Reaction products of diazotised 4,4-diaminodiphenylamine-2-sulfonic acid, subsequently coupled with 6-amino-4-hydroxynaphthalene-2-sulfonic acid, further diazotised and coupled with metaphenylendiamine, sodium salts

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

other: experimental result on similar substance

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

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 or test system and environmental conditions:

TEST ANIMALS
- Animal arrival: 14.03.2012
- Selection: 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
- Age of animals: males, females - sexually adult; 10 weeks on arrival
- Number of animals: 12 females and 12 males per group, 6 males and 6 females per satellite group
- Housing conditions: the study will proceed in SPF conditions according to SOP No.12.
pre-mating period 2 rats of the same sex in one cage
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 shavings of soft wood
- Food: complete pelleted diet for rats and mice in SPF breeding
- Water: drinking water ad libitum, quality standard ČSN 757 111
- Acclimatization: at least 5 days. During the acclimatisation period the health condition of all animals was controlled daily.

ENVIRONMENTAL CONDITIONS
- Light cycle: 12 hour light / 12 hour dark
- Temperature: 22 ± 3°C
- Humidity: relative humidity 30-70%

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

ADMINISTRATION
The test substance was administered to the stomach by gavage as the solution in aqua pro injection.
Oral way of administration was chosen according to the guideline and it was approved by sponsor.

PREPARATION OF DOSING SOLUTIONS
The concentrations of solutions at all dose levels were adjusted to ensure the administration of 1 mL per 100 g of body weight.
The application form (test substance solution in aqua pro injection) was prepared daily just before administration.

Details on mating procedure:

- Star period: animals were mated from the 15th day of study
- M/F ratio per cage: during mating period one male and one female in one cage
- Observations: each morning the females were examined for presence of spermatozoa in vaginal smears.
- Proof of pregnancy: sperm in vaginal smear; 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

Frequency of treatment:

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

Details on study schedule:

GENERAL STUDY TIME SCHEDULE

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

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:

12 males + 12 females for all tested and control group.
6 males + 6 females in satellite groups

Control animals:

yes

Details on study design:

ANIMAL
- Selection of test animals: animals were randomly divided into the control and test groups and they were marked individually.
- Identification: 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

DOSE SELECTION
1) Dose Range findings
The dose-range finding experiment with 14-day application period was performed with 4 groups of treated animals without control group.
The dose levels for the dose-range finding experiment have been chosen according to the request of sponsor and with respect to the information in Material Safety Data Sheet obtained from sponsor (LD50, oral rat - 4900 mg/kg).
Appropriate dose levels of the test substance for the 14-day dose-range finding experiment was chosen as follows:
Dose levels: 125, 250, 500 and 1000 mg/kg/day
In the dose-range finding experiment with test substance two females and one male died at the dose level 1000 mg/kg/day during the 1st week of application and two males and one female died at the dose level 500 mg/kg/day during the 2nd week of application. Four males and three females at the dose level 1000 mg/kg/day were humanely killed at the end of the first week of application and three males and four females were killed at the dose level 500 mg/kg/day during the 2nd week of application by the reason of bad health condition and prevention suffering of animals.
Marked fall of weight was recorded in males and females of the dose level 1000 mg/kg/day. The body weights and body weight increments of animals at the dose levels 500 mg/kg/day were decreased.
Clinical observation detected the impact of the test substance on the health condition of animals at the dose levels 500 and 1000 mg/kg/day.
Results of haematology examination showed that the test substance had influence on red and white blood component in both sexes mainly at the dose level 500 mg/kg/day.
Pathological examination revealed change of colour of skin, subcutis, muscle and peritoneum at the dose level 500 and 1000 mg/kg/day. Other macroscopic changes were found mainly in digestive system, in lungs and in spleen of animals at the dose levels 500 and1000 mg/kg/day.
On the basis of the results obtained the following dose levels 20, 80 and 320 mg/kg/day were chosen for the main Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test.

Parental animals: Observations and examinations:

MORTALITY: Yes
All rats during the treatment periods were examined for vitality or mortality changes daily.

HEALTH CONDITIONS: Yes
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.

CAGE SIDE OBSERVATIONS: Yes
Animals were observed in natural conditions in their cages.
Observation during the removal from cage: reaction to handling, elasticity of skin, colour of mucous membranes, salivation, lacrimation, cleanliness of fur around foramina.

DETAILED CLINICAL OBSERVATIONS: Yes
All rats were observed daily during the administration period. The 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. 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.

BODY WEIGHT: Yes
Males weekly and females weekly in premating and mating period; during pregnancy at 0, 7th, 14th, 20th day; during lactation at 0 or 1st and 4th day.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
Males weekly (except the mating period) and females weekly during premating period and after mating period; during pregnancy and lactation on the same days as body weight.

FUNCTIONAL OBSERVATION: Yes
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.

OTHER:

LABORATORY EXAMINATION: examination of Vaginal Smears, daily in mating period.
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.

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.

URINALYSIS: only males 42nd and 56th day of study. 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.
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

Sperm parameters (parental animals):

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 MORTALITY
Sperm samples were taken from one epididymis and sperm motility was assessed from these samples. The motility of sperm was determined by microscopic examination of the prepared sperm suspension. The result of observation was evaluated subjectively according to following grades: 1 - fast progressive motility, 2 - slow progressive motility, 3 - no progressive motility, 4 - non-motile sperm.

SPERM MORPHOLOGY
Sperm samples were taken from one epididymis and sperm morphology was assessed from these samples. A smear from the sperm suspension was prepared and stained (Giemsa staining). The morphology of sperm was determined by microscopic examination.
All deviations – e.g. broken tail, abnormal form of tail, double head, amorphous head, abnormal form of neck - were recorded.

Postmortem examinations (parental animals):

SACRIFICE: at the end of study the experimental animals were narcotised and sacrificed by cutting the neck spine and medulla.

GROSS NECROPSY: after the gross necropsy of the cranial, thoracic and abdominal cavities the organs for weighing and further histological examination were collected.

ORGAN WEIGHTS: 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).

HISTOPATHOLOGICAL EXAMINATION: tissue and organs reported below, 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.
The following list contains all the examined 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, para-aortal
Oesophagus
All gross lesions

Statistics:

ANOVA

Reproductive indices:

Calculated parameters Dose level
0 50 150 450
Mating index 91.67 100 100 100
Fertility index 81.82 75.00 83.33 83.33
Conception index 75.00 75.00 83.33 83.33
Gestation index 100 100 100 80
Percentage of postnatal loss 0 0 0 0

Offspring viability indices:

100 at all dose levels

Clinical signs:

no effects observed

Description (incidence and severity):

No mortality at any dose

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Not evidence any significant microscopical changes

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

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No mortality at any dose

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
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.

REPRODUCTIVE FUNCTION: OESTROUS CYCLE (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.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
The test substance had effect on sperm quality of treated males. Increased number of males with slower sperm motility was recorded at treated groups and especially at the highest dose level. Percentage portion of morphologically changed sperms was slightly increased at all treated groups comparing with control group without dependence on dose level.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Increased pre and post implantation losses at the highest dose level

LITTER OBSERVATIONS (PARENTAL ANIMALS)
Litter size was decreased at the highest 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. But the average weight of pups and the development of pups were not influenced by the test substance administration.

Dose descriptor:

NOAEL

Effect level:

ca. 80 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

mortality

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:

act. ingr.

Sex:

not specified

Basis for effect level:

mortality

Reproductive effects observed:

not specified

FURTHER DETAILS

All females (at all groups) were paired so the number of females paired was identical at all groups. 

Number of females achieving pregnancy and gestation index were decreased at the highest dose level compared to the control, because number of females with live pups born were decreased at this group. Duration of mating and pregnancy of the treated groups was similar to the control group. Average number of corpora lutea and implantation were decreased only at the highest dose level (there was lower number of pups) compared to the control.

Pre-implantation losses were increased at the highest dose level. Post-implantation losses and post-natal losses at all dose levels were well-balanced with the control group.

Table 42

Reproduction data
Observed parameters	Dose level
	0	50	150	450
Pairs started (N)	12	12	12	12
Females showing evidence of copulation (N)	11*	12	12	12
Females achieving pregnancy (N)	9	9	10	10
Females with abortion (N)	0	0	0	2
Nonpregnant females	3	3	2	2
Conceiving days (duration of mating) 1 – 5 (N)	11	12	12	12
Conceiving days (duration of mating) 6 – 13 (N)	0	0	0	0
Pregnancy = 21 days (N)	2	1	2	0
Pregnancy = 22 days (N)	6	6	8	8
Pregnancy = 23 days (N)	1	2	0	0
Females with live pups born (N)	9	9	10	8
Females with live pups at day 4 after parturition (N)	9	9	10	8
Corpora lutea/pregnant female (average)	13.78	13.44	14.90	12.20
Implantations/pregnant female (average)	12.00	11.33	13.20	9.10
Live pups/mother at birth (average)	11.44	10.22	12.60	8.88
Live pups/mother at day 4 after parturition (average)	11.44	10.22	12.60	8.88
Sex ratio (M/F) at birth (average)	5.11/6.33	5.11/5.11	5.80/6.80	4.63/4.25
Sex ratio (M/F) at day 4 after parturition (average)	5.11/6.33	5.11/5.11	5.80/6.80	4.63/4.25
Litter weight at birth (average)	70.39	66.73	75.34	55.83
Litter weight at day 4 after parturition (average)	104.74	95.66	105.41	77.95
Pup weight at birth (average)	6.27	6.90	6.10	6.41
Pup weight at day 4 after parturition (average)	9.59	10.09	8.67	9.05

Notes:(N) –Number of animals

*– Non-paired female (without presence of sperm) - not used for calculation of duration of mating.

 

Table 43

Fertility parameters
Calculated parameters	Dose level
	0	50	150	450
Mating index	91.67	100	100	100
Fertility index	81.82	75.00	83.33	83.33
Conception index	75.00	75.00	83.33	83.33
Gestation index	100	100	100	80
Percentage of postnatal loss	0	0	0	0
Viability index	100	100	100	100
LOSS OF OFFSPRING
Pre-implantation (corpora lutea minus implants)
Pregnant females with 0 (N)	4	4	5	1
Pregnant females with 1 (N)	1	1	1	2
Pregnant females with 2 (N)	1	1	2	1
Pregnant females with ≥ 3 (N)	3	3	2	6
Pre-natal/post-implantations (implants minus live births)
Pregnant females with 0 (N)	6	3	4	3
Pregnant females with 1 (N)	1	4	6	1
Pregnant females with 2 (N)	2	0	0	2
Pregnant females with ≥ 3 (N)	0	2	0	4
Post-natal (live births minus alive at post-natal day 4)
Pregnant females with 0 (N)	9	9	10	10
Pregnant females with 1 (N)	0	0	0	0
Pregnant females with 2 (N)	0	0	0	0
Pregnant females with ≥ 3 (N)	0	0	0	0

Conclusions:

The values of NOAEL for the reproduction and for developmental of pups was established to be 80 mg/kg bw/day

Executive summary:

The test substance was tested for reproduction and subacute toxicity using the OECD Test Guideline No. 422: Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, Adopted by the Council on March 22^nd1996.

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 fibres of larynx and skeletal muscle. This 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 pigment in 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 fertilize eggs and female ability to impregnate was not significantly changed.

     

The value of NOAEL for REPEATED DOSE TOXICITY was established as 80 mg/kg body weight/day both for males and females.

The value of NOAEL for the REPRODUCTIVE and DEVELOPMENTALTOXICITY was established as 80 mg/kg body weight/day

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

other: experimental result on similar substance

Adequacy of study:

supporting study

Study period:

From April 25 to July 14, 2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Reason / purpose for cross-reference:

reference to other study

Remarks:

validated analytical method

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The rat is regarded as suitable species for reproduction studies and the test guideline is designed to use the rat. The Wistar rat was selected due to large experience with this strain of rat in reproduction toxicity studies and known fertility.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Toxi-Coop Zrt. 1103 Budapest, Cserkesz u. 90. Hungary
- Hygienic level: SPF (Specific pathogen-free) at arrival and kept in good conventional environment during the study.
- Females: nulliparous, non-pregnant females.
- Age at study initiation: male animals 90 – 97 days; female animals 90 – 97 days.
- Weight at study initiation: male animals 359 – 438 g; female animals 200 – 248 g. The weight variation did not exceed ± 20 per cent of the mean weight.
- Housing: before mating 2 animals of the same sex/cage; during mating 1 male and 1 female per cage; pregnant females individually housed; males after mating 2 animals per cage. Cage type was type III polypropylene/polycarbonate (22 x 32 x 19 cm).
- Diet: animals received ssniff® SM R/M-Z+H complete diet for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany, ad libitum. Food was changed at weekly intervals. The food was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. The supplier provided an analytical certificate of the standard diet for the batch used.
- Water: animals received tap water from watering bottles, as for human consumption, ad libitum. Fresh drinking water was given daily. Water quality control analysis and microbiological assessment are performed once in every six months by Government Office of Capital Budapest Department of Public Health and Medical Officer Service (Váci út 172-174. Budapest, H-1138 Hungary).
- Acclimation period: 27 days.
- Animal health: only healthy animals were used for the study. Healthy status was certified by the breeder.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30 - 70 %
- Air changes: above 10 air-exchanges/ hour by a central air-condition system.
- Photoperiod: artificial light, from 6 a.m. to 6 p.m.

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

TREATMENT VOLUME
A constant treatment volume of 5 ml/kg body weight was administered in all groups. The individual volume of the treatment was based on the most recent individual body weight of the animals. In the first week of the pre-mating period, animals received volumes based on the actual body weight on Day 0.

FORMULATION
Test item was formulated in the vehicle (distilled water) in concentrations of 8, 24 and 70 mg/ml (corresponding to an active substance content of ca. 7.4, 22 and 64 mg/ml). Formulations were prepared in the formulation laboratory of the Test Facility beforehand not longer than for three days and stored at 5 ± 3 °C before the administration.

VEHICLE
The suitability of the chosen vehicle for the test item at the intended concentrations was analytically verified up front. Recovery of test item from the vehicle was within the acceptance criteria (relative to nominal concentrations: 98 % at ca. 1 mg/ml and 90 % at ca. 200 mg/ml).
A sufficient stability and homogeneity in the chosen vehicle was verified over the range of relevant concentrations at the appropriate frequency of preparation in a separate analytical study.Test item proved to be stable in the vehicle in a refrigerator (at 5 ± 3 °C) for three days.

Details on mating procedure:

Mating begun 2 weeks after the initiation of treatment with one female and one male of the same dose group (1:1 mating) placed in a single cage. Females remained with the same male until copulation occurred or 14 days have elapsed. For one female animal of the 120 mg/kg bw/day group (no. 332), male pair was replaced by a proven male (no. 304) until copulation occurred.
Vaginal smears were examined for the presence of vaginal plug or sperm. Presence of vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (day 0 of pregnancy as defined by OECD 422). Sperm positive females were caged individually. Mating pairs were clearly identified in the raw data.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical control of dosing formulations (control of concentration) was performed in the analytical laboratory of test facility twice during the study. Five aliquots of 5 ml of each formulation (8, 24 and 70 mg/ml) and five aliquots of control substance (vehicle) were taken and analyzed. The samples were stored at 5 ± 3 °C before the analysis.
Concentration of the test item in the dosing formulations varied between the range of 97 % and 103 % in comparison to the nominal values.

Duration of treatment / exposure:

Males: 42 days
Females: 51 – 65 days

Frequency of treatment:

Daily, 7 days/week

Details on study schedule:

- Acclimatization: 27 days, including 14 days for examination of estrous cycle.
- Mating phase: started after 14-days treatment (pre-mating) period.
- Day of birth (viz. when parturition is complete): defined as day 0 post-partum.

Dose / conc.:

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

Remarks:

Refer to formulation

Dose / conc.:

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

Remarks:

Refer to formulation

Dose / conc.:

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

Remarks:

Refer to formulation

No. of animals per sex per dose:

12 animals/sex in the control and dose groups

Control animals:

yes, concurrent vehicle

Parental animals: Observations and examinations:

MORTALITY
Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each working day).

CLINICAL OBSERVATIONS
General clinical observations were made on parental animals once a day, after the administration at approximately the same time. Detailed examinations were made at the times of weekly weighing, prior to and during the mating and until necropsy. Detailed clinical observations were made on all animals outside the home cage in a standard arena once prior to the first exposure and once weekly thereafter.
Observations were performed on the skin, fur, eyes and mucous membranes, autonomic activity (lachrymation, piloerection, pupil size, respiratory pattern, occurrence of secretions and excretions), circulatory and central nervous system, somatomotor activity and behavior pattern, changes in gait, posture and response to handling. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhea, lethargy, sleep and coma.
Sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity were conducted on five male and five female animals randomly selected from each group during the last exposure week but before the blood sampling. General physical condition and behavior of animals were tested. A modified Irwin test was performed. (Irwin, S.: Comprehensive Observational Assessment: Ia. A systematic, Quantitative procedure for Assessing the Behavioral and Physiologic State of the Mouse, Psychopharmacologia (Berl) 13 222-257 1968).

BODY WEIGHT
All parental animals were weighed with an accuracy of 1 g. Parental males were weighed on the first day of dosing (Day 0) and weekly thereafter and on the day of the necropsy.
Parental females were weighed on the first day of dosing (Day 0) then weekly, on gestation days 0, 7, 14 and 21 and on days 0 (within 24 hours after parturition), 4 and 13 post-partum. Body weight of the female animals was additionally weighed on gestational day 10 in order to give accurate treatment volumes, but these data were not valuated statistically. Body weight was measured on day of necropsy for female animals subjected to organ weighing (selected for further examinations).
Body weight data were reported individually for adult animals. Individual body weight change was calculated.

FOOD CONSUMPTION
The food consumption was determined weekly by reweighing the non-consumed diet with a precision of 1 g during the treatment period except mating phase (pre-mating days 7, 13, and post-mating days 20, 27, 34 and 41 for male animals, pre-mating days 7, 13, gestation days 0, 7, 14 and 21, lactation days 0, 4 and 13 for female animals).

PLACENTAL SIGN
All sperm positive animals were examined for vaginal bleeding (placental sign of gestation) on the gestational day 13. If the test was negative on day 13, the examination was repeated on day 14 of gestation.

OBSERVATION OF THE DELIVERY PROCESS
Females were allowed to litter and rear their offspring. Delivery process was observed as carefully as possible from gestational day 21 onwards. All observations and any evidence of abnormal deliveries were considered. The duration of gestation was recorded and was calculated from day 0 of pregnancy.
Dams were observed whether they made a nest from the bedding material and nurse their new-born or not. The sucking success was observed by the presence of milk in the pups' stomach. All observations were recorded individually for each dam.
On post-partum day 4, the size of each litter was adjusted to four pups per sex per litter, if it was feasible. Extra pups were eliminated by a random selection. Partial adjustment of litter size was performed if the number of male and female pups did not allow having four of each sex per litter (for example, five males and three females).

THYROID HORMONE
Blood samples were collected for possible determination of serum levels of thyroid hormones (T4 and TSH).
Blood samples were collected from animals as follows: from 2-7 pups per litter on post-natal day 4 (if it was feasible; samples were pooled by litter); from all dams and 5-7 pups per litter on post-partal/post-natal day 13; from all parent male animals at termination on Day 42.
Parameters: Thyroxine – free Tetra-iodothyronine and Thyroid-stimulating hormone.
Blood samples from the day 13adult males were assessed for serum levels of thyroid hormones (T4 and TSH). Further assessment of T4 and TSH in blood samples from the dams were not performed because there were no relevant changes in the examined samples (based on observations in postnatal day 13 offspring and parental male animals).
The quantitative determination of thyroid hormones (T4 and TSH) in serum samples were performed by electrochemiluminescence immunoassay with COBAS e411 immunochemistry analyzer.

Oestrous cyclicity (parental animals):

Estrous cycle was monitored by examining vaginal smears each day before the treatment started from each animal being considered for study for two weeks. Estrous cycle was evaluated and considered at randomization.
Vaginal smears were also prepared and estrous cycle was monitored daily from the beginning of the treatment period (two weeks pre-mating period) and during the mating period until evidence of mating.
Vaginal smears were also prepared on the day of the necropsy.
Vaginal smears were stained with 1 % aqueous methylene blue solution then were examined with a light microscope.

Litter observations:

Each litter were examined as soon as possible after delivery (within 24 h of parturition), to establish the number and gender of pups, stillbirths, live births, runts (pups that are significantly smaller than normal pups), and the presence of gross abnormalities.
Live pups were counted, sexed, and litters weighed within 24 hours of parturition (on the day after parturition is complete, day 0), and on days 4 and 13 post-partum with an accuracy of 0.1 g. Any abnormal behavior of the offspring was recorded.
On the day of birth, pups found dead were subjected to a lung flotation test to differentiate pups died intrauterine (stillborn) from pups died after the birth (dead pups).
All litters were checked and recorded daily for the number of viable and dead pups. Dead pups found were subjected to necropsy by macroscopic examination. All observed abnormalities were recorded.
The anogenital distance of each pup was determined on postnatal day 4. The anogenital distance was normalized to the cube root of body weight. Therefore, individual body weight of pups was also determined with an accuracy of 0.01 g on postnatal day 4 and the litter weight was calculated for evaluation on postnatal day 4.
The number of nipples/areolae in male pups was counted on postnatal day 13.

THYROID HORMONE
In addition, blood samples were collected for possible determination of serum levels of thyroid hormones (T4 and TSH).

Postmortem examinations (parental animals):

GROSS NECROPSY
Gross necropsy was performed on each animal.
One male animal at 350 mg/kg bw/day died and was subjected to necropsy immediately after founding dead on Day 22 and one female animal at 120 mg/kg bw/day died and was subjected to necropsy immediately after founding on Day 49.
All other animals were euthanized by exsanguination after verification of deep narcosis by Isofluran CP® and were subjected to gross necropsy as follows: parental male animals after the optionally extended post-mating period on Day 42; dams on post-partum day 13 or shortly thereafter (Days 51, 54, 55, 57 or 65).
After examination of the external appearance, the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed, and any abnormality was recorded including details of the location, color, shape and size. Special attention was paid to the organs of the reproductive system; the number of implantation sites was recorded.
The ovaries, uterus with cervix, vagina, testes, epididymides (total and cauda), prostate and seminal vesicles with coagulating glands, adrenal glands and pituitary of all adult animals were preserved. Testes and epididymides were preserved in modified Davidson solution, all other organs in 4 % buffered formaldehyde solution.
Thyroid gland was preserved from all adult males and females for the intended subsequent histopathological examination. Thyroid and parathyroid were preserved together with larynx.
All organs showing macroscopic lesions and the following organs were preserved in 4 % buffered formaldehyde solution (except testes and epididymides) for five male and five female animals randomly selected from each group.

ORGAN WEIGHT
At the time of termination, body weight, brain weight, weight of the testes, epididymides and prostate and seminal vesicles with coagulating glands as a whole of all male adult animals were determined. Absolute organ weight was recorded. Relative organ weight (to body and brain weight) were calculated and reported.
In addition, for five males and females randomly selected from each group, adrenals, brain, heart, kidneys, liver, spleen and thymus were weighed.
The thyroid weight will be determined – if relevant – after fixation.
Paired organs were weighed together.

HISTOPATHOLOGY
Detailed histological examination was performed on the ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland in all animals of control and high dose groups with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure and on the ovaries covering the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.
Histological examination was also performed on the testes, epididymides, prostate and seminal vesicles with coagulating gland in one not mated male in the mid dose group.
Full histopathology examinations were performed on the preserved organs and tissues of the randomly selected animals in the control and high dose (350 mg/kg bw/day) groups and in dead animals – male no. 403 at 350 mg/kg bw/day and female no. 328 at 120 mg/kg bw/day.
In addition, thymus of one male animal (no. 304 in 120 mg/kg bw/day group), kidneys of one male animal (no. 209 in 40 mg/kg bw/day group) and uterus of three female animals (no. 223 in 40 mg/kg bw/day group, no. 322 and 323 in 120 mg/kg bw/day group) were processed and examined due to macroscopic findings (hemorrhage in the thymus, pyelectasia in the kidney, hydrometra in the uterus).
The fixed tissues were trimmed, processed, embedded in paraffin, sectioned with a microtome, placed on glass microscope slides, stained with hematoxylin and eosin and examined by light microscopy.

Postmortem examinations (offspring):

GROSS NECROPSY
Gross necropsy was performed on each animal.
Offspring on postnatal day 13 were euthanized by exsanguination after verification of deep narcosis by Isofluran CP® and were subjected to gross necropsy.
Thyroid gland was preserved from one male and one female pup per litter for the intended subsequent histopathological examination. Thyroid and parathyroid were preserved together with larynx.
Dead pups and pups euthanized at day 13 post-partum, or shortly thereafter, were carefully examined for gross abnormalities.
All organs showing macroscopic lesions and the following organs were preserved in 4 % buffered formaldehyde solution (except testes and epididymides) for five male and five female animals randomly selected from each group.

Reproductive indices:

Copulatory index, measuring of animals’ ability to mate.
Fertility index, measuring of male’s ability to produce sperm that can fertilize eggs and measure of female’s ability to become pregnant.
Gestation index, measuring of pregnancy that provides at least one live pup.

Offspring viability indices:

Post-implantation mortality (intrauterine mortality).
Post-natal mortality.
Survival index.
Sex ratio.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item related clinical signs in any group, i.e. the surviving parental animals (male and female) exhibited normal behavior and physical condition with no abnormalities in the control and at 40, 120 or 350 mg/kg bw/day at the daily or at the detailed weekly clinical observations.
Piloerection and decreased activity were observed in single male animal (1/12) at 120 mg/kg bw/day group between Days 24 and 30. Alopecia on the abdomen (1 cm in diameter) was detected in another male animal (1/12) of this group.
These clinical signs - piloerection and decreased activity on Day 27 (1/12) and alopecia on the abdomen on Days 20, 27, 34 and 41 (1/12) – were also detected at the weekly detailed clinical observations.
These observations were considered to be individual signs, only detected in single animals of the mid dose group bot not in animals at 350 mg/kg bw/day.
Dark stool was detected in the bedding material in each cage of the male and female animals at 120 and 350 mg/kg bw/day groups from Day 8 until termination. The color of stools was indicative of presence of the test item or its metabolite(s) in the gastro-intestinal tract.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There was no test item related mortality at 40, 120 or 350 mg/kg bw/day groups during the course of study (male and female).
One male animal (no. 403) at 350 mg/kg bw/day was found dead on Day 22. There were no preceding clinical signs or body weigh change in this animal. Therefore, the cause of death was judged to be unknown, probably an individual lesion.
One female animal (no. 328) at 120 mg/kg bw/day was also found dead on lactation day 10 (Day 49). Individual clinical signs – abnormal head position on the right side, lateral body position on the right side and catalepsy – was noted for this dam on lactation days 8 or 9.
Dark stool was detected in the bedding material in the cages from Day 8 until the death in both cases.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weight development was undisturbed in male and female animals at 40, 120 or 350 mg/kg bw/day during the entire treatment period.
The mean body weight was comparable in the control and at 40, 120 and 350 mg/kg bw/day groups in male animals during the pre-mating, mating and post-mating periods and in female animals during the pre-mating, gestation and lactation periods.
Statistical significance with respect to the control was only detected in the female animals at 350 mg/kg bw/day at the slightly higher mean body weight gain on the first week of treatment. This minor change was considered to be toxicologically not relevant.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no test item related changes in the mean daily food consumption of male or female animals at 40, 120 or 350 mg/kg bw/day.
The mean daily food consumption was comparable in the control and test item treated animals at 40, 120 or 350 mg/kg bw/day during pre-mating and post mating periods in male animals and during the course of the pre-mating, gestation and lactation periods in female animals.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Hematological evaluation did not reveal adverse test item related changes in the examined parameters at 40, 120 or 350 mg/kg bw/day.
All examined hematological and blood coagulation parameters were comparable with the control in male animals at 40 mg/kg bw/day.
At 120 mg/kg bw/day, the mean percentage of reticulocytes (RET) was higher than in the control group in male animals.
In the male animals at 350 mg/kg bw/day, the mean red blood cell (erythrocyte) count (RBC), the mean hemoglobin concentration (HGB), the mean hematocrit value (HCT) and the mean corpuscular (erythrocyte) hemoglobin concentration (MCHC) were statistically significantly lower while the mean percentage of reticulocytes (RET) was higher compared to their control.
The examined hematological and blood coagulation parameters were comparable in female animals in the control and 40, 120 and 350 mg/kg bw/day groups.
The elevated mean percentage of reticulocytes in male animals at 120 and 350 mg/kg bw/day (dose related) and in female animals – non-statistically significant – at 350 mg/kg bw/day was not accompanied by histopathological changes in the high dose treated animals. Slightly lower mean of red blood cell parameters (HGB, HCT) in male animals at 350 mg/kg bw/day corresponded well to the historical control value; RBC values in male control animals were slightly lower than the historical control.
In two female animals at 350 mg/kg bw/day, the individual RBC values were slightly above the historical control, although the mean at 350 mg/kg bw/day resulted to be non-statistically significantly higher than in the control group; no changes were observed in the red blood cell parameters.
The elevated mean percentage of reticulocytes in male animals at 120 and 350 mg/kg bw/day and in female animals at 350 mg/kg bw/day might be in connection with a slight stimulator effect on the release of erythroid cells from the cell -store tissues but it was not accompanied by histological changes (typical on hematopoietic cell hyperplasia) in the high dose treated animals.
Therefore, these findings were considered to have little or no toxicological relevance.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

There were no test item related adverse effects on the examined clinical chemistry parameters at 40, 120 or 350 mg/kg bw/day (male or female).
In male animals at 40 mg/kg bw/day, statistical significance with respect to the control was observed at the lower mean activity of alanine aminotransferase (ALT) and at the higher mean creatinine concentration (CREA).
The mean concentration of potassium (K+) was slightly below the control value in male animals at 120 mg/kg bw/day.
Compared to their control, the mean activity of alanine aminotransferase was lower in male animals at 350 mg/kg bw/day.
All examined clinical chemistry parameters were comparable in female animals in the control and in all the test item treated groups.
The statistically significant differences in ALT, K+ and CREA with respect to their controls in male animals were considered to have little or no toxicological importance because values – mean and individual – corresponded to the historical control values, there was no dose relevance or related histopathological alterations.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Functional observation battery did not demonstrate any alterations in the behavior or reactions to different type of stimuli of selected male or female animals in the control, 40, 120 or 350 mg/kg bw/day groups at the end of the treatment period (on Day 41).
There were no changes in the physical condition, behavior or in reactions to different types of stimuli in the male or female animals of control and test item treated groups in the examined parameters during the course of the functional observations.
Alopecia on the abdomen was noted for one male animal (1/5 at 120 mg/kg bw/day) at the functional observations.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathological examinations revealed test item related centrilobular vacuolation in the hepatocytes in the liver at 350 mg/kg bw/day in male animals: mild centrilobular vacuolation was observed in the hepatocytes in three male animals at 350 mg/kg bw/day (3/5).
Centrilobular vacuolation is indicative of hepatic lipidosis and was probably due to the test item in this study. Hepatic lipidosis is considered as a slight reversible liver injury in connection with a disturbance of energy metabolism of affected hepatocytes.
In females, the histopathological examinations did not reveal any adverse test item related finding.
There were no pathologic changes in the examined reproductive organs or tissues of male or female animals (control and 350 mg/kg bw/day).

In dead male animal (1/12) at 350 mg/kg bw/day, histological investigations revealed marked purulent inflammation in the lungs and mild centrilobular vacuolation in the hepatocytes in the liver.
In dead female animal (1/12) at 120 mg/kg bw/day, severe congestion and moderate alveolar emphysema were observed in the lungs.
In details, in animals selected for full histological examinations, minimal or mild alveolar emphysema in the lungs (1/5 male and 1/5 female in the control, 1/5 male and 1/5 female at 350 mg/kg bw/day), acute hemorrhage in the thymus (1/1 male at 120 mg/kg bw/day) were detected sporadically. The pulmonary emphysema and hemorrhage in the thymus were considered as a consequence of hypoxia, dyspnea and circulatory disturbance developed during exsanguination procedure.
Hyperplasia of bronchus associated lymphoid tissue (BALT) in the lungs (1/5 control male) is a physiological immune-morphological phenomenon, occurring also in not treated rats and has no toxicological significance.
Pyelectasia (one side) was observed in some animal (1/1 male at 40 mg/kg bw/day and 2/5 female at 350 mg/kg bw/day). This finding without other pathological lesion (degeneration, inflammation, fibrosis) is a common finding in Wistar rats without toxicological significance.
Subacute lymphocytic pyelitis accompanied with pyelectasia - as a sporadic individual lesion – was noted for one male animal at 40 mg/kg bw/day (1/1) and for one female at 350 mg/kg bw/day (1/5).
There was no morphological evidence of acute or subacute injury (degeneration, inflammation, necrosis etc.) of the stomach, the small and large intestines, the pancreas, the cardiovascular system, the immune system, the hematopoietic system, the skeleton, the muscular system, the male and female reproductive system or the central, or peripheral nervous system in the animals.
The cyto-morphology of endocrine glands was the same in the control and high dose treated animals.
The macroscopically visible grey-black discoloration of some organs (kidneys, spleen, mesenteric and submandibular lymph nodes) and the intestinal content could not be associated with histologically detectable pathological findings in the affected organs.
In the male animals belonging to the treated and control groups (12/12 control; 11/11 at 350 mg/kg bw/day), the investigated organs of reproductive system (testes, epididymides, prostate seminal vesicles, coagulating glands) were histologically normal and characteristic on the sexually mature organism in all cases. The various spermatogenic cells (the spermatogonia, the spermatocytes, the spermatids and spermatozoa); representing different phases in the development and differentiation of the spermatozoons and the interstitial cells were the same in quantity and morphologically in the testes of investigated control and treated animals. The histological picture of prostate, epididymides, seminal vesicles, and coagulating glands was normal in all cases as well.
In the female animals belonging to the treated and control groups (12/12 control; 12/12 at 350 mg/kg bw/day), the ovaries, uterus, cervix and vagina had a normal structure characteristic of the species, age and phase of the active sexual cycle in all cases of control and treated groups. The cortical region of ovaries contained primary, secondary and tertiary follicles and corpora lutea, indicating the active maturation of oocytes, and ovulation. The epithelial capsule and ovarian stroma were normal in all cases as well.
Dilatation of uterine horns was observed in four dams: 1/1 at 40 mg/kg bw/day, 2/2 at 120 mg/kg bw/day and 1/12 at 350 mg/kg bw/day. This finding – without inflammatory or other pathological lesions – is a slight neuro-hormonal phenomenon and was in connection with the normal sexual cycle (pro-estrus phase) of uterus without pathological significance.

Other effects:

no effects observed

Description (incidence and severity):

THYROID HORMONE
The thyroid hormone (free T4 and TSH) levels were similar in the control and test item treated parental male animals (40, 120 and 350 mg/kg bw/day).

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

A test item influence on the estrous cycle was not detected at any dose level (40, 120 or 350 mg/kg bw/day).
There were no statistically or biologically significant differences between the control and test item treated groups (40, 120 and 350 mg/kg bw/day) in number or percentage of animals with regular or irregular cycles, in the mean number of cycles, mean length of cycles, mean number of days in pro-estrous, estrous or diestrus, in number or percentage of animals in prolonged estrous or diestrous during the pre-experimental and pre-mating periods.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

The examined parameters of reproductive performance were not affected by the test item at 40, 120 or 350 mg/kg bw/day in male or female animals.
There was a statistical difference between the control and the 120 mg/kg bw/day groups of male animals in the copulatory index as one male animal in the mid dose group did not mate. This minor change was considered to be indicative of biological variation and not related to the test item.
There was no difference between the control and test item treated groups in the number of pregnant females and dams delivered, in the fertility indices (male and female animals), gestation index (female animals), in the pre-coital interval and conceiving days.

DELIVERY OF DAMS - no effects observed
There were no toxicologically relevant differences in the evaluated parameters of delivery between the control and test item treated groups (40, 120 or 350 mg/kg bw/day).
The mean number of implantation sites per dams and the mean of post-implantation loss were comparable in the control and all test item treated groups.
There were no significant differences between the control and test item treated groups in the mean duration of pregnancy, in the mean number of total births, liveborns, stillborns or viable pups on post-partal day 0, in the percentage of dams with viable offspring on post-partal day 0 or in the live birth index.

Dose descriptor:

NOAEL

Effect level:

>= 350 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: reproductive toxicity

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Test item related clinical signs were not detected in the offspring between postnatal days 0 and 13.
The percentage of offspring with signs (cold and not suckled) at 40 and 350 mg/kg bw/day was slightly higher than in the control group on postnatal day 0. However, these signs were considered to be toxicologically not relevant in the lack of dose relevance and the signs were transient – detected only shortly after the delivery – and were not associated with depression on the development of the offspring.
Occasionally, other clinical signs were also observed: pale (1 % in the control), cachectic pup (1 % in the control), smaller than normal pup (1 % in the control; 1 % at 40 mg/kg bw/day), hemorrhage on the nose (1 % at 350 mg/kg bw/day), which however were considered to have no toxicological relevance.

Mortality / viability:

no mortality observed

Description (incidence and severity):

There was no test item related effect on offspring’s extra uterine mortality.
There were no significant differences between the control and test item treated groups (40, 120 or 350 mg/kg bw/day) in the mean number of dead (including missing) offspring per litter.
The mean number of live pups per litter and the mean number of viable pups per litter were similar in all groups on postnatal days 0, 4 and 13. There were no significant differences between the control and test item treated (40, 120 or 350 mg/kg bw/day) groups in the survival indices.
One dam at 120 mg/kg bw/day was found dead on lactation day 10, therefore its pups were euthanized on the same day.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The body weight development of the offspring was unaffected by the test item.
The mean litter weights and mean pup weights as well as the mean litter weight gains and mean pup weight gains were similar in the control and in all test item treated groups (40, 120 and 350 mg/kg bw/day) on postnatal days 0, 4 and 13.
Considering the offspring’s body weight in males and females separately, statistically significant difference with respect to the control was detected at the slightly lower mean weight of male pups at 350 mg/kg bw/day on postnatal day 4. The difference with respect to the control was minor and it was considered to be toxicologically not relevant.

Sexual maturation:

no effects observed

Description (incidence and severity):

The anogenital distances (absolute and normalized in male or female offspring) or nipple retention (male) were not adversely affected by the test item treatment at 40, 120 and 350 mg/kg bw/day.
In male pups, statistical significance was observed at the shorter mean normalized anogenital distance at 120 mg/kg bw/day. As there was no dose relevance, this difference with respect to the control was not considered to be toxicologically relevant.
Nipples/areoles were not visible in any of the examined male offspring in the control or 40, 120 or 350 mg/kg bw/day groups on postnatal day 13.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no macroscopic changes in the organs or tissues of a single stillborn offspring (1/1 at 40 mg/kg bw/day subjected to necropsy on postnatal day 0).

Other effects:

no effects observed

Description (incidence and severity):

THYROID HORMONE
The thyroid hormone (free T4 and TSH) levels were similar in the control and offspring sampled on postnatal day 13.

There were no test item related differences between the control and all test item treated groups in the ratio or in the litter means of genders on postnatal days 0, 4 or 13.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 350 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Reproductive effects observed:

no

Conclusions:

NOAEL (P0) (rat, male and female): 350 mg/kg bw/day, reproductive toxicity
NOAEL (F1): 350 mg/kg bw/day

Executive summary:

A Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test was conducted to obtain information on the toxic potential of test item, following the testing method and testing procedures outlined into the OECD guideline 422.

The substance was administered to rats orally (by gavage) once daily at 0 (vehicle only), 40, 120 and 350 mg/kg bw/day doses. The concentration of the test item in the dosing formulations was checked two times during the study and the concentrations varied within the range of 97 and 103 % in comparison to the nominal values.

All animals of the parent (P) generation were dosed prior to mating (14 days) and throughout mating. In addition, males received the test item or vehicle after mating up to the day before the necropsy (altogether for 42 days). Females were additionally exposed through the gestation period and up to lactation days 14-16, i.e. up to the day before necropsy (altogether for 51-65 days).

There was no test item related mortality at any dose level. A test item influence on the estrous cycle was not found at any dose level and there were no toxicologically significant differences between the control and test item treated male or female animals in the examined parameters of reproductive performance or in the delivery parameters of dams.

There were no test item related changes in the serum thyroid hormone (T4 and TSH) levels at any dose (parental male or 13-day offspring).

There were no toxic or other test item related lesions detectable by histological examination in the investigated reproductive organs (ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland) of male or female animals administered with 350 mg/kg bw/day.

No adverse effect on the mortality, clinical signs, body weight development or necropsy findings were detected in the offspring terminated as scheduled. The anogenital distance (male and female) or nipple retention (male) were not affected.

Conclusion

Under the conditions of the study, the substance did not adversely influence the reproductive performance in parental male and female rats.

NOAEL (P0) (rat, male and female): 350 mg/kg bw/day, reproductive toxicity

NOAEL (F1): 350 mg/kg bw/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

The read across test substance Similar substance 01 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 (Valsakova, 2012). No negative treatment-related effects were detected during functional observation of animals. Only at highest dose test-related reduction in body weight gains, some biochemical parameters and biometry of organs were significantly changed.

Nevertheless the no adverse effect level identified is sufficiently high for excluding injuries for the reproduction and for developmental of pups.

Short description of key information:

The values of NOAEL for the reproduction and for developmental of pups was established to be 80 mg/kg bw/day

Justification for selection of Effect on fertility via oral route:

Study conducted following an official method and the read across proposed is proper and reliable.

Effects on developmental toxicity

Description of key information

NOAEL for reproduction and for developmental of pups: 80 mg/kg bw/day

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

80 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

Additional information

In the Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test were not observed adverse effects on fertility and results gave no reason to suspect that the substance could have any developmental toxicant effects; therefore there are no concerns to consider further investigations.

Justification for classification or non-classification

Direct Black 22 (DBk22) does not meet the criteria to be considered toxic to reproduction, nor to development

According to CLP regulation (EC1272/2008) DBk22 is not classified toxic to reproduction.