Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
one-generation reproductive toxicity
Remarks:
based on generations indicated in Effect levels (migrated information)
Type of information:
other: read across from analogue substance
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well documented, test procedure in accordance with OECD 221 methodes, meets generally accepted scientific principles, acceptable for assessment. GLP compliant with certificate. Justification for Read Across will be provided in section 13.
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
Age of animals: males, females – sexually adult (10 weeks – on arrival)
Selection of animal species: laboratory rat has been chosen because our testing laboratory has long experience with this species
Acclimatization: at least 5 days
Number of animals: 12 females and 12 males per group, 6 males and 6 females per satellite group
Housing conditions:
SPF – 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
- Food: complete pelleted diet for rats and mice in SPF breeding
- Water: drinking water ad libitum, quality standard ČSN 757 111
- Light cycle: 12 hour light / 12 hour dark
- Microclimate: 22 ± 3°C, relative humidity 30-70%
- Bedding: sterilized shavings of soft wood
Selection of animals: random selection according to the internal rule – at the beginning of the study the weight variation of animals in groups of each sex should not exceed ± 20% of the mean weight
Identification of animals: the animals will be identified by the colour marks on their fur, each cage will be marked with the number of animals, sex, number of cage, name and dose level of the test substance
Animal housing: The study will proceed in SPF conditions according to SOP No.12.

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.

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
The test substance was administered to the stomach by gavage as the solution in aqua pro injectione. Oral way of administration was chosen according to the guideline and it was approved by sponsor. The animals were treated 7 days per week at the same time (8.00 – 10.00 am).
The concentrations of solutions at all dose levels were adjusted to ensure the administration of 1 mL per 100 g of body weight. The vehicle control group was administered by aqua pro injectione in the same volume. The application form (test substance solution in aqua pro injectione) was prepared daily just before administration.
Details on mating procedure:
Animals were mated from the 15th day of study. Mating 1 : 1 (one male to one female) was used in this study. Each morning the females were examined for presence of spermatozoa in vaginal smears. Day 0 of pregnancy was defined as the day the sperms were found.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
Parental males:
1st day – 14th day (pre-mating) - 28th day (mating) - 42nd day of study

Parental females:
1st day – 14th day (pre-mating) - 28th day (mating) - gestation - lactation - day 4 post partum

Non -pregnant femals (without evidence of copulation)
1st day – 14th day (pre-mating) - 28th day (mating) - 54th day of study

Non -pregnant femals (with evidence of copulation)
1st day – 14th day (pre-mating) - 28th day (mating) - 25th day after confirmed mating (max. 54th day of srudy)
Frequency of treatment:
The animals were treated 7 days per week at the same time (8.00-10.00 am)
Remarks:
Doses / Concentrations:
20 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
80 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
320 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
Basic groups:
1. Control 0 12 males + 12 females
2. Low dose 20 mg/kg/day 12 males + 12 females
3. Intermediate dose 80 mg/kg/day 12 males + 12 females
4. High dose 320 mg/kg/day 12 males + 12 females

Satellite groups:
5. Control – vehicle – satellite: 0 6 males + 6 females
6. High dose – satellite: 320 mg/kg/day 6 males + 6 females
Control animals:
yes
Details on study design:
Study Time Schedule

Reproduction Toxicity Main Study:
Animal arrival: 14.03.2012
Acclimatisation: at least 5 days
Administration: 20.03-15.05.2012

Urinalysis: only males – 42nd and 56th day of study

Haematology and necropsies: parental males – 43th day of study
satellite males – 57th day of study
parental females – 4th day of lactation
satellite females – 57th day of study
non-pregnant females – 55th day of study or 26th day after confirmed mating

Examination of blood and necropsies: 30.04 – 16. 05. 2012 /satellite groups: 15.05 - 16.05 2012

End of histopathological examination: 31.10.2012
Final report elaboration: 31.12.2012

-- Preparation of Experimental Animals
During the acclimatisation period the health condition of all animals was controlled daily. Then the animals were randomly divided into the control and test groups and they were marked individually.

-- 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.

-- Experimental Data Collection

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 and 4th day;
pups (litters) – 0. or 1st and 4th day;
Food consumption: males - weekly (except the mating period)
females - weekly during premating period and after mating period
during pregnancy and lactation – on the same days as body weight

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 the administration / observation period

Laboratory examinations:
- vaginal smears: daily in mating period
- pathological examination: males and nonpregnant females - after the end of administration period
parental females and pups - on the 4th day of lactation
satellite male and females - at the end of observation period
- weight of organs: during necropsy
- sperm observation: all males after necropsy
- histopathological examination: all males and females after necropsy

Parental animals: Observations and examinations:
3.9.4 Mortality Control
All rats during the treatment periods were examined for vitality or mortality changes daily.


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


3.9.6 Clinical Observations of Males and Females
All rats were observed daily during the administration period.
This observation was made in order to record possible clinical effects after application and all changes in behaviour of animals. So it was done after application at the same time every day (11.00 a.m. - 13 p.m.) – at the time of expectation of maximal effect of the test substance. Animals were observed in natural conditions in their cages.


3.9.7 Clinical Observation of Pups
All pups were observed in natural conditions in their cages daily during the lactation. Changes in behavioural abnormalities were recorded. Detailed examination of each litter was performed as soon as possible after delivery (day 0 or 1 post-partum) and the 4th day of lactation. The number and sex of pups, stillbirths, live births and presence of gross anomalies were recorded.


3.9.8 Detailed Clinical Observation
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.


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

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

Urinalysis
The rats were kept in the metabolic cages for the collection of urine for two hours. Immediately before entering metabolic cages the animals were administered 2 mL of drinking water for 100 g of body weight by gavage to the stomach.
The parameters were determined by analyser PocketChem PU-4210 (Arkray, Inc., Japan).

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

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

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

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

Sperm 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.

Biometry of Organs
At the end of study the experimental animals were narcotised and sacrificed by cutting the neck spine and medulla. After the gross necropsy of the cranial, thoracic and abdominal cavities the organs for weighing and further histological examination were collected. The absolute weights of liver, kidneys, adrenals, testes or ovaries, epididymides or uterus, prostate gland, thymus, spleen, brain, pituitary gland and heart were recorded (repeated dose toxicity part of study – 6 males and females, from each group + satellite group);
testes or ovaries, epididymides or uterus, prostate gland, pituitary gland (reproduction part of study – all animals). Afterwards the somatic indexes - SI (= relative weight of organ) were computed according to the following formula:
SI = weight of organ x 100/ body weight.

Histopathological Examination
The following list contains all the examinated organs which were collected at necropsy and fixed:

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

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

The mentioned tissue and organs were collected from all killed males and females at necropsy and fixed in neutral 4% formaldehyde solution (v/v) for further histopathological evaluation. For histopathological processing the routine histopathological paraffin technique with haematoxylin-eosin staining was used.
Detailed histological examination was performed on testes (with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure). Spermatogenesis and spermatogenic cycle were evaluated according to the publication: 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). Pathological changes were evaluated according to the publication: Creasy, D.M.; Evaluation of Testicular Toxicity in Safety Evaluation Studies: The Appropriate Use of Spermatogenic Staging (Toxicologic Pathology 25, 119-131, 1997) and Guidance Document for Histologic Evaluation of Endocrine and Reproductive Tests in Rodents, ENV/JM/MONO(2009)11.
Oestrous cyclicity (parental animals):
Examination of reproductive system of parental females did not evidence any significant microscopical changes. Only changes pertinent to previous gravidity or proceeded oestrous cycle were recorded. Biometry of organs also proved no statistically significant and no dose dependent differences in treated females.
Sperm parameters (parental animals):
Sperm motility was quite the same in control males and treated males. Presence of “non-motile sperms” was not detected.
Slightly increased presence of morphologically changed sperms was detected in males of the lowest dose level.
Litter observations:
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 and mean number of pups per litter at the dose level 320 mg/kg/day was markedly decreased in comparison with the control. The total numbers of live pups and mean number of pups per litter at the dose levels 20 and 80 mg/kg/day were similar or higher than control. The number of stillborn pups was increased at the dose level 320 mg/kg/day.
In sex ratio no significant differences were recorded in treated groups.
Mean body weights of litters at the dose levels 20 and 80 mg/kg/day were increased compared to control. Mean weight of the litterat the dose level 320 mg/kg/day was markedly decreased against control. Mean weights of pup recorded at the 1st check of litter after parturition in treated and control groups were similar. Mean body weight increment of pup (from the 1st check of litter after parturition to the 4th day of lactation) was slightly decreased at the highest dose level.
Statistics:
ANOVA
Reproductive indices:
Calculated parameters Dose level
0 20 80 320
Male mating index 91.67 100.00 100.00 100.00
Female mating index 91.67 100.00 100.00 100.00
Male fertility index 91.67 75.00 83.33 75.00
Female fertility index 100.00 75.00 83.33 75.00
Gestation index 90.91 100.00 100.00 77.78
Survival index 100.00 98.43 98.56 100.00
Offspring viability indices:
Survival index
0 mg/kg: 100.00
20 mg/kg: 98.43
80 mg/kg: 98.56
320 mg/kg: 100.00
Clinical signs:
no effects observed
Description (incidence and severity):
no mortality at any dose
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
weight increments were lower in males at highest dose levels. Females: average body weight increment was decreased at the highest dose level in the 1st week Pregnant females food consumption at highest dose level decreased during lactation.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
weight increments were lower in males at highest dose levels. Females: average body weight increment was decreased at the highest dose level in the 1st week Pregnant females food consumption at highest dose level decreased during lactation.
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not specified
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
effects observed, treatment-related
Description (incidence and severity):
slower sperm motility at the highest dose level
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
increased pre and post implantation losses at the highest dose level
Dose descriptor:
NOAEL
Effect level:
ca. 80 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: REPRODUCTION
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
decrease of total number of live pups and pups per litter at the highest dose level
Body weight and weight changes:
no effects observed
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Histopathological findings:
not specified
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
ca. 80 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
other: DEVELOPMENT
Reproductive effects observed:
not specified
Conclusions:
The values of NOAEL for the reproduction and developmental was established to be 80 mg/kg bw/day
Executive summary:

Introduction

The test substance, similar substance 2, 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 20, 80, 320 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 (320 mg/kg/day). The dose levels for study were determined on the basis of results of a dose-range finding experiment (see the Annex 2).
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 3rdday of lactation, males  after mating period – totally for 42 days, non pregnant 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. 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, 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 similar substance 2 to rats by gavage at the dose levels 20, 80 and 320 mg/kg/day did not cause any mortality. No negative treatment-related effects were detected during functional observation of animals.

Body weight, food consumption, water consumption, clinical status of animals, urinalysis, weight of organs, macroscopical and microscopical structure of organs were not seriously affected by treatment of the test substance at the dose level 80 mg/kg/day. Slight decrease of food conversionin males, change of one haematological parameter –prolonged tromboplastin time in females (above historical control), statistically significant decrease of ALP activityin males were recorded at the dose level 80 mg/kg/day.

 

Food consumption, water consumption, clinical status of animals, relative weight of organs and macroscopical structure of organs were not seriously affected by treatment of the test substance at the dose level 80mg/kg/day. Decreased body weight increments at the end of application period in males, decreased food conversion in males, changes of some haematological parameters (increase of total leucocyte count – above historical control in females), differences in biochemical blood parameters (statistically significant decrease of urea concentration, decrease ALP and concentration of inorganic phosphorus in males, statistically increase of urea concentration in females), changes of absolute weight of liver (statistically significant decrease of absolute weight of liver in males and statistically significant increase of absolute weight of liver in females), increased occurrence of microscopical changes of brain, cerebellum, spleen and thyroid gland in males (deposits of pigment), of kidneys, spleen and thyroid glandin females (deposits of pigment) and of heart (sporadical regressive changes in myocardium) were detected at the dose level 80 mg/kg/day.

 

Growth of animals at the dose level 320mg/kg/day was influenced by the test substance treatment (decrease of body weight increments or fall of body weight, food consumption and food conversion). Clinical status of animals after application was also influenced by the test substance treatment (red-coloured bedding, test-substance coloured excrements, reversible change of colour of skin and irreversible change of colour of visible mucous membranes). Haematological examination (delayed significant increase of platelets count - above historical control and change of differential leucocyte count in males; reversible increase of total leucocyte count and delayed significant increase of PT value, delayed increase of APTT value in females), blood biochemical examination (reversible statistically significant and dose dependent decrease of ALP activity and urea concentration in males; delayed statistically significant increase of urea, albumin, inorganic phosphorus and potassium ions concentration in males; irreversible significant increase of urea concentration – above historical control and decrease of total protein concentration, reversible increase of AST activity – above historical control in females, delayed statistically significant increase of ALP activity and inorganic phosphorus in females), urinalysis (change of colour of urine in males), biometry of organs (irreversible increase of relative weight of testes, reversible statistically significant and dose dependent decrease of absolute and relative weight of liver, delayed statistically significant increase of absolute and relative weight of adrenal glands and relative weight of spleen, reversible decrease of absolute weight of heart in males; irreversible statistically significant and dose dependent increase of absolute and relative weight of liver, reversible and dose dependent decrease of absolute and relative weight of thymus), gross examination of organs and tissues (irreversible change of colour of mucous membranes, muscles, brain, spleen, thyroid gland, reversible change of colour of skin, subcutis and peritoneum in males; irreversible change of colour of mucous membranes, muscles, spleen, thyroid gland, reversible change of colour of skin, subcutis, brain, organs of thoracic and abdominal cavity and peritoneum, reducement of thymus in females) andhistological examination of organs and tissues (irreversible regressive changes in myocardium accompanied by reparative fibrosis and accumulation of pigmentophages in males and females, irreversible occurrence of pigment in brain, cerebellum, heart, spinal cord, kidneys, spleen and thyroid gland in males and females, focal atrophic or necrotic changes in skeletal muscle of females; reversible dystelectasis in lungs and activation of white pulp in spleen of males; reversible inflammation or necrosis of muscle fibres in larynx, atrophy of cortex in thymus in females) of animals at the dose level 320 mg/kg/day revealed significant changes attributable to the test substance administration.    

 

Reproduction part of study:

The course of mating, pregnancy and lactation of parental animals, number of females achieving pregnancy and bearing live pups, 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 20 and 80 mg/kg/day.

The course of mating, pregnancy and lactation of parental animals, number of females achieving pregnancy, absolute weight of reproductive organs and pituitary gland and relative weight of pituitary gland, spermiogenesis and sperm parameters, macroscopical and microscopical structure of reproductive organs and pituitary gland of parental animals, number of post-implantation and post-natal losses of mothers, sex ratio and development of pups were not adversely influenced by the test substance treatment at the dose level 320 mg/kg/day

Evaluation of relative weights of reproductive organs (increase of relative weight of testes and epididymis), examination of number of pups(decrease of the total number of live pups and mean number of pups per litter), calculation of reproduction parameters (decreased number of females bearing live pups, increased number of stillborn pups), calculation of pre-implantation losses (decreased number of corpora lutea and uterus implantations) and evaluation of pup weights (decrease of mean litter weight, decrease of mean pup weight on day 4 after parturition) in animals of the dose level 320 mg/kg/day revealed adverse effects attributable to test substance.

 

Conclusion

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

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

The heart appeared to be a target organ following repeated oral exposure of the test substance, at the highest dose level. Serious irreversible regressive lesions accompanied by accumulation of pigment were diagnosed in myocardium. In addition, similar changes were observed in muscle fibers of larynx and skeletal muscle. The above mentioned findings tended to be more serious in females than in males. The test had influence on macroscopical and microscopical structure of some other organs and tissues.

Irreversible accumulation of pigmentin brain, cerebellum, spinal cord, spleen, thyroid gland and kidneys was detected in animals of the middle and the highest dose level. Atrophic changes occurred in thymus of females at the highest dose level.

 

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

     

The value of NOAEL (No Observed Adverse Effect Level) for REPEATED DOSE TOXICITY was established as 80 mg/kgbody weight/day both for MALES and FEMALES.

The value of NOAEL (No Observed Adverse Effect Level) for the REPRODUCTION and for DEVELOPMENT OF PUPS was established as 80 mg/kg body weight/day.  

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
80 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Justification for selection of Effect on fertility via oral route:

The submitted study completely assess the endpoint and is compliant with the official guideline.

Effects on developmental toxicity

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
80 mg/kg bw/day
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Based on the results obtained from the developmental part of OECD 422, made on similar substance 2, no concern can be arised forn target substance. A more detailed esplanation is provided in the Read Across attachment, in section 13.

Justification for selection of Effect on developmental toxicity: via oral route:

Based on the results obtained from the developmental part of OECD 422, made on similar substance 2, no concern can be arised for target substance.

Toxicity to reproduction: other studies

Additional information

In the framework of REACH Regulation animal testing has to be carefully evaluated and a sub-chronic toxicity study (90 days) (Annex IX, section 8.6.2) shall be proposed by the registrant only if the frequency and duration of human exposure indicates that a longer term study is appropriate. Relevant human exposure can be excluded in accordance with Annex XI section 3 where testing in accordance with sections 8.6 and 8.7 of Annex VIII, Annex IX and Annex X (Repeated dose toxicity and reproductive toxicity) may be omitted, based on the exposure scenario(s) developed in the Chemical Safety Report.

In the attachment a full Chemical Safety Report completed with exposure scenarios has been presented, where only very basic Risk management measures have been recommended to ensure Risk control for the described exposure and where the exposure has been conservatively overestimated taking into account the fact that almost no production of the substance has been performed in Europe by registrants and the use is always fractioned in hundreds of sites with very diluted solutions.

Some general effects have been reported at the highest tested doses (320 mg/Kg bw). The real exposure to the substance is by potential inhalation through a very small % that can be transported physically in aqueous vapour during formulation and application or by occasional dermal contact with a diluted solution. No release of the substance as such is forecasted from the articles, where the substance is included in a polymeric matrix and chemically transformed for better grafting resistance. As a consequence no further study is proposed on the substance, neither for reproduction, nor for teratogenicity

Justification for classification or non-classification

No classification for toxicity to reproduction is warranted under Regulation 1272/2008, Based on minimal toxic effect on 320 mg/kg bw/day.