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

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

reproductive screening from OECD 422

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 1,2010-January 20, 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Justification for type of information:

The purpose of this study was to assess the potential systemic toxicity in rats, including a screen for reproductive/developmental effects with administration of Acid Black 210 oral gavage administration

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:

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

GLP compliance:

yes

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

Sex:

male/female

Details on test animals or test system and environmental conditions:

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 injectione. 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.

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.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

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

Satellite males:
1st day → 42nd day (administration) → 56th day (observation)

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

Satellite females:
1st day → 42nd day (administration) → 56th day (observation)


Non-pregnant females (without evidence of copulation):
1st day – 14th day (pre-mating) → 28th day (mating) → 54th day of study

Non-pregnant females (with evidence of copulation):
1st day – 14th day (pre-mating) → 28th day (mating) → 25th day after confirmed mating (max. 54th day of study)

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/day
Basis:
actual ingested

Doses / concentrations 3

Remarks:

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

Doses / concentrations 4

Remarks:

Doses / Concentrations:
450 mg/kg/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.

-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.


-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

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: weekly
- Cage side observations checked in table were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: male and females daily

BODY WEIGHT: Yes
- Time schedule for examinations: males weekly,
females weekly in premating and mating period
during pregnancy 0,7,14, and 20th day
during lactation 0,1, and 4th day

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
-body weigh increment calculated: yes

WATER CONSUMPTION. yes
- Time schedule for examinations: weekly

OTHER:
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

Oestrous cyclicity (parental animals):

Examination of reproductive system of parental females did not evidence any significant microscopical changes. Only changes pertinent to previous gravidity or proceeded oestrous cycle were recorded. Biometry of organs also proved no statistically significant and no dose dependent differences in treated females.

Sperm parameters (parental animals):

The test substance had effect on sperm quality of treated males. Increased number of males with slower sperm motility was recorded at treated groups and especially at the highest dose level. Percentage portion of morphologically changed sperms was slightly increased at all treated groups comparing with control group without dependence on dose level.

Litter observations:

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.

Statistics:

ANOVA

Reproductive indices:

Calculated parameters: see tabel reported below for reproductive indices

Offspring viability indices:

100 at all dose levels

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

no mortality at any dose

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

weight increments were lower in males at highest dose levels. Females: average body weight increment was decreased at the highest dose level in the 1st week Pregnant females food consumption at highest dose level deacreased during lactation.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

weight increments were lower in males at highest dose levels. Females: average body weight increment was decreased at the highest dose level in the 1st week Pregnant females food consumption at highest dose level deacreased during lactation.

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

effects observed, treatment-related

Description (incidence and severity):

slower sperm motility at the highest dose level

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

increased pre and post implantatio losses at the highest dose level

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

reproductive function (sperm measures)

other: increased pre-post implantion losses

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

decrease of total number of live pups and pups per litter at the highest dose level

Mortality / viability:

no mortality observed

Description (incidence and severity):

100% at all dose levels

Body weight and weight changes:

no effects observed

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Histopathological findings:

not specified

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

not specified

Basis for effect level:

viability

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

 

Reproduction data

Observed parameters	Dose level
0	50	150	450
Pairs started (N)	12	12	12	12
Females showing evidence of copulation (N)	11*	12	12	12
Females achieving pregnancy (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
Pregnancy = 23 days (N)	1	2	0	0
Females with live pups born (N)	9	9	10	8
Females with live pups at day 4 after parturition (N)	9	9	10	8
Corpora lutea/pregnant female (average)	13.78	13.44	14.90	12.20
Implantations/pregnant female (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 parturition (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 parturition (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

Notes:(N) –Number of animals

*–  Nonpaired female (without presence of sperm) - not used for calculation ofduration of mating

 

 

Table 43

Fertility 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)	1	1	2	1
Pregnant females with ≥ 3 (N)	3	3	2	6
Pre-natal/post-implantations (implants minus live births)
Pregnant females with 0 (N)	6	3	4	3
Pregnant females with 1 (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 1 (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 a dose-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.

Reproduction part of study:

The course of mating, pregnancy and lactation of parental animals, number of females achieving pregnancy and bearing live pups, weights of reprodutive organs and pituitary gland, spermiogenesis and sperm parameters, macroscopical and microscopical structure of reproductive organs and pituitary gland of parental animals, number of pre-implantation, post-implantation and post-natal losses of mothers and number, weight, sex ratio and development of pups were not adversely affected by the test substance treatment at the dose levels 50 and 150 mg/kg/day.

The course of mating, pregnancy and lactation, weights of reprodutive organs and pituitary gland, spermiogenesis, macroscopical and microscopical structure of reproductive organs and pituitary gland of parental animals, number of post-natal losses of mothers, sex ratio, weight and development of pups were not adversely influenced by the test substance treatment at the dose level 450 mg/kg/day.

Examination of number of pups(decrease of the total number of live pups and average number of pups per litter),observation of sperms(slightly slower sperm motility),calculation of reproduction parameters(slightly decreased number of females achieving pregnancy) and calculation of pre-implantation and post-implantation losses (decreased number of corpora lutea, uterus implantations and pups) in animals of the dose level 450 mg/kg/day revealed adverse effects attributable to test substance.