Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
other: read across from similar substance
Adequacy of study:
key study
Study period:
2016
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

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)
Version / remarks:
Adopted by the Council on 28th July 2015
Deviations:
yes
Remarks:
see Any other information ...
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Disodium 6-hydroxy-5-[[4-[[4-(phenylamino)-3-sulphonatophenyl]azo]naphthyl]azo]naphthalene-2-sulphonate
EC Number:
228-412-8
EC Name:
Disodium 6-hydroxy-5-[[4-[[4-(phenylamino)-3-sulphonatophenyl]azo]naphthyl]azo]naphthalene-2-sulphonate
Cas Number:
6262-07-3
Molecular formula:
C32H23N5O7S2.2Na
IUPAC Name:
disodium;(5E)-5-[[4-[(4-anilino-3-sulfonatophenyl)diazenyl]naphthalen-1-yl]hydrazinylidene]-6-oxonaphthalene-2-sulfonate
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: by Sponsor, Batch No. 5008
- Expiration date of the lot/batch: 09/2017
- Storage condition of test material: The test substance was stored in dry and dark place at room temperature
- Stability under test conditions:
testing laboratory analysis of homogeneity and stability: the both application forms (1000 mg 10 mL and 10 mg /10 mL) of the test substance, Acid Black 26, at defined laboratory conditions (laboratory temperature, preparation of solution by defined manner) are homogenous and stable at least for 120 minutes from the finalization of application form preparation.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: see below Details on oral exposure

FORM AS APPLIED IN THE TEST (if different from that of starting material)
solution in water for injection

Test animals

Species:
rat
Strain:
Wistar
Remarks:
CRL (SPF quality - guaranteed)
Details on species / strain selection:
- according to guideline
- random selection according to the internal rule – at the beginning of the study the weight variation of animals in groups of each sex did not exceed ± 20% of the mean weight
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River SPF breeding, supplied via VELAZ s.r.o., Czech Republic, RČH CZ 11760500
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: sexually adult, 7-9 weeks on arrival
- Weight at study initiation: males 412 - 426 g, females 248 - 357 g
- Fasting period before study: no
- Housing: SPF conditions according to internal SOP No.12; sterilized soft wood fibers Lignocel;
Animal per cage: 2 rats of the same sex in one cage in pre-mating period, during mating period – 1 M + 1 F in one cage, pregnant females – individually, offspring – with mother, satellite animals - 2 rats of the same sex in one cage;
- Diet (e.g. ad libitum): complete pelleted diet for rats and mice in SPF breeding - Altromin for Rats/Mice (by Altromin Spezialfutter GmbH & Co. KG, Germany)
- Water (e.g. ad libitum): ad libitum; quality corresponding to the Regulation No. 252/2004 of Czech Coll. of Law
- Acclimation period: 12 days (DRFE 5 days)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3
- Humidity (%): 30-70
- Air changes (per hr): approximately 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 /12

STUDY TIME SCHEDULE
Administration (from 22.03.2016)
Parental males (totally 49 days of administration):
1st day – 14th day (pre-exposure period) → 15th day - 28th day (pre-mating period, administration ) → 29th day – 42nd day ( mating ) → 43rd day – 63rd day (administration period) → 64th day (necropsy)
Satellite males (totally 49 days of administration + 14 days of observation):
1st day – 14th day (pre-exposure period) → 15th day - 63rd day (administration period) → 64th day -77th day (observation period) → 78th day (necropsy)
Parental females:
1st day – 14th day (pre-exposure period) → 15th day - 28th day (pre-mating period, administration ) → 29th day – 42nd day (mating)→ gestation → lactation → day 12 post partum
Satellite females (totally 49 days of administration + 14 days of observation):
1st day – 14th day (pre-exposure period) → 15th day - 63rd day (administration period) → 64th day - 77th day (observation period) → 78th day (necropsy)
Non-pregnant females (without evidence of copulation):
1st day – 14th day (pre-exposure period) → 15th day - 28th day (pre-mating period, administration ) → 29th day – 42nd day (mating) → 25 days after the end of mating period
Non-pregnant females (with evidence of copulation):
1st day – 14th day (pre-exposure period) → 15th day - 28th day (pre-mating period, administration ) → 29th day – 42nd day (mating) → 25th day after confirmed mating

Observations
Urinalysis: only males – 63rd and 77th day of study
Blood collection for haematology and biochemistry:
parental males – 64th day of study
satellite males – 78th day of study
parental females - 13th day of lactation period
pups – 2 pups per litter – 4th day of lactation; 2 pups per litter – 13th day of lactation
satellite females – 78th day of study

Necropsy:
parental males – 64th day of study
satellite males – 78th day of study
parental females - 13th day of lactation period
pups – 2 pups per litter – 4th day of lactation, other pups - 13th day of lactation
satellite females – 78th day of study
non-pregnant females – 26th day after the end of mating period or confirmed mating
End of histopathological examination: till 11. 10. 2016

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was weighted into glass beaker and the beaker was replenished by water for injections. The test solution was dissolved in ultrasonic bath for a 30 minutes and then the solution was stirred by magnetic stirrer (750 rpm) for 40 minutes. Taking of the test substance was performed at reduced speed - 300 rpm.
The concentrations of solution at all dose levels were adjusted to ensure the administration of 1 mL per 100 g of body weight.
For each dose level concentration, the solution was prepared separately.
The application forms were prepared daily just before administration.
The administration of the test substance to animals was performed during one hour after preparation of application form. The stirring of solutions continued during administration.

VEHICLE
water for injection; batch no.: 1505050287; exp. 5/2017
- Concentration in vehicle:
The concentrations of solution at all dose levels were adjusted to ensure the administration of 1 mL per 100 g of body weight.
Details on mating procedure:
Animals were mated from the 29th day of study.
- M/F ratio per cage: 1:1
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
males and females – 2 weeks prior to the mating period and during the mating period,
pregnant females – during pregnancy and till the 12th day of lactation,
males – after mating period – totally for 49 days,
nonpregnant females (mated females without parturition) – for 25 days after the confirmed mating.
Frequency of treatment:
7 days per week at the same time
Doses / concentrationsopen allclose all
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
(vehicle only)
No. of animals per sex per dose:
12 females and 12 males per group,
6 males and 6 females per satellite group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels for study were determined on the basis of results of a d
ose-range finding experiment.
- Post-exposure recovery period in satellite groups: 14 days

Examinations

Parental animals: Observations and examinations:
HEALTH CONDITION CONTROL: Yes
- Time schedule: daily - during the acclimatization and the experimental part

CLINICAL OBSERVATIONS: Yes
males and females - daily during the administration period in natural conditions in cages
pups - as soon as possible after delivery and then daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before the first application and then weekly (except the mating period)

BODY WEIGHT: Yes
- Time schedule for examinations:
males - the first day of administration and then weekly,
females - the first day of administration and then weekly in premating and mating period,
during pregnancy: 0., 7th, 14th, 20th day, during lactation: 1st, 4th, 12th and 13th day,
satellite males and females - the first day of administration and then weekly.

FOOD CONSUMPTION:
- Food consumption determined and group mean daily diet consumption calculated as g food/animal/day: Yes
- Time schedule: weekly and on the same days as body weight (except the mating period); satellite males and females – weekly

FOOD EFFICIENCY:
- Body weight gain in g/food consumption in g per unit time X 100 calculated as time-weighted
averages from the consumption and body weight gain data: Yes
Time schedule: males - weekly (except the mating period), females - weekly during premating period,
during pregnancy and lactation – on the same days as body weight, satellite males and females – weekly
Oestrous cyclicity (parental animals):
yes, before beginning of treatment;
vaginal smears of all females were monitored daily for two weeks
Sperm parameters (parental animals):
Parameters examined in [P] male parental generations:
- sperm motility, sperm morphology
- in all males (except the satellite group)
Litter observations:
CLINICAL OBSERVATION OF PUPS
- Time schedule:
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 1 post-partum) and on the 4th day of lactation. The number and sex of pups, stillbirths, live births and presence of gross anomalies were recorded.
Anogenital distance measurement: 4th day of lactation
Nipples examination (the presence and number of nipples in male pups): counted on day 13 of lactation.

BODY WEIGHT
pups (litters) - 1st , 4th day, 12th day and 13th day
pups – individually – 4th day of lactation
Postmortem examinations (parental animals):
In Repeated Dose Toxicity part of study the full histopathology of the preserved organs and tissues was performed for all high dose and control animals and satellite animals. Organs with macroscopical changes and kidneys, stomach, small intestine, large intestine and caecum were examined at the lowest and middle dose level groups used for Repeated Dose Toxicity part of study.

In Reproduction Toxicity part of study the histopathology of the reproductive organs, pituitary gland and thyroid gland was performed for all high dose and control animals and organs with macroscopical changes were examined at the lowest and middle dose level groups.
Detailed histological examination was performed on testes of all high dose and control animals from Reproduction Toxicity part of study (with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure).

SACRIFICE
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.

GROSS NECROPSY
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 (buffered 4% formaldehyde). Testes and epididymides were fixed in modified Davidson’s fixative.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [5] were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
SACRIFICE
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:
The macroscopic examination was performed in all pups.
No pathological findings were recorded at the dose levels 250 and 500 mg/kg/day and in control pups.
Stomach – empty was recorded at the dose level 1000 mg/kg/day in the 16 pups from one female. These pups died after parturition.
No histopathological findings were recorded during examination of thyroid gland in pups.

GROSS NECROPSY
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 (buffered 4% formaldehyde). Testes and epididymides were fixed in modified Davidson’s fixative.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [5] were prepared for microscopic examination and weighed, respectively.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Female No.166 (at the dose level 1000 mg/kg/day) died on the 39th day of application due to intubation error.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Males:Body weight was slightly decreased in males at the dose level 1000 mg/kg/day. Body weight increment of all treated males and control males was similar.
Females: The mean body weight and increments of treated females were comparable to control females during premating period and pregnancy except the decrease of mean body weight at the dose level 250 mg/kg/day at the 14th and 20th day of pregnancy.
During lactation decreased mean body weight at the dose levels 250 and 500 mg/kg/day was detected (in the dose level 250 mg/kg/day at the 1st and 4th day of lactation period and in the dose level 500 mg/kg/day at the 4th day of lactation period).
Disbalance of mean body weight increment was recorded during lactation period.
Food consumption and compound intake (if feeding study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Haematological examination of males and females showed altered value of reticulocytes. This is probably related to adaptation organism to the test substance treatment. Others haematological parameters of red blood line were not changed.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
The concentration of thyroid hormone thyroxine (T4) in males was decreased at the middle and highest dose levels but these is values were in physiological range for rats (without toxicological importance).
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS (PARENTAL ANIMALS)
Excrements in treated animals were thinner consistency and coloured by the test substance. After the end of application of the test substance this clinical symptom disappeared.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Female with abortion was not recorded. Number of females showing evidence of copulation and number of females achieving pregnancy was not seriously affected. Number of corpora lutea, number of implantations was not significant changed.
The number of females with pups was reduced at the highest dose level but one female died during the study and this unrelated with the test substance treatment.
Male and female fertility index at the highest dose level was insignificantly decreased. Gestation and survival index was without toxicological significant changes.

ORGAN WEIGHTS (PARENTAL ANIMALS)
250 mg/kg/day:
Macroscopic examination not showed findings of reproductive organs.
Biometry of organs shows decreased relative and absolute weight of prostate gland and seminal vesicles in males, differences from control were insignificant. In females no changes of weight of organs were recorded.

500 mg/kg/day: During biometry of organs and macroscopic examination of organs no significant changes related to the test substance treatment were not detected.

1000 mg/kg/day: Biometry of organs showed significant decrease relative and absolute weight of prostate gland and seminal vesicles in males.
During the examination of microscopical structure of reproductive organs no toxicological significant changes appeared.

HISTOPATHOLOGY (PARENTAL ANIMALS)
A serious kidneys chronic progressive nephropathy was found out. The influence of the test substance on kidneys could not be excluded because the intensity of chronic progressive nephropathy in kidneys irreversibly increased with dose level.
Occurrence microscopic findings of stomach and/or intestines (eosinophile infiltration of mucosa and/or lamina propria) related with place of application (digestive tract) and disappear during recovery period.
Reproduction part:
Histopathological changes in ovaria, uterus and vagina related with reproduction cycle in females or previous pregnancy.
Examination of sperm in males was without significant changes. Only slight increase in number of affected sperm at the lowest and highest dose levels was detected but without toxicological importance. Number of females showing evidence of copulation and number of females achieving pregnancy was not seriously affected. In females, number of corpora lutea, number of implantations was not significantly changed changes. Male and female fertility index at the highest dose level was decreased.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No significant changes: in growth, during examination of sperm or microscopical structure of reproductive organs. Reproduction parameters were not significantly affected.
Remarks on result:
not determinable due to absence of adverse toxic effects

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
Mean number of pups per litter and sex ration were not affected by the test substance treatment.
No differences in postnatal developmental were observed in pups at the treated groups – presence of nipples in male pups was not recorded and anogenital distance in treated male and female pups in comparison with the control pups was similar.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
The total number of pups was lower at the higher dose level and on the contrary at the middle and lowest dose levels the total number of pups was higher. This variation is due to differences in the number of mothers in each group.
Stomach empty (without milk) in 16 pups from one mother at the highest dose level was recorded during the macroscopic examination – these pups died after parturition. These findings were probably not related with the test substance treatment.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In pups at the highest dose level the mean body weight was insignificantly decreased.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Concentration of thyroxine hormone T4 in pups was similar with the control group.
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Stomach empty (without milk) in 16 pups from one mother at the highest dose level was recorded during the macroscopic examination – these pups died after parturition. These findings were probably not related with the test substance treatment.
Other pups in all dose level were without macroscopic findings.
Histopathological findings:
no effects observed
Description (incidence and severity):
During the microscopical evaluation of thyroid gland in pups no findings were found out.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No differences in postnatal developmental were observed in pups at the treated groups. Pups in all dose level were without macroscopic findings. During the microscopical evaluation of thyroid gland in pups no findings were found out.
Remarks on result:
not determinable due to absence of adverse toxic effects

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

Deviations: During the study, short increases of temperature (22.05.2016 - for ten hour, temperature from 25.2 to 26.4°C and 23.05.2016 for two hours, temperature from 25.2 to 25.4°C) in animal room was recorded. This microclimatic deviation did not affect the welfare of animals and had no impact on the results of the study. No other deviations from study plan or test guidelines were observed during the study performance.

Applicant's summary and conclusion

Conclusions:
NOAEL for REPRODUCTION and DEVELOPMENT = 1000 mg/kg body weight/day.
Executive summary:

Introduction

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 28th July 2015.

 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 250, 500, 1000 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 (1000 mg/kg/day). The dose levels for study were determined on the basis of results of a dose-range finding experiment (see the Annex 2) and approved by Sponsor.

 

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 12th day of lactation,

males  after mating period – totally for 49 days,

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

non-mated females – for 25 days after the end of mating period

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 controls were performed daily. The body weight and food consumption were measured weekly. Detailed clinical observation was carried out weekly. The functional observation was performed at the end of application and observation period. Vaginal smears were prepared daily, 2 weeks before start of administration period (oestrous cycle monitoring), during the mating period (until the presence of spermatozoa) and at necropsy day. Reproduction parameters relevant to pups (number of pups, weight of litters, weight, sex and vitality of pups, measurement of anogenital distance, nipple retention) were also recorded.

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 and pups were removed for weighing and histopathological examination.

 

Results

Repeated oral administration of the tested substance to rats by gavage at the dose levels of 250, 500 and 1000 mg/kg/day did not cause any mortality.

 

Reproduction part of study:

Parental males and females

The dose level 250 mg/kg/day – No significant changes in growth on treated animals were recorded. Macroscopic examination not showed findings of reproductive organs.

Biometry of organs shows decreased relative and absolute weight of prostate gland and seminal vesicles in males, differences from control were insignificant. In females no changes of weight of organs were recorded. 

Examination of sperm in males was without significant changes.

The dose level 500 mg/kg/day – No significant changes in growth on treated animals were recorded.

The concentration of thyroid hormone thyroxine (T4) examined in males was decreased, but this value was in physiological range for rats.

During biometry of organs, examination of sperm in males and macroscopic examination of organs no significant changes related to the test substance treatment were not detected.

 

The dose level 1000 mg/kg/day – No significant changes in growth on treated animals were recorded.

Biometry of organs showed significant decrease relative and absolute weight of prostate gland and seminal vesicles in males.

No significant changes during examination of sperm in males were recorded.

During the examination of microscopical structure of reproductive organs no toxicological significant changes appeared.

The concentration of thyroid hormone thyroxine (T4) examined in males was significantly decreased, but this value was in physiological range for rats.

Pups

Mean number of pups per litter and sex ration were not affected by the test substance treatment. In pups at the highest dose level the mean body weight was slightly decreased. 

No differences in postnatal developmental were observed in pups at the treated groups – presence of nipples in male pups was not recorded and anogenital distance in treated pups in comparison with the control pups was similar. Concentration of thyroxine hormone T4 in pups was similar with the control group.

Pups in all dose level were without macroscopic findings.

During the microscopical evaluation of thyroid gland in pups no findings were found out.

Reproduction parameters

Female with abortion was not recorded. Number of females showing evidence of copulation and number of females achieving pregnancy was not seriously affected. Number of corpora lutea, number of implantations was not significant changed. The number of females with pups was reduced at the highest dose level but one female died during the study and this unrelated with the test substance treatment.

Male and female fertility index at the highest dose level was insignificantly decreased. Gestation and survival index was without toxicological significant changes.

Conclusion

The NOAEL (No Observed Adverse Effect Level) for REPRODUCTION and DEVELOPMENT was established as 1000 mg/kg body weight/day.