per dose:

6 females and 6 males

Control animals:

yes, concurrent no treatment

Details on study design:

The test item was given to animals by gavage in three doses (low, intermediate and high). Each dose was administered to stomach by gavage to a group of 6 males and 6 females. Females were mated with males in ratio 1:1.
Males were dosed before and during mating period and three weeks after mating up to and including the day before scheduled kill. Females were dosed throughout the study. This includes one week prior to mating, during the mating period and the duration of pregnancy.
At the end of the study females were killed on 20th day of pregnancy. Non-pregnant females and males were killed after the end of last application.
During the study and after euthanasia, the animals were examined by examination methods (mortality/viability, body weight, food consumption, health condition control, clinical observation and laboratory examinations).

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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):

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.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

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

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

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

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

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.

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

Reproductive function / performance (P0)

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

No significant changes in sperm motility and morphology of treated group of males were found during the observation of sperms.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

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.

Effect levels (P0)

Dose descriptor:

LOAEL

Effect level:

ca. 150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Target system / organ toxicity (P0)

Critical effects observed:

no

Lowest effective dose / conc.:

0

Results: F1 generation

General toxicity (F1)

Mortality / viability:

no mortality observed

Description (incidence and severity):

Dead foetuses were not recorded in females at the dose levels 150 and 450 mg/kg/day, and control group. The total number of live foetuses in group was decreased at the dose level 450 mg/kg/day compared with group of control females.
Average number of foetuses per litter at all dose levels was comparable with control group. The highest number of live foetuses was recorded in the control group

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The mean body weight of foetuses was slightly decreased at the middle dose level compared to the control group. Male foetuses were heavier than females in all groups.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No macroscopic changes of soft tissues and external alteration were found during the pathological examination of the foetuses at all dose levels

Effect levels (F1)

Key result

Dose descriptor:

LOAEL

Generation:

F1

Effect level:

ca. 150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

yes

Relation to other toxic effects:

not specified

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*
Number of pregnant dams	6/6	6/6	5/6	0/6	-
Implantations (mean ± SD)	17.17± 2.04	16.67 ± 1.51	16.80 ± 2.68	-	-
Resorptions (mean ± SD)	0.33 ± 0.52	1.00 ± 1.10	1.00 ± 1.00	-	-
Corpora lutea (mean ± SD)	17.67 ± 1.51	16.67 ± 1.51	17.20 ± 2.28	-	-

For foetuses (per dose)	Dose levels (mg/kg/day)				Notes
	Control	150	450	1000*
Total number of live foetuses	101	94	79	-
Total number of dead foetuses	0	0	0	-
External, soft tissue and skeletal malformations and other relevant alterations	none	none	none	-

* Females No. 131-136 – died during 1st week of application.

Applicant's summary and conclusion

Conclusions:

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.

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.

Study performance
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.

Dose levels: 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
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.