Reaction products of diazotised m-phenylendiamine, subsequently coupled with diazotised 4-aminobenzene-1-sulfonic acid, sodium salts

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From October 15,2012 to April 30,2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: SPF breeding, VELAZ
- Age at study initiation: 10 weeks on arrival
- Weight at study initiation: body weight - group mean ± standard deviation (g)

MALE
Group code 0 15 mg/kg bw 60 mg/kg bw 240 mg/kg bw
248.58 ± 10.33 246.23 ± 13.77 255.17 ± 11.91 243.32 ± 6.97
FEMALE
Group code 0 15 mg/kg bw 60 mg/kg bw 240 mg/kg bw
201.92 ± 6.38 200.73 ± 9.63 205.87 ± 5.15 199.53 ± 8.19
- Housing: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 fibers (Lignocel)
- Diet: complete pelleted diet for rats and mice in SPF breeding
- Water: drinking water ad libitum, quality corresponded to Regulation No. 252/2004 Czech Coll. of Law, Health Ministry
- Acclimation period:6 days
- Health chech: 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.

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

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Aqua pro injectione

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Concentration Level 50 mg/10mL
Ca. 0.5 g of the test substance was weighted with wider end of glass Pasteur pipette into a 150mL glass beaker calibrated to 100 mL and the beaker was replenished by the vehicle. The solution was stirred by magnetic stirrer (800 rpm) for 60 minutes.

Concentration Level 1000 mg/10 mL
Ca. 10 g of the test substance was weighted with wider end of glass Pasteur into a 150mL glass beaker calibrated to 100 mL and the beaker was replenished by the vehicle and disslolved in ultrasonic bath for a 15 min. This solution was stirred by magnetic stirrer (800 rpm) for 30 minutes.

Details on mating procedure:

Animals were mated from the 15th day of study. Mating 1 : 1 (one male to one female) was used in this study. Each morning the females were examined for presence of spermatozoa in vaginal smears. Day 0 of pregnancy was defined as the day the sperms were found.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability and the homogeneity were determined by means of measuring of a peak area of the test substance by high-performance liquid chromatography based on the method developed at the test facility.

The stability of the application form
The stability of the application form was checked by determination of a concentration of the test substance within 120 min (at the time 0 min, 30 min, 60 min and 120 min). The samples were taken from the middle of the beaker content after required time intervals.

The 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 solution (at the bottom, in the middle and at the surface).

Results of analysis
From the results of analyses (homogeneity and stability) follows that the solution of the test substance in vehicle in concentration level 50 mg/10 ml prepared at defined laboratory conditions (laboratory temperature, preparation of solution by defined manner) is homogenous and stable at least for 120 minutes starting with preparation of the application form.
From the results of analyses (homogeneity and stability) follows that the solution of the test substance in vehicle in concentration level 1000 mg/10 ml prepared at defined laboratory conditions (laboratory temperature, preparation of solution by defined manner) is homogenous and stable at least for 60 minutes from starting with analysis of the application form.

Duration of treatment / exposure:

Parental males:
1st day – 14th day (pre-mating) → 28th day (mating) → 42nd day of study
Satellite males:
1st day → 42nd day (administration) → 56th day (observation)
Parental females:
1st day – 14th day (pre-mating) → 28th day (mating) → gestation → lactation → day 4 post partum
Satellite females:
1st day → 42nd day (administration) → 56th day (observation)
Non-pregnant females (without evidence of copulation):
1st day – 14th day (pre-mating) → 28th day (mating) → 54th day of study
Non-pregnant females (with evidence of copulation):
1st day – 14th day (pre-mating) → 28th day (mating) → 25th day after confirmed mating (max. 54th day of study)

Frequency of treatment:

7 days per week at the same time (8.00 – 10.00 am). The vehicle control group was administered by aqua pro injectione in the same volume.

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: twice 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: the 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 (exc. satellite males)

Remarks:

Doses / Concentrations:
0, 15, 60 and 240 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

1. Control 0 mg/kg bw day 12 males + 12 females
2. Low dose 15 mg/kg/day 12 males + 12 females
3. Intermediate dose 60 mg/kg/day 12 males + 12 females
4. High dose 240 mg/kg/day 12 males + 12 females
Satellite groups:
5. Control – vehicle – satellite: 0 mg/kg bw day 6 males + 6 females
6. High dose – satellite: 240 mg/kg/day 6 males + 6 females

Control animals:

yes, concurrent vehicle

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
males and females - daily during the administration period
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.

DETAILED CLINICAL OBSERVATIONS: Yes
before the first application and then weekly (except the mating period)
Functional observation: at the end of administration/observation 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;
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 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:
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 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. (Numbers of females for repeated dose toxicity part of study were chosen at the end of study.)

WATER CONSUMPTION: Yes
satellite males and females – twice a week
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.

OTHER:
CLINICAL CHEMISTRY: Yes
at the end of administration/observation period
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: Yes
the last day of administration/observation period – only males
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 by analyser PocketChem PU-4210 (Arkray, Inc., Japan).
Volume
Colour
Cloud
Odour
Glucose
Protein
Bilirubin
Urobilinogen
pH
Specific gravity
Blood
Ketones
Nitrite
Leucocytes

Sperm parameters (parental animals):

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

Litter observations:

CAGE SIDE OBSERVATIONS: yes
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.


BODY WEIGHT: Yes
pups (litters) – 0. or 1st, 3rd and 4th day;

Postmortem examinations (parental animals):

GROSS PATHOLOGY: Yes
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
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: Yes
after necropsy
Samples of the following tissues and organs were collected at necropsy and fixed:
Repeated dose toxicity part of study (6 males and 6 females from each main group + 6 males and 6 females from satellite groups)
Adrenal glands
Aorta
Brain (incl. cerebellum and med. oblongata)
Caecum
Coagulating gland
Colon
Duodenum
Pancreas
Rectum
Salivary glands
Sciatic nerve
Seminal vesicles
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
Reproduction part of study (12 males and 12 females from each main group)
Pituitary gland
Ovaries
Uterus
Cervix of uterus
Vagina
Epididymis/Epididymides
Prostate gland
Seminal vesicles and coagulating gland
Testes
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.
Detailed histological examination was performed on testes of males from Reproduction Toxicity part of study – for all high dose and control animals (with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure).
Spermatogenesis and spermatogenic cycle were evaluated according to the following publications:

1. Hess, R.A.; Quantitative and qualitative Characteristics of the Stages and Transitions in the Cycle of the Rat Seminiferous Epithelium: Light Microscopic Observation of Perfusion-Fixed and Plastic-Embedded Testes (Biology of Reproduction 43, 525-542, 1990);
2. Creasy, D.M.; Evaluation of Testicular Toxicity in Safety Evaluation Studies: The Appropriate Use of Spermatogenic Staging (Toxicologic Pathology 25, 119-131, 1997);
3. Guidance Document for Histologic Evaluation of Endocrine and Reproductive Tests in Rodents, ENV/JM/MONO(2009)11;
4. Lonnie D. Russell et al.: Histological and histopathological evaluation of the testis, 1st ed., Published 1990 by Cache River Press in Clearwater.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at [#?] days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]

HISTOPATHOLOGY / ORGAN WEIGTHS
parental females and pups - on the 4th day of lactation

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 – number of live born pups, number of corpora lutea, number of implantations, mean weight of pup on the 0./1st day and mean weight of pup on the 4th day. 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

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

coloured faeces and orange urine

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

decrease of body weight in males and females (statistically significant in males and females in the 2nd week of application period and in females at the end of observation period. The test substance affected growth of parental females of the highest dose level in pregnancy: the mean body weight increment in the 3rd week of pregnancy was decreased. In lactation period the mean body weight increment was slightly increased against control but the body weight of mothers of the highest dose level remained decreased compared to control

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

decrease of food consumption at the highet dose

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

decrease of food conversion at the highest dose

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

reversible increased water consumption

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Haematological examination showed irreversible effect on one haemocoagulation parameter in both sexes: APTT value was increased at the highest dose level. Change of red blood component in females had delayed character – haemoglobin concentration, haematocrite and mean corpuscular volume were significantly increased at the highest dose level. White blood component was not significantly changed in parental treated animals.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Significant changes in clinical biochemistry were observed only at the highest dose level: increase of ALP activity in females was irreversible vice versa in males it was changed insignificantly and reversibly. Activity of ALT was significantly increased in males at the end of application period. Other changes of biochemical parameters had delayed character: significant increase of total bilirubin concentration and decrease of calcium and creatinine concentration in males and females. Changes in biochemical parameters of parental animals were not dependent on dose level and did not exceed the limits of historical control.

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:

effects observed, treatment-related

Description (incidence and severity):

Microscopical evaluation of the highest dose level elicited irreversible accumulation of the test substance (or its metabolites) in gastric and intestinal mucosa, mesenteric lymph nodes, in epithelial cells of renal cortical tubules and also in neurons of brain and spinal cord of males and females and in spleen, ovaries and uterus in females.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

spermiogenesis and sperm parameters were not affected by the test substance

Reproductive performance:

no effects observed

Description (incidence and severity):

The course of mating, pregnancy and lactation of parental animals, number of females achieving pregnancy, macroscopical and microscopical structure of reproductive organs in males and pituitary gland of parental males and females, number of post-natal losses of mothers and sex ratio and postnatal development of pups were not adversely affected by the test substance treatment.

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
There were no unscheduled deaths during the whole study.

MALE
At the lowest and the middle dose level no clinical changes after application of the test substance were recorded. The following clinical changes after application of the test substance were detected in all males of the highest dose level: test substance-coloured excrements and orange-brown colour of urine (changed colour of bedding: shavings) from the 1st week to the 6th week of application period.
FEMALE
There were no unscheduled deaths during the whole study. One female of the middle dose level was euthanasied before the scheduled time of necropsy by the reason of bad health status (on the 24th day of pregnancy).
At the lowest dose level no clinical changes after application of the test substance were recorded. In one female of the middle dose level piloerrection, disquietness and discharge
from vulva was observed in the 6th week. The following clinical changes after application of the test substance were detected in all females of the highest dose level: test substance-
coloured excrements and orange-brown colour of urine (changed colour of bedding: shavings) from the 1st week to the 6th week of application period.

Health Condition Control
MALE
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 of the lowest and the middle dose level no changes of health condition were found out before, during and immediately after application of the test substance. In males of the highest dose level the following changes were observed during the application period: orange-brown colour of urine (changed colour of bedding: shavings) and test substance-coloured excrements.
FEMALE
In control females and treated females of all dose levels no signs of diseases were recorded during the check-in and acclimatisation period.
In treated females of the lowest no changes of health condition were found out before, during and immediately after application of the test substance. In one female of the middle dose level piloerrection, disquietness and discharge from vulva was observed in the 6th week (for two days). In females of the highest dose level the following changes were observed during the application period: orange-brown colour of urine (changed colour of bedding: shavings) and test substance-coloured excrements.


BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
MALE
Statistically significantly decreased body weight was found in males of the highest dose level in the 2nd week of application.
The body weight of males of the lowest dose level was similar compared to control. In males of the middle and the highest dose level decreased body weight was recorded in comparison with control. This difference was dependent on dose level.
FEMALE
Pre-mating mating period
Statistically significantly decreased body weight was found in females of the highest dose level in the 2nd week of application.
The mean body weight increments of the control females and females at the lowest and the middle dose level were quite balanced whilst weight increments of females of the highest dose level slightly decreased in the pre-mating period.
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 all dose levels were similar to control mothers in the 1st and in the 2nd week of pregnancy. In the 3rd week of pregnancy body weight increment of pregnant females of the highest dose level was decreased.
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 and the middle dose level and control mothers were similar. The mean body weight increment of mothers at the highest dose level was slightly increased against control but the body weight of mothers of the highest dose level remained decreased compared to control.


FOOD CONSUMPTION:
MALE
The mean food consumption of males of the lowest and the middle dose level was analogous to control males. The mean food consumption of males of the highest dose level was slightly decreased in the 1st week, from the 2nd to the 6th week it was similar to control.
FEMALE
Pre-mating period
The mean food consumption of females treated by the lowest and the middle dose levels was balanced with control females in pre-mating period. The food consumption of females of the highest dose level was decreased.
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 lowest and the middle dose levels was similar to control. The mean food consumption of pregnant females of the highest dose level was decreased in the 3rd week of pregnancy.
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 similar to control mothers.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
Sperm motility was similar in control males and treated males. Presence of “non-motile sperms” was not recorded.
Examination of sperm morphology did not reveal marked differences between control and treated males.


REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
FEMALE
All females of control group and the lowest and the middle dose level were mated. Evidence of copulation was not found in one female of the highest dose level.
The number of females achieving pregnancy was analogous in treated and control groups. Abortions occurred at the middle and the highest dose level. The duration of mating of females at the lowest and the middle dose level was similar to the control group. The duration of mating of females at the highest dose level was longer against control. The duration of pregnancy of females at all dose level was comparable to the control.
The number of females bearing live pups was well-balanced in the control and the lowest dose level. In females of the middle and the highest dose level this number was apparently decreased compared to control.
Mean number of corpora lutea was decreased at the highest dose level. Vice versa mean number of implantations was decreased at the middle dose level. Mean number of live pups at birth and at day 4 after parturition in treated mothers was similar to control.
Mean weight of the litter at the highest dose level was markedly decreased against control. Mean weights of pup recorded at the 1st check of litter after parturition and on 4th day of lactation was slightly decreased at the highest dose level.
Macroscopical findings were recorded in stomach of pups of treated mothers: sporadically at the lowest and the middle dose level and in more than 25% of pups at the highest dose level.
No significant differences of mating indexes were observed. Fertility indexes were decreased at the middle and the highest dose level. At the middle and the highest dose level gestation index was markedly decreased. Survival indexes of treated groups were analogous to control.
Pre-implantation losses were slightly increased at the middle dose level and post-implantation losses were increased at the middle and the highest dose level. No significant differences were detected in post-natal losses.

ORGAN WEIGHTS (PARENTAL ANIMALS)
MALE
The statistical analysis of the data revealed no significant intergroup differences in absolute and relative weight of reproductive organs and pituitary gland.
Absolute and relative weight of prostate gland was slightly decreased in males of the highest dose level. Relative weight of testes was increased in treated males in a dose dependent manner.

FEMALE
The statistical analysis of the data did not reveal significant intergroup differences in absolute and relative weights of ovaries, uterus and pituitary gland.
Nonpregnant females and females with abortion were not used for calculation of means and evaluation of biometry results.
Slight increase of absolute and relative weight of ovaries at the middle dose level and of relative weight at the highest dose level was described.
Absolute and relative weights of examined organs were conformable to the control group.

GROSS PATHOLOGY (PARENTAL ANIMALS)
MALE
The incidence of affected males is expressed in numeric form and ranged in sequence of
the dose levels 0-15-60-240 mg/kg/day further in the text.
No macroscopical findings were recorded in 11-11-11-0 males. In 11-12-12-12 males no macroscopical changes were observed in testes, epididymides, seminal vesicles, coagulation glands and pituitary gland.
Change of colour of organs and tissues (or contents of organs) were observed in males of the highest dose level: dark colour subcutis and skeletal muscle in 0-0-0-2 males, brown colour of stomach mucosa membrane and chyme in 0-0-0-11 males, brown colour of stomach chyme in 0-0-1-1 males. Other macroscopical changes were described sporadically.

FEMALE
The incidence of affected females is expressed in numeric form and ranged in sequence of the dose levels of 0-15-60-240 mg/kg/day further in the text.
No macroscopical findings were recorded in 12-12-6-1 females. Change of colour of organs and tissues (or contents of organs) were observed in females of the middle and the highest dose level: dark or brown colour of stomach mucous membrane and chyme in 0-0-0-4 females, dark or brown colour of stomach chyme in 0-0-3-7 females, brown colour of mammary gland in 0-0-2-4 females, yellow or brown colour of fat tissue in 0-0-2-5 females, yellow or brown colour of subcutis in 0-0-2-11 females, yellow or brown colour of skeletal muscle in 0-0-0-11 females, brown colour of heart in 0-0-0-2 females, dark red or brown-red colour of spleen in 0-0-0-10 females, dark brown colour of liver and brown-red colour of kidneys in 0-0-0-3 females. Enlargement of spleen was observed in 0-0-0-4 females. Discharge from vagina was found out in 0-0-2-0 females and late resorptions or dead foetuses was detected in uterus of 0-0-3-1 females.


HISTOPATHOLOGY (PARENTAL ANIMALS)
MALE
Microscopic examination of testes, epididymis, seminal vesicles, coagulation glands and pituitary gland did not reveal presence of treatment related changes.

FEMALE
The incidence of affected females is expressed in numeric form and ranged in sequence of the dose levels 0-15-60-240 mg/kg/day further in the text. In 4-1-1-0 females no histological changes were detected. During microscopical examination of reproductive organs the following possible treatment related changes were registered. Presence of brown pigment was found out in ovaries of 0-0-0-12 females and in uterus of 0-1-3-11females. Inflammation was diagnosed in uterus of 0-0-4-1 females. In uterus the changes related to previous pregnancy were found: accumulation of siderophages in mesometrium or/and in endometrium in 8-9-4-5 females and haemorrhages or haematoma in mesometrium in 2-2-1-2 females.
Hydrometra - the change associated with oestrous cycle was registered in uterus of 0-0-0-2 females.
Other microscopical changes observed in reproductive organs occurred only sporadically or they did not relate to test substance treatment

Dose descriptor:

NOAEL

Effect level:

ca. 240 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive function (oestrous cycle)

reproductive function (sperm measures)

reproductive performance

Dose descriptor:

NOAEL

Effect level:

ca. 60 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

haematology

clinical biochemistry

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Description (incidence and severity):

sex ratio was not influenced

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Macroscopical findings were observed at pups of treated mothers: yellow colour of milk, empty stomach or flatulence of stomach ath the highest dose level

Histopathological findings:

not examined

VIABILITY (OFFSPRING)
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 the middle and the highest dose levels was markedly decreased in comparison with the control. The total numbers of live pups per litter at the lowest dose level was slightly higher than control. Mean number of pups per litter in treated mothers was similar to control.
In sex ratio no significant differences were recorded in treated groups.


CLINICAL SIGNS (OFFSPRING)
no data

BODY WEIGHT (OFFSPRING)
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 and the middle dose levels were slightly increased compared to control. Mean weight of the litter at the highest dose level was markedly decreased against control. Mean weights of pup recorded at the 1st check of litter after parturition at lowest and the middle dose levels and control were similar. Mean weights of pup recorded at the 1st check of litter after parturition at the highest dose level was slightly decreased. Mean body weight of pup on the 4th day of lactation was also slightly decreased at the highest dose level.

GROSS PATHOLOGY (OFFSPRING)
macroscopic examination was performed in all pups. Macroscopical findings were observed at pups of treated mothers: yellow colour of milk, empty stomach or flatulence of stomach.

OTHER FINDINGS (OFFSPRING)
Development of Pups
Prenatal mortality was increased at the middle and the highest dose level.
Number of stillborn pups was negligible at control group and at the lowest and the highest dose levels. Number of stillborn pups at the middle dose level was not exactly determined by the reason of total cannibalism of pups in one female.
Mortality of pups in lactation period was sporadically detected only at the middle and the highest dose level.
No differences in postnatal development of pups were observed at the control and treated groups.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

ca. 60 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

gross pathology

Reproductive effects observed:

not specified

Reproduction data
Observed parameters	Dose level
	0	15	60	240
Pairs started (N)	12	12	12	12
Females showing evidence of copulation (N)	12	12	12	11
Females achieving pregnancy (N)	11	11	10	10
Females with abortion (N)	0	0	3	2
Conceiving days (duration of mating) 1 – 5 (N)	12	12	12	10
Conceiving days (duration of mating) 6 – 10 (N)	0	0	0	1
Mean duration of mating (days)	1.25	1.67	1.83	3.27
Pregnancy ≤ 21 days (N)	3	1	2	5
Pregnancy = 22 days (N)	4	7	4	3
Pregnancy ≥ 23 days (N)	4	3	0	0
Mean duration of pregnancy (days)	22.09	22.18	21.33	21.13
Females with live pups born (N)	11	11	6^C	8
Females with live pups at day 4 after parturition (N)	11	11	6	8
Number of corpora lutea (mean)*	14.36	15.64	14.50	13.00
Number of implantations (mean)*	12.27	14.18	10.60	11.50
Number of live born pups - at birth (mean/mother)	11.45	13.18	12.67	11.38
Number of live pups - at day 4 after parturition (mean/mother)	11.45	13.18	12.33	11.25
Number of stillborn pups (total number/mean)	0/0.00	0/0.00	1^C/0.17	0/0.00
Sex ratio (M/F) at birth (mean)**	5.73/5.73	6.18/7.00	6.00/6.83	5.63/5.75
Sex ratio (M/F) at day 4 after parturition (mean)	5.73/5.73	6.18/7.00	5.83/6.50	5.63/5.63
Mean litter weight at birth (g)	75.19	84.47	82.30	64.61
Mean litter weight at day 4 after parturition (g)	115.28	124.99	121.70	100.19
Mean pup weight at birth (g)	6.68	6.46	6.48	5.90
Mean pup weight at day 4 after parturition (g)	10.44	9.56	9.96	9.23
ABNORMAL PUPS
Mothers with 0 (N)	11	7	4	0
Mothers with 1 (N)	0	3	0	1
Mothers with ≥ 2 (N)	0	1	2	7

Note:(N)–number of animals, * - calculated in pregnant females, ** - including stillborn pups,^C-

female No. 143 – cannibalism of all pups (number of stillborn pups was not recorded)

Fertility parameters
Calculated parameters	Dose level
	0	15	60	240
Male mating index	100.00	100.00	100.00	91.67
Female mating index	100.00	100.00	100.00	91.67
Male fertility index	91.67	91.67	83.33	83.33
Female fertility index	91.67	91.67	83.33	90.91
Gestation index	100.00	100.00	60.00	80.00
Survival index	100.00	100.00	97.32	98.86
LOSS OF OFFSPRING
Pre-implantation (corpora lutea minus implants) - mean	2.09	1.45	3.90	1.50
Pregnant females with 0 (N)	4	4	3	2
Pregnant females with 1 (N)	1	4	0	5
Pregnant females with 2 (N)	1	0	1	0
Pregnant females with ≥ 3 (N)	5	3	6	3
Post-implantation (implants minus live births) - mean	0.82	1.00	3.00	2.40
Pregnant females with 0 (N)	5	5	3	2
Pregnant females with 1 (N)	3	3	4	4
Pregnant females with 2 (N)	3	2	1	1
Pregnant females with ≥ 3 (N)	0	1	2	3
Post-natal (live births minus alive at day 4) - mean	0.00	0.00	0.33	0.13
Mothers with 0 (N)	11	11	5	7
Mothers with 1 (N)	0	0	0	1
Mothers with 2 (N)	0	0	1	0
Mothers with ≥ 3 (N)	0	0	0	0

Effects on reproduction at the dose level 240 mg/kg/day:

-         moderate increase of relative weight of testes in males (dose dependent)

       -    slight increase of relative weight of ovaries (without dose dependence)

-         decrease of body weight increments of females in pregnancy period

-         increase occurrence of abortions (increase of prenatal mortality)

-         decrease of number of females bearing live pups

-         decrease of mean number of corpora lutea

-         decrease of mean weight of litter

-         increase of post-implantation losses

-        increased occurrence of macroscopical findings in pups: change of colour of milk

-        increased occurrence of pathological findings in females: dead foetuses andinflammation in uterus[fd2] [RV3] – in aborted females

 

Effects on reproduction at the dose level 60 mg/kg/day:

-         euthanasia of one female before the scheduled time of necropsy by the reason of bad health status (abortion)

      -     slight increase of absolute and relative weight of ovaries (without dose dependence)

-         increase occurrence of abortions (with not clear dose/effect dependence: the effect diminishes at the highest dose)

-         decrease of number of females bearing live pups (consequence of decreasing number of mothers)

-        sporadical occurrence of macroscopical findings in pups: change of colour of milk

 

Effects on reproduction at the dose level 15 mg/kg/day:

- minimal occurence of macroscopiacal findings in pups: change of colours in milk

Conclusions:

The NOAEL (No Observed Adverse Effect Level) for REPRODUCTION and DEVELOPMENT was proposed as 15 mg/kg body weight/day. The value was set as a conservative value, determined on the basis of decreased number of females bearing live pups, increased prenatal mortality (increased number of females with abortions, but dose/response relationship is not clear) and decreased mean litter weight. Male ability to produce sperm that can fertilise eggs and female ability to be impregnated were not significantly changed. Therefore the value of 60 mg/Kg body weght/day has been used for risk assessment purposes

Executive summary:

The test substance, Direct Brown 44, 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 15, 60, 240 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 (240 mg/kg/day). The dose levels for study were determined on the basis of results of a dose-range finding experiment.

 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.

    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. 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

    Repeated oral administration of Direct Brown 44 to rats by gavage at the dose levels 15, 60 and 240 mg/kg/day did not cause any mortality. One female of the dose level 60 mg/kg/day was euthanasied before the scheduled time of necropsy by the reason of bad health status (abortion on the 24th day of pregnancy)

The course of mating, pregnancy and lactation of parental animals, number of females achieving pregnancy, absolute weight and relative weight of pituitary gland, spermiogenesis and sperm parameters, macroscopical structure of reproductive organs and pituitary gland of parental animals, number of post-natal losses of mothers, sex ratio and early postnatal development of pups were not adversely influenced by the test substance treatment.

    Evaluation of body weight of parental animals (decrease of body weight increments of females in pregnancy – at the dose level 240 mg/kg/day) and relative weights of reproductive organs (dose dependent increase of relative weight of testes – at the dose level 240 mg/kg/day), examination of number of live pups (decrease of the total number of live pups – at the dose levels 240 mg/kg/day), calculation of reproduction parameters (increased number of females with abortion and decreased number of females bearing live pups - at the dose levels 240 mg/kg/day), calculation of prenatal losses (increase of pre-implantation loss at the dose level 240 mg/kg/day), evaluation of pup weights (decrease of mean litter weigh - at the dose level 240 mg/kg/day), macroscopical examination of pups (change of colour of milk – at the dose level 240 mg/kg/day, sporadically at the dose levels 15 and 60 mg/kg/day), microscopical examination of reproductive organs of parental females (deposits of pigment in ovaries and uterus – at the dose level 240 mg/kg/day and inflammation in uterus in females with abortion – at the dose levels 60 and 240 mg/kg/day) revealed effects attributable to test substance.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

60 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

Effects on developmental toxicity

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

60 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

Reproductive toxicity includes adverse effects on sexual function and fertility in sexually adult males and females animals, as well as developmental toxicity in the offspring. However, developmental toxicity essentially means all the adverse effects induced during pregnancy that can be manifested at any point of the life span of the animal, which might in turn bring to structural abnormality, altered growth and/or organs development, functional deficiency, even death.

Table 3.7.1(a) of Annex I of EC Regulation 1272/2008 states that to classify compounds "for category 2 suspected human reproductive toxicant, reproductive effects shall have been observed in the absence of other toxic effects, or if occurring together with other toxic effects the adverse effect on reproduction is considered not to be a secondary non-specific consequence of the other toxic effects". To this extent the screening study OECD422 performed on the target substance does not provide any indication of direct adverse effect on reproduction up to the highest tested dose. In fact no reproduction performance or function is impared by the substance treatment while adverse effects are mainly due to general toxicity of the animals.

The pregnancy is greatly influenced by the mother intoxication which adversly affected the number of abortions, gestation indexes and weights of litters at 1st and 4th day PND. However no malformations were recorded for the skeletal part while for the gross pathology content of orange milk was detected in the stomach of offsprings of the highets tested dose mothers. Moreover, a test equivalent to OECD 414 performed on analogue substance 4 did no show any adverse effect up to highest tested dose.

Finally, considering the available information, the substance is not classified for toxicity to reproduction and development.