Chromate(3-), [3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-2-hydroxy-5-nitrobenzenesulfonato(3-)][3-hydroxy-4-[(2-hydroxy-1-naphthalenyl)azo]-7-nitro-1-naphthalenesulfonato(3-)]-, sodium

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

The No Observed Adverse Effect Level (NOAEL) for reproductive and development was established as 80 mg/kg body weight/day. The value was determined mainly on the basis of changed sperm motility and morphology, decreased weight of prostate gland, microscopical findings in reproductive organs and decreased number of females bearing live pups.

Link to relevant study records

Reference

Endpoint:

one-generation reproductive toxicity

Type of information:

other: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From February 12 to August 01, 2014

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Combined Repeated Dose and Reproductive / Developmental Toxicity Screening Test (Precursor Protocol of GL 422)

Deviations:

yes

Remarks:

no impact on the results of the study (details below)

GLP compliance:

yes (incl. QA statement)

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: SPF breeding, VELAZ s.r.o., Únětice, Czech Republic, RČH CZ 21760118.
- Age at study initiation: males, females: sexually adult, 9 weeks on arrival; dose-range finding experiment: 9 weeks on arrival.
- Weight at study initiation: males 363-510 g and females 224 - 382 g.
- Housing: SPF conditions according to internal SOP No.12. 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.
- Bedding: sterilized soft wood fibres Lignocel.
- Diet: complete pelleted diet for rats and mice in SPF breeding.
- Water: drinking water ad libitum, quality corresponding to the Regulation No. 252/2004 of Czech Coll. of Law.
- Acclimation period: at least 5 days (DRFE – at least 5 days).

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30 - 70 %
- Photoperiod: 12 hour light / 12 hour dark.

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Aqua pro injection

Details on exposure:

PREPARATION OF DOSING SOLUTIONS
The application form for analysis was prepared in the same manner as for application to animals – i.e. suspension in water for injection.
- Concentration Level 50 mg/10 ml: ca. 0.5 g of the test substance was weighted with wider end of glass Pasteur pipette into a 150 ml glass beaker calibrated to 100 ml and the beaker was replenished by the water for injection and dissolved in ultrasonic bath for 15 min. The suspension was stirred by magnetic stirrer (600 rpm) for 15 minutes.
- Concentration Level 1000 mg/10 ml: ca. 5 g of the test substance was weighted with wider end of glass Pasteur pipette into a 150 ml glass beaker calibrated to 50 ml and the beaker was replenished by the vehicle and dissolved in ultrasonic bath for a 30 min. The suspension was stirred by magnetic stirrer (850 rpm) for 30 minutes.
- Stability of the application form: the samples were taken from the middle of the beaker content at required time intervals (0, 30, 60, 90 and 120 minutes) for the determination of stability of both application forms. Two samples were taken at all time intervals.
- Homogeneity of the application form: the homogeneity of the application form was checked by determination of a concentration of the test substance in three places of suspension (at the bottom, in the middle and at the surface).
- Results of analysis: from the results of analyses (homogeneity and stability) follows that the suspension of the test substance in vehicle in concentration level 50 mg/10 ml prepared at defined laboratory conditions (laboratory temperature, preparation of suspension by defined manner) is homogenous and stable at least for 120 minutes starting with preparation of the application form.

DIET PREPARATION
The test substance was weighted into glass beaker and the beaker was replenished by water for injections. The test suspension was dissolved in ultrasonic bath for a 20 minutes and then the suspension was stirred by magnetic stirrer (800 rpm) for 30 minutes. The concentrations of suspension 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 suspension 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 suspensions continued during 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.

Duration of treatment / exposure:

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 3rd day of lactation
males – after mating period – totally for 42 days
nonpregnant females (mated females without parturition) – for 25 days after the confirmed mating
non-mated females – to the 54th day of study

Frequency of treatment:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

12 females and 12 males per dose per group, 6 males and 6 females per satellite group.
Dose-range finding experiment: 5 males and 5 females per group.

Control animals:

yes, concurrent vehicle

Details on study design:

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.

Parental animals: Observations and examinations:

HEALT CONDITION CONTROL
- Time schedule for examinations: daily - during the acclimatization and the experimental part.
- Method of investigation: 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, during application and immediately after application.

CLINICAL OBSERVATIONS
- Time schedule for examinations:
males and females: daily during the administration period.
pups: as soon as possible after delivery and then daily.
- Method of investigation: males and Females: all rats were observed daily during the administration period. This 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 – 13.00 p.m.) – at the time of expectation of maximal effect of the test substance. Animals were observed in natural conditions in their cages.
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 on the 4th day of lactation. The number and sex of pups, stillbirths, live births and presence of gross anomalies were recorded.

DETAILED CLINICAL OBSERVATIONS
- Time schedule for examinations: before the first application and then weekly (except the mating period).
- Method of investigation: 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.
The second part was the observation during the removal from cage: reaction to handling, elasticity of skin, colour of mucous membranes, salivation, lacrimation, cleanliness of fur around foramina.

MORTALITY
- Time schedule for examinations: twice daily.
- Method of investigation: all rats during the treatment periods were examined for vitality or mortality twice daily.

BODY WEIGHT
- Time schedule for examinations:
males: weekly.
females: weekly in premating and mating period; during pregnancy at the 0., 7th, 14th, 20th day; during lactation: 0. or 1st, 3rd and 4th day.
pups (litters): 0. or 1st, 3rd and 4th day.
satellite males and females: weekly.
- Method of investigation: the body weight of animals was recorded on automatic balances with group mean computing module on specified days. All animals were weighed immediately before euthanasia too. Weight increment was computed as an mean per group (in grams). Non-pregnant females (females without parturition) were not included in calculation of means in pregnancy and lactation period.

FOOD CONSUMPTION AND COMPOUND INTAKE
- Time schedule for examinations:
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.
- Method of investigation: in a specified day the remainder of pellets was weighed in each cage, the new food was weighed out and the food consumption for the previous week was computed. In males mean values were calculated for each week of the study (except the mating period). Food consumption for animal/day was calculated from mean values of each group. The same way of calculation of mean food consumption was used for females in pre-mating period. In pregnancy and lactation period mean individual values (grams/animal/day) were calculated for each week of the study. Mean food consumption for each group was calculated from individual values. Nonpregnant females (females without parturition) were not included in calculation of mean food consumption in pregnancy and lactation period.
Food conversion in % (weight increment/food consumption x 100) was calculated for animals of Repeated Dose Toxicity part of study. In pre-mating period the food consumption and conversion of females was calculated from values of all females.

WATER CONSUMPTION AND COMPOUND INTAKE
- Time schedule for examinations: satellite males and females: twice a week.
- Method of investigation: the drinking water consumption was recorded in satellite males and females. The mean values in groups (water consumption per animal and per day) were calculated for each week of the study.

FUNCTIONAL OBSERVATION
- Time schedule for examinations: at the end of administration/observation period.
- Method of investigation: this observation was done at the end of administration period (only in 6 males and 6 females of each group) 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.

URINALYSIS
- Time schedule for examinations: the last day of administration/observation period – only males.
- Method of investigation: this examination was performed only in 6 males of each group and in satellite males. In females this examination was not performed (dams should not be removed from the pups for long time). 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 following parameters were determined: volume, colour, cloud, odour, glucose, protein, bilirubin, urobilinogen, pH, specific gravity, blood, ketones, nitrite, leukocytes.

HAEMATOLOGY
- Time schedule for examinations: at the end of administration/observation period.
- Method of investigation: this examination was performed only in 6 males and 6 females of each group and in satellite males and females. 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.
The following parameters were determined: total erythrocyte count, mean corpuscular volume, haematocrit, haemoglobin concentration, total leucocyte count, total platelets count, partial thromboplastin time, prothrombin time, granulocytes, lymphocytes, monocytes.

BIOCHEMISTRY
- Time schedule for examinations: at the end of administration/observation period.
- Method of investigation: this examination was performed only in 6 males and 6 females of each group and in satellite males and females. 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 following parameters were determined: glucose, cholesterol total, urea, bilirubin total, aspartate aminotransferase, alanine amonitransferase, alkaline phosphatase, calcium, phosphorus, protein total, protein albumin, creatinine, sodium, potassium, chloride.

PATHOLOGICAL EXAMINATION
- Time schedule for examinations:
males and nonpregnant females: at the end of administration period
parental females and pups: on the 4th day of lactation
satellite males and females - at the end of observation period.
- Method of investigation: 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.

Sperm parameters (parental animals):

- Time schedule for examinations: all males after necropsy (exc. satellite males).
- Method of investigation: in all males (except the satellite group) the following sperm parameters were examined: sperm motility and sperm morphology. Sperm specimens were prepared and examined according to internal SOP No. M/45.
- Sperm motility: 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):

ORGANS WEIGHT
- Time schedule for examinations: during necropsy.
- Method of investigation: at the end of study the experimental animals were narcotised and sacrificed by cutting the neck spine and medulla. After the gross necropsy of the cranial, thoracic and abdominal cavities the organs for weighing and further histological examination were collected. The absolute weights of liver, kidneys, adrenals, testes or ovaries, epididymis/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 groups); testes or ovaries, epididymis or uterus, prostate gland, pituitary gland (Reproduction part of study – all animals). Afterwards the somatic indexes - SI (= relative weight of organ) were computed according to the following formula: SI = weight of organ x 100/ body weight.

HISTOPATHOLOGICAL EXAMINATION
- Time schedule for examinations: after necropsy.
- Samples of the following tissues and organs were collected at necropsy and fixed:
Reproduction part of study: pituitary gland, ovaries, uterus, cervix of uterus, vagina, epididymis, prostate gland, seminal vesicles and coagulating gland, testes and all gross lesions.
Repeated Dose Toxicity part of study: adrenal glands, aorta, brain (incl. cerebellum and med. oblongata), caecum, colon, duodenum, pancreas, rectum, salivary glands, sciatic nerve, skeletal muscle, skin, spinal cord - thoracic, spleen, stomach, thymus, thyroid gland incl. parathyroid, trachea, urinary bladder, female mammary gland area, femur, heart, ileum, jejunum, kidneys, liver, lungs, lymph nodes – mesenteric, paraaortal, oesophagus, all gross lesions.
The mentioned tissues and organs were collected from all killed males and females at necropsy and fixed in buffered 4 % formaldehyde solution (v/v) for further histopathological evaluation. For histopathological processing the routine histopathological paraffin technique with haematoxylin-eosin staining was used.
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 demonstrating treatment-related changes: mesenteric lymph nodes, large intestine, caecum, liver, adrenal glands, kidneys, testes, epididymis, prostate gland, seminal vesicles, ovaries, uterus and organs with macroscopical changes were examined at the lowest and middle dose level groups used for Repeated Dose Toxicity part of study.
Detailed histological examination was performed on testes of all males from Reproduction Toxicity part of study (with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure).

VAGINAL SMEARS
- Time schedule for examinations: daily in mating period.
- Method of investigation: 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.

Statistics:

For statistical evaluation the software Statgraphic ® Centurion (version XV, USA) was used. Males/females from control group were compared with males/females from three treated groups. Satellite males/females from control group were compared with satellite males/females from treated group.

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical changes after application of the test substance were recorded

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

statistically significantly increased body weight of females treated with 500 and 180 mg/kg bw/day during the pregnancy and lactation

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

statistically significantly increased body weight of females treated with 500 and 180 mg/kg bw/day during the pregnancy and lactation

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

differences from the control recorded at all the doses tested and both, in males and females

Reproductive function: sperm measures:

effects observed, treatment-related

Description (incidence and severity):

at 500 and 180 mg/kg bw/day markedly changed sperm motility and morphology

PARENTAL MALES
MORTALITY
There were no unscheduled deaths during the whole study.

CLINICAL OBSERVATION
No clinical changes after application of the test substance were recorded in all treated animals; only the test substance-coloured excrements were recorded in different time-interval in all treated groups of animals.

BODY WEIGHT
Decreased body weight of males of the middle dose level was recorded during the whole application period. This decreasing was caused by lower body weight of animals in the beginning of application period, thus it seems not to be related with treatment. Body weight of males of the lowest and highest dose levels was similar to control group.
The body weight increment of males of all treated groups was increased during the 1st and 2nd week of application, in the 3rd week was the body weight increment increased only in the lowest dosed-males and then the body weight increments were similar.

FOOD CONSUMPTION
The mean food consumption of treated males was slightly increased and quite well balanced in comparison with the control animals.

HEALT CONDITION CONTROL
In control males and treated males of all dose levels no signs of diseases were recorded during the check-in and acclimatisation period.
In treated males, the test-substance coloured excrements were recorded in different time-intervals in all treated groups.

REPRODUCTIVE FUNCTION: SPERM MEASURES
Sperm motility was similar in control males and males of the dose level 80 mg/kg. Increased presence of sperms with changed motility was recorded in treated males of the dose level 180 and 500 mg/kg.
Presence of “no progressive motility” was detected in 2 males of the dose level 180 mg/kg.
Examination of sperm morphology revealed marked differences between control and treated males. The dose-dependent increase of morphologically changed sperms at treated males was recorded.

PATHOLOGICAL EXAMINATION
The incidence of affected males is expressed in numeric form and ranged in sequence of the dose levels 0-80-180-500 mg/kg/day further in the text.
No macroscopical findings were recorded in 12-0-0-0 males.
In 12-12-10-12 males no macroscopical changes were observed in testes, epididymides, seminal vesicles, coagulation glands and pituitary gland.
The main changes were related to colour of the test substance such as content of gastrointestinal tract coloured by the test substance in all treated animals, mesenteric lymph nodes coloured in males of the middle and the highest dose levels. Orange colour of pancreas in animals of the highest dose level was recorded.
Atrophy and soft testes were noted in two males of the middle dose level.

BIOMETRY of REPRODUCTIVE ORGANS
The statistical analysis of the data revealed statistical significant intergroup differences in absolute and relative weight of prostate gland in males of the middle and the highest dose levels. Relative weight of testes was significantly decreased in males of the middle dose level.
Absolute weight of epididymis was dose-dependently decreased in treated males.

HISTOPATHOLOGY
Microscopic examination of testes, epididymis, seminal vesicles, prostate gland, coagulation glands and pituitary gland revealed presence of treatment related changes.
Acinar atrophy of prostate gland was observed in 2-5-6-4 males. Focal interstitial mononuclear infiltration and/or oedema was reported in prostate gland of 3-2-5-6 males. Tubular degeneration/atrophy in testes was observed in 0-3-1-5 males (at the lowest and the highest dose level less than 10 % affected tubules, at the middle dose level 75 – 100 % affected tubules).
Findings in epididymis were sporadic.

PARENTAL FEMALES
MORTALITY
There were no unscheduled deaths during the whole study.

CLINICAL OBSERVATION
No clinical changes after application of the test substance were recorded.

BODY WEIGHT
- Pre-mating mating period: The mean body weight increments of the females of the dose levels 180 and 500 were increased in comparison with the control females. Slightly decreased mean body weight was noted in females of the lowest dose level in the pre-mating period. Mean body weight increment was increased in all treated females compared to control females.
- Pregnancy: Females without parturition (non-pregnant or aborted females) were not included in the evaluation of mean body weight increments during pregnancy. The mean body weight increments of treated pregnant females at the lowest dose level were similar to control group. Statistically significant increasing of mean body weight was reported in females of the middle and highest dose level compared to control group. Mean body weight increment was increased in all treated females compared to control females.
- Lactation: Only mothers (females with live pups born) were included in the evaluation of body weight increments during lactation period.
The mean body weight increments of treated mothers at the lowest dose level were slightly increased compared to control animals. Statistically significant increase of mean body weight was reported in mothers of the middle and highest dose levels. The mean body weight increment was lower in treated mothers than in mothers of the control group.

FOOD CONSUMPTION
- Pre-mating period: The mean food consumption of treated females was in pre-mating period increased in comparison with the control animals.
- Pregnancy: Females without parturition (non-pregnant or aborted females) were not included in the evaluation of food consumption during pregnancy. The mean food consumption of pregnant females treated by the test substance was increased compared to control females.
- Lactation: Only mothers (females with live pups born) were included in evaluation of food consumption during lactation period. The mean food consumptions of treated mothers were lower compared to control mothers

HEALT CONDITION CONTROL
In control females and treated females of all dose levels no signs of diseases were recorded during the check-in and acclimatisation period.
No changes of health condition were found out before, during and immediately after application of the test substance. Only test substance-coloured excrements were recorded in all treated females in different time period.

PATHOLOGICAL EXAMINATION
The incidence of affected females is expressed in numeric form and ranged in sequence of the dose levels of 0-80-180-500 mg/kg/day further in the text.
No macroscopical findings were recorded in 11-0-0-0 females.
The changes related to colour of the test substance – content of gastrointestinal tract coloured by the test substance in all treated females, also coloured mesenteric lymph nodes in females of middle and the highest dose levels and also orange colour of pancreas in animals of the highest dose level were recorded.
Dead fully developed foetuses were found in uterus in one female of the control group and in one female of the lowest dose level. One female of the lowest dose had cyst on ovaries.

BIOMETRY of REPRODUCTIVE ORGANS
The statistical analysis of the data did not reveal significant intergroup differences in absolute and relative weights of ovaries, uterus and pituitary gland.
Non-pregnant females and females with abortion were not used for calculation of means and evaluation of biometry results.
Slight increase of absolute weight of ovaries in females at all treated groups was described.
Absolute and relative weights of uterus were decreased in females of the lowest dose level.
Absolute weight of pituitary gland of females at the dose level 180 and 500 was increased compared to control group.

HISTOPATHOLOGY
The incidence of affected females is expressed in numeric form and ranged in sequence of the dose levels 0-80-180-500 mg/kg/day further in the text.
In 1-4-1-1 females no histological changes were detected.
During microscopical examination of reproductive organs the following possible treatment related changes were registered. Squamous metaplasia of endometrial epithelium in uterus of 0-1-2-2 females and follicular cysts in 0-1-3-3 ovaries of females were detected.
In uterus the changes related to previous pregnancy were found in both controls and treated animals: accumulation of siderophages in mesometrium or/and in endometrium in 5-6-5-6 females and haemorrhages or haematoma in mesometrium in 4-0-2-2 females.
Other microscopical changes observed in reproductive organs occurred only sporadically or they did not relate to test substance treatment – see the table No. 52.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: The value was determined mainly on the basis of changed sperm motility and morphology, decreased weight of prostate gland and microscopical findings in reproductive organs.

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

post-implantation and post-natal losses were increased at 500 and 180 mg/kg bw/day

Body weight and weight changes:

no effects observed

Gross pathological findings:

no effects observed

NUMBER and SEX RATIO of PUIPS
The statistical evaluation of the number of live born pups/per litter, number of corpora lutea and number of implantations was performed. No statistically significant intergroup differences were recorded.
The total number of live pups at first check of litter after parturition at the lowest and the highest dose levels was decreased in comparison with the control. The total number of live pups per litter at the middle dose level was higher than control.
At 4th day of lactation, decreased numbers of pups at the lowest and highest dose levels were recorded.
Mean number of pups per litter in treated mothers at the lowest dose level was decreased compared to control mothers and on contrary was increased in mothers of the middle and highest dose levels.

BODY WEIGHT
The statistical evaluation of mean weight of pup on the 0./1st day and mean weight of pup on the 4th day was performed. No statistically significant intergroup differences were recorded.
Mean body weights of litters at the lowest dose level were decreased compared to control; mean body weights of litter of the middle and highest dose level were comparable and higher in comparison with the control group.
Mean weights of pup recorded at the 1st check of litter after parturition at treated groups were slightly increased at the lowest dose level and slightly increased at the middle and the highest dose levels. Mean body weight of pup on the 4th day of lactation was also increased at the lowest dose level and similar to control at the middle and highest dose levels.

DEVELOPMENT of PUPS
No stillborn pups were found at control and the lowest dose level. Number of stillborn pups was negligible at middle and the highest dose levels.
Mortality of pups in lactation period was detected in all treated groups; increased mortality was observed at the middle and the highest dose levels.
No differences in postnatal development of pups were observed at the control and treated groups.

PATHOLOGICAL EXAMINATION
The macroscopic examination was performed in all pups. Macroscopical findings were observed sporadically at pups of treated mothers: empty stomach or flatulence of stomach.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: The value was determined mainly on the basis of decreased number of females bearing live pups.

Reproductive effects observed:

not specified

REPRODUCTIVE PARAMETERS

Evidence of copulation was not found in one female of the control, the lowest and the middle dose levels. All females of the highest dose level were mated.

The number of females achieving pregnancy was analogous in control and the lowest dose levels.Decreased number of females achieving pregnancy was recorded in the middle dose level and the lowest number of females achieving pregnancy was noted in the highest dose level. Abortions occurred at the control, the lowest and the middle dose levels.

The duration of mating of females of the highest dose level it was shorter in comparison with the control group. 

The number of females bearing live pups was the same in the control and the lowest dose level. The number of mothers with live pups at the middle and the highest dose level was decreased compared to control. Mean number of corpora luteawas increased in all treated groups against the control group. Vice versa mean number of implantations was decreased at the lowest dose level.

Mean number of live pups at birth and at day 4 after parturition in mothers was decreased in females at the lowest dose level.

Mean weight of the litter at the lowest dose level was decreased against control. Vice versa the mean weights of pup at the 1stcheck of litter after parturition and on 4th day of lactation were increased at the lowest dose level.

No treatment-related findings were observed in pups at macroscopical examination.

FERTILITY PARAMETERS

Fertility indexes and survival index were decreased at the middle dose level and the highest dose level. Male fertility index was decreased in the middle dose level and markedly decreased in males of the highest dose level. Female fertility index was markedly decreased in females of the highest dose level.

Pre-implantation losses were increased at the lowest and the middle dose level.

Post-implantation losses increased with the dose level.

Post-natal losses were increased in females at the middle and the highest dose levels.

Conclusions:

NOAEL reproduction and developmental: 80 mg/kg bw/day. The value was determined mainly on the basis of changed sperm motility and morphology, decreased weight of prostate gland, microscopical findings in reproductive organs and decreased number of females bearing live pups.

Executive summary:

Method

The substance was tested for reproduction and subacute toxicity using the OECD Test Guideline No. 422. Wistar rats of SPF quality were used for testing and the test substance was administered in the form of suspension in water for injection. Oral application by stomach tube was performed daily. The study includes four main groups and two satellite groups of animals. Main groups contained 3 treated groups (doses 80, 180, 500 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 (500 mg/kg/day).  

During the study clinical observation and health status controls 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. The functional observation was performed at the end of application and observation period. 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, weight, sex and vitality of pups) 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 were removed for weighing and histopathological examination.

Results

The number of females achieving pregnancy, sperm parameters and microscopical structure of reproductive organs parental males and females, number of post-implatation and post-natal losses of mothers were adversely affected by the test substance treatment.

The test substance affected sperm motility and morphology of parental males. Markedly changed sperm motility and morphology were recorded in males of the middle and the highest dose level. Significantly decreased absolute and relative weight of prostate gland was recorded in males of the middle and the highest dose levels. Negative effect of the test substance treatment on prostate gland was confirmed by microscopical findings – acinar atrophy and focal interstitial mononuclear infiltration and oedema of prostate gland were recorded. Also tubular degeneration or atrophy of testes (with minimal severity) was observed in males of the highest dose level. Changed weight of prostate is the most sensitive indicator of low testosterone and antiandrogenic activity. Organ weight is also more sensitive than histopathologic assessment of this organ. This finding could be related with worse ability parental males to make parental females pregnant. 

All findings mentioned above related to test-substance treatment were confirmed at evaluation of reproduction parameters. Reproduction performance of males and females was evaluated according to the male and female reproduction data and calculated reproduction parameters. Male ability to produce sperm that can fertilise eggs and female ability to achieve pregnancy was changed – number of females achieving pregnancy was decreased in females at the middle and the highest dose level. Number of females bearing live pups at the middle and the highest dose levels was reduced. Post-implantation and post-natal losses were increased in females of the middle and the highest dose levels which confirmed negative effect of the test substance on reproduction organs of females. The findings in uterus and ovaries - increased count of follicular cysts in ovaries and squamous metaplasia of endometrial epithelium in uterus were observed in nonpregnant females of the middle and the highest dose levels.

Conlusion

NOAEL reproduction and developmental: 80 mg/kg bw/day. The value was determined mainly on the basis of changed sperm motility and morphology, decreased weight of prostate gland, microscopical findings in reproductive organs and decreased number of females bearing live pups.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

43.2 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

No repeated dose toxicity data are available on Acid Brown 355 (ABr355); nevertheless a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test was conducted on the structural analogue Acid Brown 282 (ABr282 - Similar Substance 01).

ABr282 was tested for reproduction and subacute toxicity using the OECD Test Guideline No. 422. Wistar rats of SPF quality were used for testing and the test substance was administered in the form of suspension in water for injection. Oral application by stomach tube was performed daily at the following doses: 80, 180, 500 mg/kg of body weight /day (adjusted on the basis of the substance purity: 43.2, 97.2 and 270 mg/kg bw/day, respectively).

The number of females achieving pregnancy, sperm parameters and microscopical structure of reproductive organs parental males and females, number of post-implantation and post-natal losses of mothers were adversely affected by the test substance treatment.

The test substance affected sperm motility and morphology of parental males. Markedly changed sperm motility and morphology were recorded in males of the middle and the highest dose level. Significantly decreased absolute and relative weight of prostate gland was recorded in males of the middle and the highest dose levels. Negative effect of the test substance treatment on prostate gland was confirmed by microscopical findings – acinar atrophy and focal interstitial mononuclear infiltration and oedema of prostate gland were recorded. Also tubular degeneration or atrophy of testes (with minimal severity) was observed in males of the highest dose level. Changed weight of prostate is the most sensitive indicator of low testosterone and antiandrogenic activity. Organ weight is also more sensitive than histopathologic assessment of this organ. This finding could be related with worse ability parental males to make parental females pregnant. 

All findings mentioned above related to test-substance treatment were confirmed at evaluation of reproduction parameters. Reproduction performance of males and females was evaluated according to the male and female reproduction data and calculated reproduction parameters. Male ability to produce sperm that can fertilise eggs and female ability to achieve pregnancy was changed – number of females achieving pregnancy was decreased in females at the middle and the highest dose level. Number of females bearing live pups at the middle and the highest dose levels was reduced. Post-implantation and post-natal losses were increased in females of the middle and the highest dose levels which confirmed negative effect of the test substance on reproduction organs of females. The findings in uterus and ovaries - increased count of follicular cysts in ovaries and squamous metaplasia of endometrial epithelium in uterus were observed in nonpregnant females of the middle and the highest dose levels.

The No Observed Adverse Effect Level (NOAEL) for reproductive and development was established as 80 mg/kg body weight/day. The value was determined mainly on the basis of changed sperm motility and morphology, decreased weight of prostate gland, microscopical findings in reproductive organs and decreased number of females bearing live pups.

Nevertheless, the substance purity of the lot tested was about 54 %, thus the NOAEL can be adjusted at 43.2 mg/kg bw/day.

ABr282 is a disodium salt and the main structural difference with the ABr355 is that it presents one sulphonated group less than the ABr355. It is expected that these differences have not a significant impact on the repeated dose toxicity.

The read across approach has been further detailed in the report attached to the IUCLID section 13.

Justification for selection of Effect on fertility via oral route:

Study conducted on structural analogue, according to internationally accepted testing guidelines and performed according to GLP.

The NOAEL recorded in the study was 80 mg/kg bw/day, nevertheless the effect level here indicated is corrected on the basis of the substance purity.

Justification for classification or non-classification

According to the CLP Regulation (EC 1272/2008), 3.7 Reproductive toxicity section, reproductive toxicity includes adverse effects on sexual function and fertility in adult males and females, as well as developmental toxicity in the offspring.

The substance is not classified for reproductive toxicity according to the CLP Regulation (EC 1272/2008).