Reaction products of diazotised 4,4-diaminodiphenylamine-2-sulfonic acid, subsequently coupled with 6-amino-4-hydroxynaphthalene-2-sulfonic acid, further diazotised and coupled with metaphenylendiamine, sodium salts

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

other: experimental result on similar substance

Adequacy of study:

key study

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:

2012

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-3,6-bis[[4-[(2,4-diaminophenyl)azo]phenyl]azo]-5-hydroxynaphthalene-2,7-disulphonate

EC Number:

229-208-1

EC Name:

Disodium 4-amino-3,6-bis[[4-[(2,4-diaminophenyl)azo]phenyl]azo]-5-hydroxynaphthalene-2,7-disulphonate

Cas Number:

6428-31-5

Molecular formula:

C34H29N13O7S2.2Na

IUPAC Name:

disodium 4-amino-3,6-bis({4-[(2,4-diaminophenyl)diazenyl]phenyl}diazenyl)-5-hydroxynaphthalene-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:

TEST ANIMALS
- Animal arrival: 14.03.2012
- Selection: 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
- Age of animals: males, females - sexually adult; 10 weeks on arrival
- Number of animals: 12 females and 12 males per group, 6 males and 6 females per satellite group
- Housing conditions: the study will proceed in SPF conditions according to SOP No.12.
pre-mating period 2 rats of the same sex in one cage
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
- Bedding: sterilized shavings of soft wood
- Food: complete pelleted diet for rats and mice in SPF breeding
- Water: drinking water ad libitum, quality standard ČSN 757 111
- Acclimatization: at least 5 days. During the acclimatisation period the health condition of all animals was controlled daily.

ENVIRONMENTAL CONDITIONS
- Light cycle: 12 hour light / 12 hour dark
- Temperature: 22 ± 3°C
- Humidity: relative humidity 30-70%

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

ADMINISTRATION
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.

PREPARATION OF DOSING SOLUTIONS
The concentrations of solutions at all dose levels were adjusted to ensure the administration of 1 mL per 100 g of body weight.
The application form (test substance solution in aqua pro injection) was prepared daily just before administration.

Details on mating procedure:

- Star period: animals were mated from the 15th day of study
- M/F ratio per cage: during mating period one male and one female in one cage
- Observations: each morning the females were examined for presence of spermatozoa in vaginal smears.
- Proof of pregnancy: sperm in vaginal smear; 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

Frequency of treatment:

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

Details on study schedule:

GENERAL STUDY TIME SCHEDULE

Main Study:
Animal arrival: 14.03.2012
Acclimatisation: at least 5 days
Administration: 20.03-15.05.2012
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
Examination of blood and necropsies: 30.04 – 16. 05. 2012 /satellite groups: 15.05 - 16.05 2012
End of histopathological examination: 31.10.2012
Final report elaboration: 31.12.2012

Doses / concentrations

Doses / concentrations 1

Remarks:

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

Doses / concentrations 2

Remarks:

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

Doses / concentrations 3

Remarks:

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

No. of animals per sex per dose:

12 males + 12 females for all tested and control group.
6 males + 6 females in satellite groups

Control animals:

yes

Details on study design:

ANIMAL
- Selection of test animals: animals were randomly divided into the control and test groups and they were marked individually.
- Identification: 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

DOSE SELECTION
1) Dose Range findings
The dose-range finding experiment with 14-day application period was performed with 4 groups of treated animals without control group.
The dose levels for the dose-range finding experiment have been chosen according to the request of sponsor and with respect to the information in Material Safety Data Sheet obtained from sponsor (LD50, oral rat - 4900 mg/kg).
Appropriate dose levels of the test substance for the 14-day dose-range finding experiment was chosen as follows:
Dose levels: 125, 250, 500 and 1000 mg/kg/day
In the dose-range finding experiment with test substance two females and one male died at the dose level 1000 mg/kg/day during the 1st week of application and two males and one female died at the dose level 500 mg/kg/day during the 2nd week of application. Four males and three females at the dose level 1000 mg/kg/day were humanely killed at the end of the first week of application and three males and four females were killed at the dose level 500 mg/kg/day during the 2nd week of application by the reason of bad health condition and prevention suffering of animals.
Marked fall of weight was recorded in males and females of the dose level 1000 mg/kg/day. The body weights and body weight increments of animals at the dose levels 500 mg/kg/day were decreased.
Clinical observation detected the impact of the test substance on the health condition of animals at the dose levels 500 and 1000 mg/kg/day.
Results of haematology examination showed that the test substance had influence on red and white blood component in both sexes mainly at the dose level 500 mg/kg/day.
Pathological examination revealed change of colour of skin, subcutis, muscle and peritoneum at the dose level 500 and 1000 mg/kg/day. Other macroscopic changes were found mainly in digestive system, in lungs and in spleen of animals at the dose levels 500 and1000 mg/kg/day.
On the basis of the results obtained the following dose levels 20, 80 and 320 mg/kg/day were chosen for the main Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test.

Examinations

Parental animals: Observations and examinations:

MORTALITY: Yes
All rats during the treatment periods were examined for vitality or mortality changes daily.

HEALTH CONDITIONS: Yes
All rats were observed pre-experimentally to ensure that only the animals exhibiting normal behavioural activity would be entered into the study. During the administration period they were examined for behaviour changes each day before and during application.

CAGE SIDE OBSERVATIONS: Yes
Animals were observed in natural conditions in their cages.
Observation during the removal from cage: reaction to handling, elasticity of skin, colour of mucous membranes, salivation, lacrimation, cleanliness of fur around foramina.

DETAILED CLINICAL OBSERVATIONS: Yes
All rats were observed daily during the administration period. The 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 (11.00 a.m. - 13 p.m.) at the time of expectation of maximal effect of the test substance. This observation was carried out before the first application and then weekly. At the first part of observation the behaviour of animals in the cage was monitored: piloerection, posture, position of eyelids, breathing, tonic or clonic movements, stereotypes or bizarre behaviour.

BODY WEIGHT: Yes
Males weekly and females weekly in premating and mating period; during pregnancy at 0, 7th, 14th, 20th day; during lactation at 0 or 1st and 4th day.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
Males weekly (except the mating period) and females weekly during premating period and after mating period; during pregnancy and lactation on the same days as body weight.

FUNCTIONAL OBSERVATION: Yes
This observation was done at the end of administration period and recovery period.
During functional examination, the sensory reactivity on auditory, visual, proprioceptive stimuli and pupillary reflex were evaluated and motor activity assessment was conducted. Moreover the individual observations of grip strength were performed using grip strength meter. Measurements were made on: 1) pectoral legs, 2) pelvis legs. Grip power was expressed in Newtons.

OTHER:

LABORATORY EXAMINATION: examination of Vaginal Smears, daily in mating period.
The pregnancy was determined by the presence of spermatozoa in vaginal smear. The vaginal smears were carried out daily in the morning during mating period. The smears were stained and the presence of sperm was evaluated. Day 0 of pregnancy was defined as the day when sperms were found.

BIOCHEMICAL EXAMINATION: the animals starved approximately for 18 hours before blood collection but they were supplied by drinking water ad libitum.
The blood samples were collected from the orbital plexus under the light ether narcosis. Biochemical parameters were measured in serum.
The test parameters were determined by automatic biochemical analysers SPOTCHEMTM EZ SP-4430 and SPOTCHEMTM EL SE-1520 (Arkray, Inc., Japan).

PATHOLOGICAL EXAMINATION: during the necropsy a revision of the external surface of the body, of all orifices and the cranial, thoracic and abdominal cavities were carried out. Organs for consequent histopathological examination were taken out and stored in containers with fixative (buffer 4% formaldehyde). Testes and epididymides were fixed in modified Davidson’s fixative.

URINALYSIS: only males 42nd and 56th day of study. The rats were kept in the metabolic cages for the collection of urine for two hours. Immediately before entering metabolic cages the animals were administered 2 mL of drinking water for 100 g of body weight by gavage to the stomach. The parameters were determined by analyser PocketChem PU-4210 (Arkray, Inc., Japan).

HAEMATOLOGICAL EXAMINATION: The blood samples were collected from the orbital plexus by glass micropipette under the light ether narcosis into the PVC test tubes containing anticoagulation systems. Haematology analysers Coulter AC.T diffTM, Celltac alfa and Coagulometer ACL 200 were used for examination.
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

Sperm parameters (parental animals):

In all males of all groups surviving to scheduled necropsy the sperm parameters were examined: sperm motility and sperm morphology. Sperm specimens were prepared and examined according to the SOP No. M/45.

SPERM MORTALITY
Sperm samples were taken from one epididymis and sperm motility was assessed from these samples. The motility of sperm was determined by microscopic examination of the prepared sperm suspension. The result of observation was evaluated subjectively according to following grades: 1 - fast progressive motility, 2 - slow progressive motility, 3 - no progressive motility, 4 - non-motile sperm.

SPERM MORPHOLOGY
Sperm samples were taken from one epididymis and sperm morphology was assessed from these samples. A smear from the sperm suspension was prepared and stained (Giemsa staining). The morphology of sperm was determined by microscopic examination.
All deviations – e.g. broken tail, abnormal form of tail, double head, amorphous head, abnormal form of neck - were recorded.

Postmortem examinations (parental animals):

SACRIFICE: at the end of study the experimental animals were narcotised and sacrificed by cutting the neck spine and medulla.

GROSS NECROPSY: after the gross necropsy of the cranial, thoracic and abdominal cavities the organs for weighing and further histological examination were collected.

ORGAN WEIGHTS: the absolute weights of liver, kidneys, adrenals, testes or ovaries, epididymides or uterus, prostate gland, thymus, spleen, brain, pituitary gland and heart were recorded (repeated dose toxicity part of study - 6 males and females, from each group + satellite group); testes or ovaries, epididymides or uterus, prostate gland, pituitary gland (reproduction part of study - all animals).

HISTOPATHOLOGICAL EXAMINATION: tissue and organs reported below, were collected from all killed males and females at necropsy and fixed in neutral 4% formaldehyde solution (v/v) for further histopathological evaluation. For histopathological processing the routine histopathological paraffin technique with haematoxylin-eosin staining was used.
The following list contains all the examined organs which were collected at necropsy and fixed:

Reproduction part of study (12 males and 12 females from each main group)
Pituitary gland
Ovaries
Uterus
Cervix of uterus
Vagina
Epididymis
Prostate gland
Testes
All gross lesions

Repeated dose toxicity part of study (6 males and 6 females in each main group + 6 males and 6 females in each satellite group)
Adrenal glands
Aorta
Brain (incl. cerebellum and med. oblongata)
Caecum
Coagulating gland
Colon
Duodenum
Pancreas
Rectum
Salivary glands
Sciatic nerve
Seminal vesicle
Skeletal muscle
Skin
Spinal cord – thoracic
Spleen
Stomach
Thymus
Thyroid gland incl. parathyroid
Trachea
Urinary bladder
Female mammary gland area
Femur
Heart
Ileum (incl. Peyer´s patches)
Jejunum (incl. Peyer´s patches)
Kidneys
Liver
Lungs
Lymph nodes – mesenteric, para-aortal
Oesophagus
All gross lesions

Statistics:

ANOVA

Reproductive indices:

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

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

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Not evidence any significant microscopical changes

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 implantation losses at the highest dose level

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No mortality at any dose

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
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 decreased during lactation.

REPRODUCTIVE FUNCTION: OESTROUS CYCLE (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.

REPRODUCTIVE FUNCTION: SPERM MEASURES (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.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Increased pre and post implantation losses at the highest dose level

LITTER OBSERVATIONS (PARENTAL ANIMALS)
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.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

ca. 80 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

mortality

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

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. 80 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

not specified

Basis for effect level:

mortality

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

FURTHER DETAILS

All females (at all groups) were paired so the number of females paired was identical at all groups. 

Number of females achieving pregnancy and gestation index were decreased at the highest dose level compared to the control, because number of females with live pups born were decreased at this group. Duration of mating and pregnancy of the treated groups was similar to the control group. Average number of corpora lutea and implantation were decreased only at the highest dose level (there was lower number of pups) compared to the control.

Pre-implantation losses were increased at the highest dose level. Post-implantation losses and post-natal losses at all dose levels were well-balanced with the control group.

Table 42

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

*– Non-paired female (without presence of sperm) - not used for calculation of duration 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 80 mg/kg bw/day

Executive summary:

The test substance 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.

Conclusion

Administration of the test substance, had not adverse effect on mortality, parameters of functional observation and on some reproduction parameters - course of mating, pregnancy and lactation, weights of reproductive organs and pituitary gland, spermiogenesis, macroscopical and microscopical structure of reproductive organs and pituitary gland of parental animals, number of post-implantation and post-natal losses of mothers, sex ratio and development of pups.  

Test-related reduction in body weight gains (reduced body weight increments in both sexes, fall of body weight in females, decreased food consumption and conversion in both sexes) were noted at the dose of 320 mg/kg/day. Some biochemical parameters(changes of enzymes activity, urea, inorganic phosphorus, total protein and albumin concentration) and biometry of organs (changes of thymus, heart and spleen weight) were significantly changed especially in animals of the highest dose level.

The heart appeared to be a target organ following repeated oral exposure of the test substance at the highest dose level. Serious irreversible regressive lesions accompanied by accumulation of pigment were diagnosed in myocardium. In addition, similar changes were observed in muscle fibres of larynx and skeletal muscle. This findings tended to be more serious in females than in males. The test had influence on macroscopical and microscopical structure of some other organs and tissues. Irreversible accumulation of pigment in brain, cerebellum, spinal cord, spleen, thyroid gland and kidneys was detected in animals of the middle and the highest dose level. Atrophic changes occurred in thymus of females at the highest dose level.

Daily oral administration of the test substance at the dose level 320 mg/kg/day also affected the number of live pups (decrease of the number of females bearing live pups, decrease of total number of live pups and mean number of pups per litter), early prenatal development of organism in uterus (increased pre-implantation losses), growth of pups(decreased mean litter weight, decreased mean pup weight on day 4 after parturition). However, male ability to produce sperm that can fertilize eggs and female ability to impregnate was not significantly changed.

     

The value of NOAEL for REPEATED DOSE TOXICITY was established as 80 mg/kg body weight/day both for males and females.

The value of NOAEL for the REPRODUCTIVE and DEVELOPMENTALTOXICITY was established as 80 mg/kg body weight/day