Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From May,30. 05. 2012 to December 14,2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant with international guideline

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)
Deviations:
yes
Remarks:
The increase of humidity up to 74.6% (OECD 422 limit was 70%) and decrease of temperature to 17.8 ºC (OECD 422 range was 19-25ºC) in SPF animal room was observed for a short-term. Deviations did not influence the wellness of animals and the study results.
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Reference substance name:
Acid Brown 75
IUPAC Name:
Acid Brown 75
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder

Test animals

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., Koleč u Kladna Czech Republic
- Age at study initiation: 10 weeks
- Weight at study initiation: 356.38 ± 32.41 g
- Housing: Animals were housed in SPF animal room, 2 rats of the same sex in one plastic cage (40x25x20 cm) containing sterilised clean shavings of soft wood. in pre-mating period, during mating period – one male and one female in one cage, pregnant females – individually, offspring – with moth er, satellite animals - 2 rats of the same sex in one cage
- Diet: Complete pelleted diet for rats in SPF breeding - ST 1 BERGMAN, manufacturer; Diet was sterilised before using.
Composition of diet: Wheat, Oats, Fish meal powder, Dried snail-clover, Soya extracted groats, Wheat sprouts, Dehydrated yeast, Calcium carbonate, Vitamin and Mineral complex. Nutrient content of the diet: Crude protein – min. 21%, Drip – max. 14%, Fat – min. 3%, Fiber – max. 4.1%, Ash – max. 7 %, Calcium – min. 1%, Phosphorus – min. 0.8%, Magnesium – min. 0.2%, Sodium – max. 0.25%.
- Water: Free access to drinking water (water ad libitum). Water quality corresponded to Regulation No. 252/2004 Czech Coll. of Law, Health Ministry.
- Acclimation period: 11 days
- health check: daily during acclimatation period

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

OTHER:
The standard pelleted laboratory animal diet is analysed for nutrients (once a year) and bacteriological contaminants every 2 months on a regular basis.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
The application form was prepared by mixing with aqua pro injectione. Two concentrations of application form were prepared (50 mg/10 mL and 1000 mg/10 mL).
Details on mating procedure:
Animals were mated from the 15th day of study. Mating schedule 1 : 1 (one male to one female) was used in this study. Each morning the females were examined for presence of spermatozoa in vaginal smears. Day 0 of pregnancy was defined as the day the sperms were found.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The determination of Acid Brown 75 was performed by high-performance liquid chromatography based on a method developed at the test facility.
Duration of treatment / exposure:
56 days
Frequency of treatment:
7 days per week
Details on study schedule:
Health condition control: daily - during the acclimatization and the experimental part
Body weight: males - weekly
females - weekly in premating and mating period,
during pregnancy: 0., 7th, 14th, 20th day,
during lactation: 0. or 1st, 3rd and 4th day;
pups (litters) – 0. or 1st, 3rd and 4th day;
satellite males and females - weekly
Food consumption: 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
Water consumption: satellite males and females – twice a week
Clinical observations: males and females - daily during the administration period
pups - as soon as possible after delivery and then daily
Mortality control: daily
Detailed clinical observation: before the first application and then weekly (except the mating
period)
Functional observation: at the end of administration/observation period
Laboratory examinations:
- vaginal smears: daily in mating period
- urinalysis: last day of administration/observation period – only males
- haematology: at the end of administration/observation period
- biochemistry: at the end of administration/observation period
- pathological examination: 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
- weight of organs: during necropsy
- sperm observation: all males after necropsy
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 70, 250, 630
Basis:
actual ingested
No. of animals per sex per dose:
Basic groups:
1. Control 0 12 males + 12 females
2. Low dose 70 mg/kg/day 12 males + 12 females
3. Intermediate dose 250 mg/kg/day 12 males + 12 females
4. High dose 630 mg/kg/day 12 males + 12 females
Satellite groups:
5. Control – vehicle – satellite: 0 6 males + 6 females
6. High dose – satellite: 630 mg/kg/day 6 males + 6 females
Control animals:
yes, concurrent no treatment

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes , daily during the acclimatization and the experimental part.
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 (12.00 - 14 p.m.) – at the time of expectation of maximal effect of the test substance. Animals were observed in natural conditions in their cages.
Detailed clinical observation: before the first application and then weekly (except the mating period)
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.
Functional Observation
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.

BODY WEIGHT: Yes
males - weekly
females - weekly in premating and mating period, during pregnancy: 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
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 a mean per group per day (in grams). Non-pregnant females (females without parturition) were not included in calculation of means in pregnancy and lactation period.

FOOD CONSUMPTION yes
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
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 of 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 premating 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 of pregnant females.


FOOD EFFICIENCY:yes
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain

WATER CONSUMPTION: yes
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.

HAEMATOLOGY: Yes , 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
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.
Haematology analysers Coulter AC.T diffTM, Celltac alfa and Coagulometer ACL 200 were used for examination and 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

Biochemical Examination
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 by automatic biochemical analysers SPOTCHEMTM EZ SP-4430 and SPOTCHEMTM EL SE-1520 (Arkray, Inc., Japan).
Glucose
Cholesterol, total
Urea
Bilirubin, total
Aspartate aminotransferase
Alanine amonitransferase
Alkaline phosphatase
Calcium
Phosphorus
Protein, total
Protein, albumin
Creatinine
Sodium
Potassium
Chloride

URINALYSIS: only males – 42nd and 56th day of study.
This examination was performed only in 6 males of each group and in satellite males. 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 by analyser PocketChem PU-4210 (Arkray, Inc., Japan).
Volume
Colour
Cloud
Odour
Glucose
Protein
Bilirubin
Urobilinogen
pH
Specific gravity
Blood
Ketones
Nitrite
Leucocytes

OTHER:
Mortality control: daily
Laboratory examinations:
- vaginal smears: daily in mating period
- biochemistry: at the end of administration/observation period
- haematology: at the end of administration/observation period
- pathological examination: 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
- weight of organs: during necropsy
- sperm observation: all males after necropsy
- histopathological examination: after necropsy
Sperm parameters (parental animals):
In all males (except the satellite group) the following sperm parameters were examined: sperm motility and sperm morphology.
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.
Litter observations:
All pups were observed in natural conditions in their cages daily during the lactation. Changes in behavioural abnormalities were recorded. Detailed examination of each litter was performed as soon as possible after delivery (day 0 or 1 post-partum) and the 4th day of lactation. The number and sex of pups, stillbirths, live births and presence of gross anomalies were recorded.
Statistics:
The ANOVA test - Analysis of Variance (QC.Expert 2.5) at significance level 0.05 was used for the statistical analysis (the raw data were used for statistical analysis). This statistical analysis was used for the results of body weight, results of haematology, blood biochemistry, urinalysis, biometry of organs and selected reproduction parameters. 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.
Reproductive indices:
- Pre-implantation loss:
Number of corpora lutea – number of implantations
- Post-implantation loss:
Number of implantations – number of live births
- Post-natal loss:
Number of live births – number of alive at postnatal day 4

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Excrements and tail in all treated groups were coloured
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
at the highest dose level
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Water consumption was observed only in satellite animals. The water consumsption of dosed group increased during dosing form males and females. During the recovery period the water consumption became comparable to control
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
above 250 mg/kg bw (primarily on the red blood cells - decreased value of total erythrocyte count, haematocrit and haemoglobin, increased value of mean corpuscular volume)
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
above 250 mg/kg bw for parametrs connected to spleen and kidney
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
above 250 mg/kg bw for urine volume, colours, specific weight, leucocyte, proteins and ketones presence (only at the highest dose).
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
above 250 mg/kg for intestine and rectum (ulcer or erosion, focal inflammation of mucosa and/or submucosa); Extramedullar haemopoiesis and hemosiderin in spleen and/or liver
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, treatment-related
Description (incidence and severity):
above 250 mg/kg for corpora lutea (decrease), pre-implantation loss and posti implantation loss (decreae), number of female with live pups and at day 4 (decrease)
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
The fertility index was decreased in all treated groups, markedly at the highest dose level and gestation index were decreased in all treated groups

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
General Clinical Observation
General Clinical Observation was performed in all 12 animals from each group and satellite animals and it is described in this part. General Clinical Observation was not performed in satellite animals during recovery period (it was performed only after application).
Males
Excrements and tail of males in all treated groups and satellite treated group were coloured by the test substance during the whole study. Other symptoms of clinical status were not observed.
Females
Excrements and tail of females in all treated groups and satellite treated group were coloured by the test substance during the whole study. Other symptoms of changed clinical status were not observed.
Mortality Control
Males
There were no unscheduled deaths during the study in all animals.
Females
There were no unscheduled deaths during the study in all animals.
Health Condition Control
Health Condition Control was performed in all 12 animals from each group and satellite animals and it is described in this part.
Males
Excrements and tail of males in all treated groups were coloured by the test substance during the whole study. Other changes of health condition were not observed.
Satellite males
Excrements and tail of males in treated group were coloured by the test substance during application and recovery period. Other changes of health condition were not observed.
Females
Excrements and tail of females in all treated groups were coloured by the test substance during the whole study. Other changes of health condition were not observed.
Satellite females
Excrements and tail of females in treated group were coloured by the test substance during application and recovery period. Other changes of health condition were not observed.
Detailed Clinical Observation
Detailed Clinical Observation was performed in all 12 animals from each group and satellite animals and it is described in this part.
Males
The activity (poise, gait, reaction to handling) of all males of all treated groups was similar
during the study and it was not different from the activity of males of the control groups.
No significant changes were found at all dose levels during the examinations of skin, hair, eyes, lacrimation, visible mucous membrane, secretion and respiration. Only excrements and tail of males in all treated groups were coloured by the test substance during the whole study.
Satellite males
The activity (poise, gait, reaction to handling) of all males of treated group was similar during the study the activity of males of the control groups.
No significant changes were found at all dose levels during the examinations of skin, hair, eyes, lacrimation, visible mucous membrane, secretion and respiration. Excrements and tail of males in treated group were coloured by the test substance during application period. During recovery period only tail of males in treated group was coloured by the test substance.
Females
The activity (poise, gait, reaction to handling) of all females of all treated groups was similar during the study and not different from the activity of females of the control groups.
No significant changes were found at all dose levels during the examinations of skin, hair, eyes, lacrimation, visible mucous membrane, secretion and respiration. Only excrements and tail of females in all treated groups were coloured by the test substance during the whole study.
Satellite females
The activity (poise, gait, reaction to handling) of all females of treated group was similar during the study and not different from the activity of females of the control groups.
No significant changes were found at all dose levels during the examinations of skin, hair, eyes, lacrimation, visible mucous membrane, secretion and respiration. Excrements and tail of females in treated group were coloured by the test substance during application period. During recovery period only tail of females in treated group was coloured by the test substance.
Functional Observation
Males
Reactions to contact, to noise, to pain and pupillary reflex of treated males and satellite treated group were the same as in the control group.
Results of upstanding were similar in males of all dose level in comparison with control. Results of emiction and defecation in treated males were not the same as in control males but the variation was within the range of the physiological reaction of animals. The values of grip strength of pectoral legs and pelvic legs in males did not show any difference between control
Females
Reactions to contact, to noise, to pain and pupillary reflex of treated females and satellite treated group were the same as in the control group.
Results of upstanding were similar in females of all dose level in comparison with control. Results of emiction and defecation in treated females were not the same as in control females but the variation was within the range of the physiological reaction of animals. The values of grip strength of pectoral legs and pelvic legs in females did not show any difference between control and the treated dose levels.
Pathological Examination
Affections of genital organs were observed only sporadically (reduced prostate gland in one male at the dose level 70 mg/kg/day).
Examination of the external surface of animal bodies showed brown colour of tail and anus in all animals at the dose level 250 and 630 mg/kg/day.
In the abdominal cavity macroscopic changes in stomach (congested mucous membrane and/or dark colour of chymus) were recorded in all treated groups. At the dose level of 250 and 630 mg/kg/day macroscopic changes in spleen (dark colour and/or enlargement) were recorded.



BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Males
Decreased body weight at the dose level 250 and 630 mg/kg/day was recorded from the 2nd week of application period to the end of study. The body weight increment of animals at the dose level 70 and 250 mg/kg/day was relatively well-balanced with the control group during application period (except the 1st week of application period when it was decreased). At the dose level 630 mg/kg/day body weight increment was decreased in the 1st, 2nd and 4th week of study.
No statistically significant differences were detected.
Females
Statistically significant differences were not found.
Before mating period
The body weight of treated females was relatively well-balanced with respect to the control
group. The body weight increment at the dose level of 250 and 630 mg/kg/day was negative in the 1st week of application, but it reached normal values during the second week of application.
Pregnancy
Females without parturition (non-pregnant or aborted females) were not included in the evaluation of body weight increments during pregnancy.
The body weight in mothers of treated groups was decreased in comparison with control group from the 14th day of pregnancy. The body weight increments in treated mothers were decreased compared to control group.
Lactation
Only mothers (females with live pups) were included in the evaluation of body weight increment during lactation period.
Mean body weight of treated mothers at all dose levels was decreased. The body weight increments in mothers of the dose level 630 mg/kg/day was negative .


Food Consumption
Males
Food consumption of males at all treated dose levels and control group was relatively well-balanced during the whole study.
Females
Pre-mating period
The mean food consumption of all treated groups and control group was similar.
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 mothers at all dose levels and control group was similar.
Lactation
Only mothers (females with live pups) were included in evaluation of food consumption during lactation period.
Mean food consumption of treated females was lower than in control females.


REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
Acute colpitis in vagina was recorded in two females of the control group. Lobular hyperplasia in mammary gland was detected in all groups. These changes were possible related with previous gravidity pregnancy or proceeded oestrous cycle. Biometry of organs also proved statistically significant decrease of relative and absolute weight of uterus.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
Sperm motility and sperm morphology in treated males and control group was similar.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Females
females with abortion were recorded in all treated groups. Decreased number of corpora lutea and number of implantations were recorded at the middle dose level. Pre-implantation losses were decreased at the middle dose level and slightly decreased at the highest dose level. Post-implantation losses were similar. Number of pups per litter was markedly decreased at the middle dose level. The number of females bearing live pups and females with live pups at day 4 after parturition was decreased at the middle and highest dose level. The fertility index was decreased in all treated groups, markedly at the highest dose level and gestation index were decreased in all treated groups.
Decreased of fertility index observed at the highest dose level is regarded as biologically significant.


ORGAN WEIGHTS (PARENTAL ANIMALS)
Males
Biometry of Reproductive Organs
The statistical analysis of the data revealed no significant intergroup difference.
Absolute and relative weight of prostate glands was slightly increased at the dose level 630 mg/kg/day. Decreased absolute and relative weight of pituitary gland at the dose level 70 mg/kg/day was found.
Females
Non-pregnant females and females with abortion were not used for calculation of means and evaluation of organs weight.
Significant increase of relative and absolute weights of uterus at the dose level of 630 mg/kg/day and slight increased relative weight of pituitary gland was found at the dose level 250 and 630 mg/kg/day.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Males
The incidence of affected males is expressed in numeric form and ranged in sequence of the dose levels 0-70-250-630 mg/kg/day further in the text.
Histopathological changes in male reproductive system were detected at the control group and all dose levels.
In 12-12-12-12 males no histopathological changes were detected in seminal vesicles.
Exfoliation of germ cells into the lumen of tubules in testes in 7-4-5-7 males was recorded (it was recorded only in sporadic - less than 10% tubules).
In the epididymis, focal infiltration of interstitium in 3-1-2-1 males; germ cells in lumen in 0-0-0-2 males and siderophages in 1-1-0-0 males were found out.
Focal mononuclear infiltration of the interstitium and/or oedema in 10-0-0-5 males and cell detritus in lumen in 2-4-0-2 males in prostate gland was detected.
In the spleen, the following microscopical changes were found: venostasis in 0-0-1-11 males, extramedullary haematopoiesis in 0-1-11-9 males and hemosiderin in 0-1-5-4 males.
Females
Modulations of genital organs were observed only sporadically (cervix filled with gelatinous mass in one female at the dose level 250 mg/kg/day and dead foetuses in uterus in one female
at the dose level 630mg/kg/day). Dilatation of uterus (accompanied oestrus) in all treated groups was recorded.
Examination of the external surface of animal bodies revealed no macroscopic changes in treated and control animals. In the abdominal cavity macroscopic changes in stomach (congested mucous membrane and/or dark colour of chymus) were recorded in all treated groups. Dark colour of chymus in GIT was recorded at the dose level 250 and 630 mg/kg/day. At the dose levels 250 and 630 mg/kg/day macroscopic changes in spleen (dark colour and/or enlargement) were recorded. Dark colour of kidneys at the dose level 630 mg/kg/day was also observed.
Females
The incidence of affected females is expressed in numeric form and ranged in sequence of the dose levels 0-70-250-630 mg/kg/day further in the text.
In the reproductive system affections were recorded in females at the control group and at all dose levels.
In 12-12-12-12 females no histopathological changes were detected in ovaries and pituitary gland.
In the uterus, the following microscopical changes were found: focal accumulation of lipophages and siderophages in mesometrium in 5-5-4-4 females; siderophages in mucosa in 4-1-2-1 females; organizing hematoma in 2-0-1-0 females and hydrometra in 0-2-3-2 females. In the mammary gland lobular hyperplasia in 6-6-6-6 females was recorded. In the vagina acute colpitis was found in two control animals.
In the spleen, the following microscopical changes were found: venostasis in 0-0-6-4 females, extramedullary haematopoiesis in 1-0-3-12 females and hemosiderin in 0-0-8-12 females.
The incidence of affected females is expressed in numeric form and ranged in sequence of the dose levels 0-70-250-630 mg/kg/day further in the text.
In the reproductive system affections were recorded in females at the control group and at all dose levels.
In 12-12-12-12 females no histopathological changes were detected in ovaries and pituitary gland.
In the uterus, the following microscopical changes were found: focal accumulation of lipophages and siderophages in mesometrium in 5-5-4-4 females; siderophages in mucosa in 4-1-2-1 females; organizing hematoma in 2-0-1-0 females and hydrometra in 0-2-3-2 females. In the mammary gland lobular hyperplasia in 6-6-6-6 females was recorded. In the vagina acute colpitis was found in two control animals.
In the spleen, the following microscopical changes were found: venostasis in 0-0-6-4 females, extramedullary haematopoiesis in 1-0-3-12 females and hemosiderin in 0-0-8-12 females.



Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
ca. 70 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
food efficiency
haematology
clinical biochemistry
urinalysis
organ weights and organ / body weight ratios
reproductive performance
other: see 'Remark'

Results: F1 generation

General toxicity (F1)

Clinical signs:
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
the maen litter weight was affected fom 250 mg/kg
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
change of the colour of milk possibly due to the increased exposure to the test substance, or stomach without milk possibly due to decreased production of milk in exposed mothers, and stomach flatulence (seconday effects due to maternal toxicity)
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
total number of ups was decreased from 250 mg/kg

Details on results (F1)

VIABILITY (OFFSPRING)
Statistical evaluation was performed on the number of live pups. No significant changes were recorded.
The total number of live pups (on the day of parturition/1st day after parturition and the 4th day after parturition) at all dose levels was lower than at the control but it was markedly decreased at the dose level 250 and 630 mg/kg/day. Mean number of pups per litter at the dose level 250 mg/kg/day was lower than at the control.
Sex ratio of pups was balanced in treated and control groups.

CLINICAL SIGNS (OFFSPRING)
Total number of pups was decreased at the middle and highest dose level (at the middle 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. The mean weight of litters was decreased at the middle and highest dose level. No significant differences in development of all pups were observed. During pathological examination higher numbers of affected pups were recorded at the middle and highest dose level: change of the colour of milk possibly due to the increased exposure to Acid brown 75, stomach without milk (possibly due to decreased production of milk in exposed mothers), and stomach flatulence.

BODY WEIGHT (OFFSPRING)
The mean weight of the litters at the dose levels 250 and 630 mg/kg/day was decreased compared to control in both interval of weighting.
The mean weight of pups at the dose level 70 and 630 mg/kg/day and control group was relative well-balanced. At the dose levels 250 mg/kg/day, the mean weight of pups was slightly increased in comparison with control group.
No significant changes were recorded.

GROSS PATHOLOGY (OFFSPRING)
The macroscopic examination was performed in all pups (except one pup in the control group – cannibalism of mother).
Stomach without milk in pups, which died during lactation, was recorded at the dose level 70 mg/kg/day and in the control group.
At the dose level of 250 mg/kg/day change of colour of milk and flatulence in stomach were recorded in all pups from two litters.
Stomach without milk in pups of four litters, change of colour of milk in pups of three litters and flatulence in stomach of pups in all litters were recorded at the dose level 630 mg/kg/day.


OTHER FINDINGS (OFFSPRING)
Three pups from two mothers
of the control group, one pup at the dose level of 70 mg/kg/day and two pups from two mothers at of the dose level of 630 mg/kg/day died during lactation period. One stillborn pup was found at the dose level of 70 mg/kg/day. No significant differences in development of treated and control pups were observed.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
ca. 70 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
other: see 'Remark'

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Reproduction Parameters

All treated and control females were mated. Presence of sperm was not found in one female at the control group. Females with abortion were recorded in all treated groups (one female at the lowest dose level, two females at the middle dose level and one female at the highest dose level). 

The number of females achieving pregnancy, duration of mating and pregnancy of treated groups were similar to the control group.

The number of females bearing live pups and females with live pups at day 4 after parturition was decreased at the dose level 250 and 630 mg/kg/day.

The number of corpora lutea was slightly decreased at the dose level 250 mg/kg/day. Number of implantations at the dose level 250 mg/kg/day was lower than at the control group.

Number of pups per litter was slightly lower in all treated groups but significant at the dose level 250 mg/kg. Mean weight of litters was decreased at the dose level 250 and 630 mg/kg/day and mean weight of pups at the dose level 250 mg/kg/day was increased. Abnormal pups were found in all groups mostly at the highest dose level.

Mating index of treated males and treated females was well-balanced with control group. Fertility index of treated males and treated females was lower than at the control group markedly at the highest dose level. Gestation index of treated groups was decreased in comparison with the control group. Survival index of treated groups was well-balanced with the control group.

Pre-implantation losses were slightly increased at the dose level 250 and 630 mg/kg/day. Post-implantation losses of treated groups were relative similar with the control group. No significant differences in post-natal losses were detected between the control and treated mothers.

Applicant's summary and conclusion

Conclusions:
The test substance administration had not adverse effect on mortality, parameters of functional observation and some reproduction parameters - course of mating and lactation, spermiogenesis, macroscopical and microscopical structure of reproductive organs and pituitary gland of parental animals and number of post-natal losses of mothers.
The test substance treatment affected the number of pups (decrease of the total number of live pups and mean weight of litters and affected pups, pre-implantation losses (decreased number of corpora lutea, uterus implantations and pups). The highest incidence of statistically or biologically significant effects was recorded at the middle and highest dose levels while most of changes which were found at the lowest dose level had only mild intensity without adverse alteration of animal organism.
The NOAEL (No Observed Adverse Effect Level) for REPRODUCTION for MALES and FEMALES was established as 70 mg/kg body weight/day. The value is based on the effect in females: decrease of the total number of live pups and mean weight of litters, decreas of pre-implantation losses and fertility parameters (decreased number of corpora lutea and uterus implantations). In males no effect on reproduction was observed. The NOAEL (No Observed Adverse Effect Level) for DEVELOPMENT was established as 70 mg/kg body weight/day. The values were established on the basis of decrease of the total number of live pups and mean weight of litters, decrease of pre-implantation losses and fertility parameters (decreased number of corpora lutea and uterus implantations).
Executive summary:

The 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 22nd 1996.

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 70, 250, 630 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 (630 mg/kg/day). The dose levels for study were determined on the basis of results of a dose-range finding study phase.

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.

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

During the study clinical observation and health status control were performed daily. The body weight and food consumption were measured weekly or in the specified time intervals. Detailed clinical observation was carried out weekly. 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, sex or vitality) 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

Repeated dose toxicity part of study:

Repeated oral administration of Acid brown 75 to rats by gavage at the dose levels 70, 250 and 630 mg/kg/day did not cause mortality. 

Reproduction part of study:

The course of mating, pregnancy and lactation of parental animals, weights of reprodutive organs and pituitary gland, spermiogenesis and sperm parameters, macroscopical and microscopical structure of reproductive organs and pituitary gland of parental animals, number of pre-implantation, post-implantation and post-natal losses of mothers and number, weight, sex ratio and development of pups were not adversely affected by the test substance

treatment at the dose levels 70 mg/kg/day. Only fertility and gestation index were slightly decreased.

The course of mating, pregnancy and lactation, weights of reprodutive organs and pituitary gland, spermiogenesis, macroscopical and microscopical structure of reproductive organs and pituitary gland of parental animals, number of post-natal losses of mothers were not adversely influenced by the test substance treatment at the dose level of 250 and 630 mg/kg/day.

Examination of number and weight of pups (decrease of the total number of live pups, mean number of pups per litter and mean weight of litters), pathological examination of pups (flatulence and changed colour of milk), calculation of pre-implantation losses (decreased number of corpora lutea and uterus implantations) and fertility parameters (decreased fertility and gestation index) in animals of the dose level 250 mg/kg/day revealed adverse effects attributable to test substance.

Examination of number and weight of pups (decrease of the total number of live pups, mean number of pups per litter and mean weight of litters), pathological examination of pups (flatulence and changed colour of milk), calculation of pre-implantation losses and fertility parameters (decreased fertility and gestation index) in animals of the dose level 630 mg/kg/day revealed adverse effects attributable to test substance.

Conclusion

The test substance administration had not adverse effect on mortality, parameters of functional observation and some reproduction parameters - course of mating and lactation, spermiogenesis, macroscopical and microscopical structure of reproductive organs and pituitary gland of parental animals and number of post-natal losses of mothers.

The test substance, Acid Brown 75, during Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test caused changes in clinical status (excrement and tail were coloured by the test substance), haematological parameters (primarily on the red blood cells - decreased value of total erythrocyte count, haematocrit and haemoglobin, increased value of mean corpuscular volume), biochemical parameters (primarily increased value of sodium ions, phosphorus, bilirubin, total protein and creatinine, decreased value of potassium ions), biometry of organs (changes weight of spleen, kidneys, heart and brain weight) and urine parameters (urine volume, colour, specific weight, occurrence of leucocytes, protein and ketones) at the highest dose level. 

The test substance had influence on macroscopical and microscopical structure of some organs and tissues (occurrence of pigment in kidneys, rectum, intestines and stomach, reversible increased occurrence of ulcer or erosion in large intestines and rectum, extramedular haemopoiesis and hemosiderin in spleen and liver, hyaline droplets in kidneys).

The test substance treatment affected the number of pups (decrease of the total number of live pups and mean weight of litters and affected pups, pre-implantation losses (decreased number of corpora lutea, uterus implantations and pups).

The highest incidence of statistically or biologically significant effects was recorded at the middle and highest dose level while most of changes which were found at the lowest dose level had only mild intensity without adverse alteration of animal organism.

The value of NOAEL (No Observed Adverse Effect Level) for REPEATED DOSE TOXICITY was established as 70 mg/kg body weight/day both for MALES and FEMALES. This value was established on the basis of haematology parameters (mainly - decreased value of total erythrocyte count, haematocrit and haemoglobin, increased value of mean corpuscular volume) and biochemistry findings (mainly - increased value of sodium ions, phosphorus, bilirubin, total protein and creatinine, decreased value of potassium ions) Histopathological evaluation revealed specific target organ toxicity effect on the spleen, kidneys and intestines.

The NOAEL (No Observed Adverse Effect Level) for REPRODUCTION for MALES and FEMALES was established as 70 mg/kg body weight/day. The value is based on the effect in females: decrease of the total number of live pups and mean weight of litters, decreased of pre-implantation losses and fertility parameters (decreased number of corpora lutea and uterus implantations). In males no effect on reproduction was observed. 

The NOAEL (No Observed Adverse Effect Level) for DEVELOPMENT was established as 70 mg/kg body weight/day. The values were established on the basis of decrease of the total number of live pups and mean weight of litters, decreased of pre-implantation losses and fertility parameters (decreased number of corpora lutea and uterus implantations). Other effects were recorded such as

changed colour of milk, flatulence, stomach without milk. These modulations though observed in pups are secondary effects displayed on offspring after exposure of pregnant mice to Acid Brown.