disodium 4-amino-6-((4-((4-(2,4-diaminophenyl)azo)phenylsulfamoyl)phenyl)azo)-5-hydroxy-3-((4-nitrophenyl)azo)naphthalene-2,7-disulfonate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 1,2010-January 20, 2011

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: 422/Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

GLP compliance:

yes

Limit test:

no

Test material

Test material information

1

Reference substance name:

-

EC Number:

421-880-6

EC Name:

-

Cas Number:

201792-73-6

Molecular formula:

C34H25N11Na2O11S3

IUPAC Name:

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

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

FOR THE PARENTS
Age of animals: males, females – sexually adult (10 weeks – on arrival)
Selection of animal species: laboratory rat has been chosen because our testing laboratory has long experience with this species
Acclimatization: at least 5 days
Number of animals: 12 females and 12 males per group, 6 males and 6 females per satellite group
Housing conditions: SPF – 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, offspring – with mother, satellite animals - 2 rats of the same sex in one cage
- Food: complete pelleted diet for rats and mice in SPF breeding
- Water: drinking water ad libitum, quality standard ČSN 757 111
- Light cycle: 12 hour light / 12 hour dark
- Microclimate: 22 3°C, relative humidity 30-70%
- Bedding: sterilized shavings of soft wood
Selection of animals: random selection according to the internal rule – at the beginning of the study the weight variation of animals in groups of each sex
should not exceed + 20% of the mean weight
Identification of animals: the animals will be identified by the colour marks on their fur, each cage will be marked with the number of animals, sex, number of
cage, name and dose level of the test substance
Animal housing: The study will proceed in SPF conditions according to SOP No.12.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

The test substance was administered to the stomach by gavage as the solution in aqua pro injection. Oral way of administration was chosen according to the guideline and it was approved by sponsor. The animals were treated 7 days per week at the same time (8.00 – 10.00 am).
The concentrations of solutions at all dose levels were adjusted to ensure the administration of 1 mL per 100 g of body weight. The vehicle control group was administered by aqua pro injectione in the same volume. The application form (test substance solution in aqua pro injectione) was prepared daily just before administration.

Analytical verification of doses or concentrations:

no

Details on mating procedure:

Animals were mated from the 15th day of study. Mating 1 : 1 (one male to one female) was used 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.

Duration of treatment / exposure:

Parental males:
1st day – 14th day (pre-mating) → 28th day (mating) → 42nd day of study

Parental females:
1st day – 14th day (pre-mating) → 28th day (mating) → gestation → lactation → day 4 post partum

Frequency of treatment:

The animals were treated 7 days per week at the same time (8.00-10.00 am)

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0 mg/kg/day
Basis:

Doses / concentrations 2

Remarks:

Doses / Concentrations:
50 mg/kg bw /day
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
150 mg/kg bw /day
Basis:
actual ingested

Doses / concentrations 4

Remarks:

Doses / Concentrations:
450 mg/kg bw /day
Basis:
actual ingested

No. of animals per sex per dose:

Basic groups:
1. Control: 0 (12 males + 12 females)
2. Low dose: 50 mg/kg/day (12 males + 12 females)
3. Intermediate dose: 150 mg/kg/day (12 males + 12 females)
4. High dose: 450 mg/kg/day (12 males + 12 females)

Satellite groups:
5. Control – vehicle – satellite: 0 6 males + 6 females
6. High dose – satellite: 450 mg/kg/day 6 males + 6 females

Control animals:

yes

Details on study design:

Study Time Schedule
Main Study:
Animal arrival: 01. 09. 2010
Acclimatisation: at least 5 days
Administration: 08. 09. – 22. 10. 2010
Urinalysis: only males – 42nd and 56th day of study
Haematology and necropsies: parental males – 43th day of study
satellite males – 57th day of study
parental females – 4th day of lactation
satellite females – 57th day of study
non-pregnant females – 55th day of study or 26th day after confirmed mating

-Preparation of Experimental Animals
During the acclimatisation period the health condition of all animals was controlled daily.
Then the animals were randomly divided into the control and test groups and they were marked individually.

- Experimental Data Collection
Health condition control: daily - during the acclimatization and the experimental part
Body weight: males - weekly
females - weekly in premating and mating period,
during pregnancy: 0., 7th, 14th, 20th day,
during lactation: 0. or 1st and 4th day;
pups (litters) – 0. or 1st and 4th day;
Food consumption: males - weekly (except the mating period)
females - weekly during premating period and after mating period
during pregnancy and lactation – on the same days as body weight

Clinical observations: males and females - daily during the administration period
pups - as soon as possible after delivery and then daily
Mortality control: daily
Laboratory examinations:
- vaginal smears: daily in mating period
- pathological examination: males and nonpregnant females - after the end of administration period
parental females and pups - on the 4th day of lactation
- weight of organs: during necropsy
- sperm observation: all males after necropsy
- histopathological examination: all males and females after necropsy

Clinical Observation of Pups
All pups were observed in natural conditions in their cages daily during the lactation. Changes in behavioural abnormalities were
recorded. Detailed examination of each litter was performed as soon as possible after delivery (day 0 or 1 post-partum) and the
4th day of lactation. The number and sex of pups, stillbirths, live births and presence of gross anomalies were recorded.

Examinations

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No data
- Number of corpora lutea: Yes, average number of corpora lutea were decreased only at the highest dose level
- Number of implantations: Yes, average number of corpora lutea were decreased only at the highest dose level
- Number of early resorptions: No data
- Number of late resorptions:No data
- Other:

Fetal examinations:

- External examinations: No data
- Soft tissue examinations: No data
- Skeletal examinations: No data
- Head examinations: No data

Indices:

Calculated parameters see table reported in next section for indices

Results and discussion

Results: maternal animals

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

pre and post implantation loss

other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no data

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

not specified

Basis for effect level:

changes in litter size and weights

Remarks on result:

other: only litter size was influenced while weights remained unaffected

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Number and sex ratio of pups

The total number of live pups and average number of pups per litter (in the day of parturition/1st day and the 4th day after parturition) at the highest dose leveles were decreased compared to control. Sex ratio (males/females) of pups was relatively well balanced at all dose levels.

Body weight

the average body weights of pups at all dose levels were well-balancesd with the control group and had an increasing trend during the whole lactation period.

Development of pups

No pups died at the control group and at all dose levels during lacation period. No differences in development of pups were observed at the control group and at all treated groups.

Pathological examination

The macroscopic examination was perfomed in all pups. Withish strata in liver were observed in one female at the control. Pick of knucle bones of rioght front limb were observed in one female at the middle dose level.

Reproduction data
	Dose level
Observed parameters
	0	50	150	450
Pairs started (N)	12	12	12	12
Females showing evidence of emulation (N)	11*	12	12	12
Females achieving pres ancy (N)	9	9	10	10
Females with abortion (N)	0	0	0	2
Nonpregnant females	3	3	2	2
Conceiving days (duration of mating) 1 - 5 (N)	11	12	12	12
Conceiving days (duration of mating) 6 - 13(N)	0	0	0	0
Pregnancy -== 21 days (N)	2	1	2	0
Pregnancy = 22 days (N)	6	6	8	8
Pre iancy = 23 days (N)	1	2	0	0
Females with live pups lxini(N)	9	9	10	8
Females with live pups at day 4 after parturition (N)	9	9	10	8
Corpora lutea/pregniant female (average)	13.78	13.44	14.90	12.20
Implantations//pregnant feniale (average)	12.00	11.33	13.20	9.10
Live pups/mother at birth (average)	11.44	10.22	12.60	8.88
Live pups/mother at day 4 after parturition (average)	11.44	10.22	12.60	8.88
Sex ratio (M/F) at birth (average)	5.11/6.33	5.11/5.11	5.80/6.80	4.63/4.25
Sex ratio ("M/F) at day 4 after parsurition (average)	5.11/6.33	5.11/5.11	5.80/6.80	4.63/4.25
Litter weight at birth (average)	70.39	66.73	75.34	55.83
Litter weight at day 4 after partuntion (average)	104.74	95.66	105.41	77.95
Pup weight at birth (average)	6.27	6.90	6.10	6.41
Pup weight at day 4 after parturition (average)	9.59	10.09	8.67	9.05

Fertilty parameters
Calculated parameters	Dose level
	0	50	150	450
Mating index	91.67	100	100	100
Fertility index	81.82	75.00	83.33	83.33
Conception Index	75.00	75.00	83.33	83.33
Gestation index	100	100	100	80
Percentage of postnatal loss	0	0	0	0
Viability index	100	100	100	100
LOSS OF OFFSPRING
Pre-implantation (corpora lutea minus implants
Pregnant females with 0 (N)	4	4	5	1
Pregnant females with 1 (N)	1	1	1	2
Pregnant females with 2 (N)   f.	1	1	2	1
Pregnant females with >=3 (N )	3	3	2	6
Pre-nataUpost-implantations (implants minus live births)
Pregnant females with 0 (N)	6	3	4	3
Pregnant females with l (N)	1	4	6	1
Pregnant females with 2 (N)	2	0	0	2
Pregnant females with >= 3 (N)	0	2	0	4
Post-natal (live births minus alive at post-natal day 4)
Pregnant females with 0 (N)	9	9	10	10
Pregnant females with I (N)	0	0	0	0
Pregnant females with 2 (N)	0	0	0	0
Pregnant females with >=3 (N)	0	0	0	0

Applicant's summary and conclusion

Conclusions:

The values of NOAEL for the reproduction and for developmental of pups was established to be 150 mg/kg bw/day

Executive summary:

Introduction

The test substance, Acid Black 210, was tested for reproduction and subacute toxicity using the OECD Test Guideline No. 422:Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, Adopted by the Council on March 22^nd1996.

 

Methods

 Wistar rats of SPF quality were used for testing. The test substance was administered in the form of solution in water for injection. Oral application by stomach tube was performed daily. The study includes four main groups and two satellite groups of animals. Each main group consisted of 12 males and 12 females; each satellite group consisted of 6 males and 6 females.Main groups contained 3 treated groups (doses 50, 150, 450 mg/kg of body weight /day) and one control group (vehicle only). The satellite groups contained one control group (vehicle only) and one treated group (450 mg/kg/day).The dose levels for study were determined on the basis of results of adose-range finding study phase (see the Annex 2).

The treated groups were administered daily for the following periods:

males and females – 2 weeks prior to the mating period and during the mating period,

pregnant females – during pregnancy and till the 3^rdday of lactation,

males – after mating period – totally for 42 days,

nonpregnant females (mated females without parturition) – for 25 days after the confirmed mating.

After the end of administration period the animals of main groups were sacrificed and satellite animals were observed for the next 14 days without treatment.

   

   During the study clinical observation and health status control were performed daily. The body weight and food consumption were measured weekly or in the specified time intervals. Detailed clinical observation was carried out weekly. Water consumption was measured twice a week. Vaginal smears were prepared daily during the mating period (until the presence of spermatozoa). Reproduction parameters relevant to pups (number of pups, weight of litters, sex or vitality) were also recorded. Before the end of study the functional observation was accomplished.

The study was finished by urinalysis, haematological and biochemical analysis and gross necropsy of animals. In all males of main groups the sperm parameters, sperm motility and sperm morphology were examined. The selected organs from parental animals were removed for weighing and histopathological examination.

Observation of pups- litter size was decreased at the highest dose level (accompanied by lower number of corpora lutea and implantations) and this difference can be considered as an adverse effect of the test substance treatment because the litter size is an important indicator of overall reproductive performance. But the average weight of pups and the development of pups were not influenced by the test substance administration.