[2-[[2-cyano-3-[4-(diethylamino)phenyl]-1-oxoallyl]oxy]ethyl][3-[[2-cyano-3-[4-(diethylamino)phenyl]-1-oxoallyl]oxy]propyl]dimethylammonium chloride

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and OECD testing guideline compliant study with well-characterized test material

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sulzfeld, Germany
- Age at study initiation: 10-11 weeks old
- Housing: During the study period, the rats were housed individually in Makrolon type M III cages supplied by Becker & Co., Castrop-Rauxel, Germany (floor area of about 800 cm²), with the following exceptions:
- For motor activity (MA) measurements the animals were housed individually in polycarbonate cages supplied by TECNIPLAST, Hohenpeißenberg, Germany with wire covers from Ehret, Emmendingen, Germany (floor area of about 800 cm²) and small amounts of bedding material.
- Diet: ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature : 20-24°C
- Humidity: 30-70%
- Air changes: 15 air per hour
- Photoperiod: day/night cycle was 12 hours

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was applied as a suspension. To prepare this suspension, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, drinking water was filled up to the desired volume, subsequently released with a magnetic stirrer. During administration of the test substance, preparations were kept homogeneous by stirring with a magnetic stirrer. The test substance preparations were prepared daily.

Details on mating procedure:

- M/F ratio per cage: 1:1 during overnight matings, male and female mating partners were housed together in Makrolon type M III cages.
- Pregnant animals and their litters were housed together until PND 4 (end of lactation).
- Length of cohabitation: overnight for max. 2 weeks.
- Proof of pregnancy: sperm in vaginal smear referred as day 0 of gestation (GD0).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test substance in drinking water for a period of 4 hours at room temperature was proven during the study. Homogeneity and concentration control analyses of the test-substance preparations were performed in all concentrations at the start of the administration period. Additionally, samples from all concentrations as reverse samples for concentration control analysis were taken at the end of the study.

Duration of treatment / exposure:

The duration of treatment covered a 2-week pre-mating and mating period (maximum: 2 weeks) in both sexes, approximately 1 week post-mating in males, and the entire gestation period as well as 4 days of lactation in females followed by an additional treatment until one day before sacrifice. Based on this, for male animals the administration period was maximum 31 days and for female animals the administration period was maximum 52 days.

Frequency of treatment:

daily at the same time in the morning

Remarks:

Doses / Concentrations:
100, 300 and 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

10 rats

Control animals:

yes, concurrent vehicle

Parental animals: Observations and examinations:

DETAILED CLINICAL OBSERVATIONS:
- Time schedule:
1.) At least once daily: morbidity, pertinent behavioral changes, signs of overt toxicity, littering and lactation behavior of the dams
2.) On weekdays: the parturition behavior of the dams
3.) The day of littering was considered the 24-hour period from about 15.00 h of one day until about 15.00 h of the following day.

BODY WEIGHT:
Body weight was determined before the start of the administration period in order to randomize the animals. During the administration period body weight was determined on study day 0 (start of the administration period) and thereafter once a week at the same time of the day (in the morning). The body weight change of the animals was calculated from these results.

The following exceptions are notable for the female animals:

- During the mating period the parental females were weighed on the day of positive evidence of sperm on gestational day(GD) 0 and on GD 7, 14 and 20.
- Females with litter were weighed on the day of parturition on postnatal day(PND) 0 and on PND 4.
- Females without a litter and without positive evidence of sperm in the vaginal smear were weighed weekly. These body weight data were solely used for the calculations of the dose volume.

FOOD CONSUMPTION:
Generally, food consumption was determined once a week for male and female parental animals, with the following exceptions:
- Food consumption was not determined during the mating period (male and female F0 animals).
- Food consumption of the F0 females with evidence of sperm was determined on GD 0-7, 7-14, 14-20.
- Food consumption of F0 females, which gave birth to a litter, was determined for PND 1-4.

Food consumption was not determined in females without positive evidence of sperm (during the mating period of dams used in parallel) and females without litter (during the lactation period of dams used in parallel).

WATER CONSUMPTION:
Drinking water consumption was monitored by daily visual inspection of the water bottles for any changes in volume.

FUNCTIONAL OBSERVATION BATTERY:
A functional observational battery (FOB) was performed in five animals per sex and group at the end of the administration period. The FOB started with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable. The observations were performed at random.

MOTOR ACTIVITY ASSESSMENT:
The motor activity assessment (MA) was carried out in five animals per sex and group at the end of the administration period. Motor activity was measured on the same day as FOB was performed.

MALE REPRODUCTION DATA:
FThe pairing partners, the number of mating days until vaginal sperm was detected in the female animals, and the gestational status of the females were recorded for F0 breeding pairs. For the males, mating and fertility indices were calculated for F1 litters.

FEMALE REPRODUCTION DATA:
The pairing partners, the number of mating days until vaginal sperm were detected and gestational status were recorded for F0 females. For the females, mating, fertility and gestation indices were calculated for F1 litters. The total number of pups delivered and the number of liveborn and stillborn pups were noted, and the live birth index was calculated for F1 litters.

Litter observations:

PUP NUMBER AND STATUS AT DELIVERY:
All pups delivered from the F0 parents (F1 litter) were examined as soon as possible on the day of birth to determine the total number of pups, the sex and the number of liveborn and stillborn pups in each litter. At the same time, the pups were also examined for macroscopically evident changes. Pups, which died before this initial examination, were defined as stillborn pups.

PUP VIABILITY/MORTALITY:
In general, a check was made for any dead or moribund pups twice daily on workdays (once in the morning and once in the afternoon) or as a rule, only in the morning on Saturdays, Sundays or public holidays. The number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PND 1-4 (lactation period) were determined. Pups which died accidentally or were sacrificed due to maternal death were not included in these calculations. The number of live pups/litter was calculated on the day after birth, and on lactation day 4. The viability index was calculated.

SEX RATIO:
On the day of birth (PND 0) the sex of the pups was determined by observing the distance between the anus and the base of the genital tubercle; normally, the anogenital distance is considerably greater in male than in female pups. The sex of the pups was finally confirmed at necropsy.

PUP CLINICAL OBSERVATION:
The live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams.

PUP BODY WEIGHT DATA:
The pups were weighed on the day after birth (PND 1) and on PND 4. Pups' body weight change was calculated from these results. The individual weights were always determined at about the same time of the day (in the morning). Pup body weights and pup body weight change were listed for males, females and males + females. “Runts” were defined on the basis of the body weights on PND 1. "Runts" are pups that weigh less than 75% of the mean weight of the respective control pups.

Postmortem examinations (parental animals):

CLINICAL PATHOLOGY:
In the morning blood was taken from the retroorbital venous plexus from overnight fasted animals. The animals were anaesthetized using isoflurane. The blood sampling procedure and subsequent analysis of blood and serum samples were carried out in a randomized sequence. For urinalysis the individual animals were transferred to metabolism cages (withdrawal of food and water) and urine was collected overnight. Urine samples were evaluated in a randomized sequence
The assays of blood and serum parameters were performed under internal laboratory quality control conditions with reference controls to assure reliable test results.
The results of clinical pathology examinations were expressed in International System (SI) units.
The following examinations were carried out in the first 5 surviving parental males and the first 5 surviving females with litter (in order of delivery) per group:

HEMATOLOGY
The following parameters were determined:

- Leukocyte count (WBC),
- Erythrocyte count (RBC),
- Hemoglobin (HGB),
- Hematocrit (HCT),
- Mean corpuscular volume (MCV),
- Mean corpuscular hemoglobin (MCH),
- Mean corpuscular hemoglobin concentration (MCHC),
- Platelet count (PLT),
- Differential blood count,
- Reticulocytes (RET),
- Prothrombin time (Hepato Quick’s test) (HQT).

CLINICAL CHEMISTRY:
The following parameters were determined:

- Alanine aminotransferase (ALT),
- Aspartate aminotransferase (AST),
- Alkaline phosphatase (ALP),
- Gamma-Glutamyltransferase (GGT),
- Sodium (NA),
- Potassium (K),
- Chloride (CL),
- Inorganic phosphate (INP),
- Calcium (CA),
- Urea (UREA),
- Creatinine (CREA),
- Glucose (GLUC),
- Total bilirubin (TBIL),
- Total protein (TPROT),
- Albumin (ALB),
- Globulins (GLOB),
- Triglycerides (TRIG),
- Cholesterol (CHOL),
- Bile acids (TBA).

URINANALYSIS:
The following parameters were determined:

- pH,
- Protein,
- Glucose,
- Ketones,
- Urobilinogen,
- Bilirubin,
- Blood,
- Specific gravity,
- Sediment,
- Color, turbidity,
- Volume.

NECROPSY OF THE PARENTAL ANIMALS:
All parental animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology; special attention was given to the reproductive organs.

Weight PARAMETERS:

The following weights were determined in all animals sacrificed on schedule:

1. Anesthetized animals
2. Epididymides
3. Testes

The following weights were determined in 5 animals per sex/test group sacrificed on schedule (females with litters only, same animals as used for clinical pathological examinations):

1. Adrenal glands
2. Brain
3. Heart
4. Kidneys
5. Liver
6. Spleen
7. Thymus

ORGAN/TISSUS FIXATION:

The following organs or tissues of all parental animals were fixed in 4% buffered formaldehyde solution or modified Davidson’s solution:

1. All gross lesions
2. Adrenal glands
3. Aorta
4. Bone marrow (femur)
5. Brain
6. Cecum
7. Cervix
8. Coagulating glands
9. Colon
10. Duodenum
11. Eyes with optic nerve
12. Esophagus
13. Extraorbital lacrimal glands
14. Epididymides (modified Davidson’s solution)
15. Femur with knee joint
16. Heart
17. Ileum
18. Jejunum (with Peyer’s patches)
19. Kidneys
20. Larynx
21. Liver
22. Lungs
23. Lymph nodes (axillary and mesenteric)
24. Mammary gland (male and female)
25. Nose (nasal cavity)
26. Ovaries (modified Davidson’s solution)
27. Oviducts
28. Pancreas
29. Parathyroid glands
30. Pharynx
31. Pituitary gland
32. Prostate gland
33. Rectum
34. Salivary glands (mandibular and sublingual)
35. Sciatic nerve
36. Seminal vesicles
37. Skeletal muscle
38. Spinal cord (cervical, thoracic and lumbar cord)
39. Spleen
40. Sternum with marrow
41. Stomach (forestomach and glandular stomach)
42. Testes (modified Davidson’s solution)
43. Thymus
44. Thyroid glands
45. Trachea
46. Urinary bladder
47. Uterus
48. Vagina

HISTOPATHOLOGY:
Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings in the following tissues and organs:

Adrenal glands
All gross lesions
Bone marrow (femur)
Brain
Cecum
Cervix
Coagulating glands
Colon
Duodenum
Epididymides
Heart
Ileum
Jejunum
Kidneys
Liver
Lungs
Lymph nodes (axillary and mesenteric)
Ovaries
Oviducts
Prostate gland
Peyer’s patches
Rectum
Sciatic nerve
Seminal vesicles
Spinal cord (cervical, thoracic, lumbar)
Spleen
Stomach (forestomach and glandular stomach)
Testes
Thymus
Thyroid glands
Trachea
Urinary bladder
Uterus
Vagina

Special attention was given to stages of spermatogenesis in the male gonads. A correlation between gross lesions and histopathological findings was attempted.

Postmortem examinations (offspring):

NECROPSY OBSERVATION OF THE PUPS:
All pups with scheduled sacrifice on PND 4 were sacrificed under isoflurane anesthesia with CO2. All pups were examined externally and eviscerated; their organs were assessed macroscopically. All stillborn pups and all pups that died before PND 4 were examined externally, eviscerated and their organs were assessed macroscopically. All pups without notable findings or abnormalities were discarded after their macroscopic evaluation. Animals with notable findings or abnormalities were evaluated on a case-by-case basis, depending on the type of finding noted.

Statistics:

Please refer to any other information on material and methods incl. tables

Reproductive indices:

Male: male mating index, male fertility index
Female: female mating index, female fertility index, gestation index

Offspring viability indices:

Live birth index, viability index, sex ratio

CLINICAL SIGNS AND MORTALITY:
No parental animal died prematurely in the present study. Brightly yellowish discolored urine and light yellow discolored feces were observed during the administration period at detailed clinical observations (DCO) in all dosed animals starting at DCO on study days 7 (urine) and 14 (feces).
The effects were related to the test substance but assessed as being non-adverse.

FOOD CONSUMPTION:
During the premating period food consumption was significantly decreased in female animals of test group 3 (1000 mg/kg bw/d), i.e. on study day 7 (-15%) and in mean of means (-11%). However, as the body weight gain of these animals did not show any impairment during the first week of treatment the finding was assessed as being spontaneous.

WATER CONSUMPTION:
No test substance-related, adverse findings were noted.

BODY WEIGHT DATA:
No test substance-related changes in body weight or body weight gain were observed for male and female animals of test groups 1-3 (100, 300 and 1000 mg/kg bw/d) when compared to control groups.

FUNCTIONAL OBSERVATION BATTERY:
Deviations from "zero values" were obtained in several rats. However, as most findings were equally distributed between test-substance treated groups and controls, were without a dose-response relationship or occurred in single rats only, these observations were considered to have been incidental.

MOTOR ACTIVITY MEASUREMENT:
There were no significant deviations concerning the overall motor activity (summation of all intervals) and regarding the single intervals in male and female animals of all test groups in comparison to the concurrent control group.

MALE REPRODUCTION DATA:
For all F0 parental males, which were placed with females to generate F1 pups, mating was confirmed. Thus, the male mating index was 100% in all test groups (0, 100, 300, 1000 mg/kg bw/d).

Fertility was proven for most of the F0 parental males within the scheduled mating interval to produce F1 litter. Two male animals of test group 1 (100 mg/kg bw/d), 2 male animals of test group 2 (300 mg/kg bw/d) and 1 male animal of test group 3 (1000 mg/kg bw/d) did not generate F1 pups. Thus, the male fertility index ranged between 80% and 100% . These values reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.

FEMALE REPRODUCTION AND DELIVERY DATA:
The female mating index calculated after the mating period for F1 litter was 100% for all test groups (0, 100, 300, 1000 mg/kg bw/d). The mean duration until sperm was detected in vaginal smears (GD 0) was 2.3, 2.3, 3.9 and 3.3 days in test groups 0-3 (0, 100, 300, 1000 mg/kg bw/d, respectively).

All sperm positive rats delivered pups with the exception of two females of test group 1 (100 mg/kg bw/d), two females of test group 2 (300 mg/kg bw/d) and one female of test group 3 (1000 mg/kg bw/d), which were mated with males but did not become pregnant. The female fertility index varied between 80% and 100%. The mean duration of gestation was between 22.0 and 22.2 days and did not show significant differences. The gestation index reached 100% in all test groups including the control group. The rate of liveborn pups was not affected by the test substance, as indicated by live birth indices of 99.2% (control group), 100.0% (test groups 1 and 2) and 99.0% (test group 3). Single stillborn pups were seen in control group 0 and test group 3 (1000 mg/kg bw/d). A relation to treatment was excluded.


HEMATOLOGY:
No treatment-related changes among hematological parameters were observed.

CLINICAL CHEMISTRY:
In rats of both sexes of test group 3 (1000 mg/kg bw/d) creatinine values and, additionally, in females of the same test group urea levels were increased. Potassium levels were higher in females of test groups 1 and 3 (100 and 1000 mg/kg bw/d) but the potassium mean in test group 1 was within the historical control range. Therefore, the potassium level alteration in females of test group 1 (100 mg/kg bw/d) was regarded as incidental and not treatment-related.

URINALYSES:
No treatment-related changes among urinalysis parameters were observed.

ABSOLUTE WEIGHTS:
When compared to the control group 0 (set to 100%), none of the mean absolute weight parameters in male and female animals showed significant differences.

RELATIVE ORGAN WEIGHTS:
When compared to the control group 0 (set to 100%), the mean relative weight of the kidneys was significantly increased in females of test group 3 (1000 mg/kg bw/d). All other mean relative weight parameters in females and all weight parameters in males did not show significant differences when compared to the control group 0. For the increased relative kidney weight in female animals of test group 3 a treatment-related effect could not be ruled out.

GROSS LESIONS:
A treatment-related yellow discoloration of contents was observed in the forestomach, glandular stomach, and the cecum. Two males of test group 3 (1000 mg/kg bw/d) showed a yellow discoloration of the seminal vesicles. All further findings occurred individually. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

FERTILITY:
The female animals, which were not pregnant as well as the male mating partners, did not show relevant gross lesions. In male mating partners of test group 3 (1000 mg/kg bw/d) the size of testes and of epididymides were reduced. One male animal of test group 3 (1000 mg/kg bw/d) showed an extreme (grade 5) diffuse degeneration of the testes that caused an aspermia in the epididymides. Therefore, the corresponding female animal was not pregnant. The occurrence of these findings was regarded to be incidental. All other females that were not pregnant as well as their male mating partners were not investigated histopathologically.

HISTOPATHOLOGY:

LIVER
In the centrilobular regions of the liver, the occurrence of single cell necrosis or apoptosis was minimally increased in male and female animals of test group 3 (1000 mg/kg bw/d). The increased number of single cell necroses or apoptotic bodies in animals of test group 3 (1000 mg/kg bw/d) was considered to be treatment-related.

SEMINAL VESICLES
For the yellow discoloration of the seminal vesicle in two males of test group 3 (1000 mg/kg bw/d), there were no histopathological correlates. All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Dose descriptor:

NOAEL

Remarks:

(fertility)

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No reproduction effects were noted.

Dose descriptor:

NOAEL

Remarks:

(systemic toxicity)

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on effects on kidneys and liver noted at 1000 mg/kg bw/day

PUPS NUMBER AND STATUS AT DELIVERY:
The mean number of delivered pups per dam and the rate of liveborn and stillborn pups reflect the normal range of biological variation inherent in the strain used in this study.

PUPS VIABILITY/MORTALITY:
The viability index as indicator for pup mortality between postnatal day (PND) 0 and 4 was 100% for test groups 1, 2 and 3 (100, 300 and 1000 mg/kg bw/d). In the control group the viability index was decreased down to 99.2% because 1 pup was found dead. This decreased viability index was within the historical control data and reflected the normal range of biological variation inherent in the strain used in this study.

SEX RATIO:
The sex distribution and sex ratios of live F1 pups on the day of birth and on PND 4 did not show biologically relevant differences between test groups.

PUP CLINICAL OBSERVATION:
The surviving F1 pups of any test group did not show adverse clinical signs up to scheduled sacrifice on PND 4.

PUP BODY WEIGHT DATA:
No test substance-related significant changes in body weight or body weight change were observed for male and female pups of test groups 1-3 (100, 300 and 1000 mg/kg bw/d) when compared to control groups.
Two male runts were seen in 1 litter of the control group 0 on PND 1. Also, 1 male runt was observed in test group 2 (300 mg/kg bw/d) on PND 1 and each 2 female runts were detected in test groups 1 (100 mg/kg bw/d) and 3 (1000 mg/kg bw/d). All values were within the range of the biological variation inherent in the strain of rats used for this study.

PUPS NECROPSY OBSERVATIONS:
No test substance-related effects were observed.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No developmental effects were noted.

Reproductive effects observed:

not specified

STABILITY ANALYSES:

The stability of the test substance indrinking water was demonstrated over a period of 4 hours at room temperature. As the mixtures were stored no longer than this time period, the stability was guaranteed.

HOMOGENEITY CONTROL ANALYSES:

Considering the low relative standard deviation in the homogeneity analysis, it can be concluded that the test item was distributed homogeneously in drinking water.

CONCENTRATION CONTROL ANALYSES:

The concentration control analyses of all concentrations revealed that the values were in the expected range of the target concentrations, i.e. were always in a range of about 98-100% of the nominal concentrations. Taken together, the results demonstrate the correctness of the concentrations of the test item.

FOOD ANALYSES:

On the basis of duration of use and the analytical findings with respect to chemical and microbiological contaminants the diet was found to be suitable.

DRINKING WATER ANALYSES:

On the basis of the analytical findings the drinking water was found to be suitable.

BEDDING AND ENRICHMENT ANALYSES:

On the basis of the analytical findings the bedding and the enrichment are found to be suitable.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

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

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

In a repeated dose oral toxicity study (BASF SE, 2013, OECD 422) the registered dye was administered to Wistar rats (4 groups of 10 male and 10 female animals each) via oral gavage at dose levels of 0, 100, 300 or 1000 mg/kg bw/day. The treatment period covered a 2-week pre-mating and mating period (maximum: 2 weeks) in both sexes, approximately 1 week post-mating period in males, and the entire gestation period as well as 4 days of lactation in females followed by an additional treatment until one day before sacrifice. Based on this, for male animals the administration period was maximum 31 days and for female animals the administration period was maximum 52 days. Mortality, clincal signs, body weight and food consumption were assessed at regular intervals. At the end of the study functional observation battery and motor activity parameters as well as haematology, urinalysis and clinical chemistry parameters were determined. At necropsy, selected organs were weighed and the animals were examined macroscopically and histopathologically. Relevant reproductive parameters and indices were determined. Litter observations and examination of pups were conducted. Daily clinical observations during the premating, gestation and lactation period did not reveal any treatment-related changes in the parental animals appearance, general condition or behavior. Brightly yellowish discolored urine and light yellow discolored feces were observed during the administration period in all dosed animals. The effect was related to the test substance but assessed as being non-adverse. No treatment-related effects were observed in mean body weight, body weight changes and food consumption of exposed animals throughout the study. Neurological testing did not indicate any neurotoxic potential of the test item. There were no substance-induced effects on the male and female fertility indices. The mean duration of gestation and the gestation index did no show significant differences to the control group. The rate of liveborn pups was not affected by the test substance. Single stillborn pups were seen in control and test group (1000 mg/kg bw/day) animals, however, without relation to treatment. At macroscopic examination treatment related yellow discoloration of contents was observed in the forestomach, glandular stomach, and the cecum. Two male animals of the highest dose group (1000 mg/kg bw/day) showed a yellow discoloration of the seminal vesicles. All further findings occurred individually. The results of the clinical chemistry showed that in rats of both sexes of the highest dose group (1000 mg/kg bw/day) creatine values and, additionally, in females of the same test group urea level and potassium levels were increased. The mean relative weight of kidneys was significantly increased in females of the high dose group (1000 mg/kg bw/day) and a treatment-related effect could not be ruled out. Further, histopathology examinations of the sampled organs revealed a minimally increased number of single cell necroses or apoptotic bodies (grade 1) in 6/10 male and 3/10 female animals in the highest dose group (1000 mg/kg bw/day).The female animals, which were not pregnant as well as the male mating partners, were histopathologically investigated. Therefore, no treatment related effects could be observed.

The number of pups, status at delivery and the pups viability/mortality reflected the normal range of biological variation inherent in the strain used in this study. The sex ratio did not show biologically relevant differences between the test groups. No treatment related effects on the pups were observed for clinical signs, body weight and necropsy observation.

 

The present study indicated the kidney and the liver as specific target organs triggered at a dose level of 1000 mg/kg bw/day. The effect in the kidney was supposed to be test item related but revealed no high toxicological hazard. The effect in the liver was considered to be test item related and adverse. However, due to the high dose level at which the effect was seen, a serious health risk for humans is not expected. In conclusion, based on the systemic effects at a dose level of 1000 mg/kg bw/day the No Observed Adverse Effect Level (NOAEL) for parental males and females was established at 300 mg/kg bw/day.

Since no treatment related effect could be observed for male and female reproduction parameters, female delivery and for any endpoint assessed during examination of the pups the NOAEL for reproduction and the NOAEL for developmental effects was determined to be 1000 mg/kg bw/day.

Short description of key information:
Based on an OECD Guideline 422 study in compliance with GLP with the registered dye, the NOAEL for reproductive toxicity was considered to be 1000 mg/kg bw/day for all relevant endpoints, which was the highest dose tested.

Justification for selection of Effect on fertility via oral route:
only available reliable study

Effects on developmental toxicity

Description of key information

In an OECD Guideline 422 and GLP study with the registered dye in rats, the NOAEL for development of offspring was at the highest test dose of 1000 mg/kg/day. 

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and OECD testing guideline compliant study with well-characterized test material

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sulzfeld, Germany
- Age at study initiation: 10-11 weeks old
- Housing: During the study period, the rats were housed individually in Makrolon type M III cages supplied by Becker & Co., Castrop-Rauxel, Germany (floor area of about 800 cm²), with the following exceptions:
- For motor activity (MA) measurements the animals were housed individually in polycarbonate cages supplied by TECNIPLAST, Hohenpeißenberg, Germany with wire covers from Ehret, Emmendingen, Germany (floor area of about 800 cm²) and small amounts of bedding material.
- Diet: ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature : 20-24°C
- Humidity: 30-70%
- Air changes: 15 air per hour
- Photoperiod: day/night cycle was 12 hours

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was applied as a suspension. To prepare this suspension, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, drinking water was filled up to the desired volume, subsequently released with a magnetic stirrer. During administration of the test substance, preparations were kept homogeneous by stirring with a magnetic stirrer. The test substance preparations were prepared daily.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test substance in drinking water for a period of 4 hours at room temperature was proven during the study. Homogeneity and concentration control analyses of the test-substance preparations were performed in all concentrations at the start of the administration period. Additionally, samples from all concentrations as reverse samples for concentration control analysis were taken at the end of the study.

Details on mating procedure:

- M/F ratio per cage: 1:1 during overnight matings, male and female mating partners were housed together in Makrolon type M III cages.
- Pregnant animals and their litters were housed together until PND 4 (end of lactation).
- Length of cohabitation: overnight for max. 2 weeks.
- Proof of pregnancy: sperm in vaginal smear referred as day 0 of gestation (GD0).

Duration of treatment / exposure:

The duration of treatment covered a 2-week pre-mating and mating period (maximum: 2 weeks) in both sexes, approximately 1 week post-mating in males, and the entire gestation period as well as 4 days of lactation in females followed by an additional treatment until one day before sacrifice. Based on this, for male animals the administration period was maximum 31 days and for female animals the administration period was maximum 52 days.

Frequency of treatment:

daily at the same time in the morning

Remarks:

Doses / Concentrations:
100, 300 and 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

10 rats

Control animals:

yes, concurrent vehicle

Maternal examinations:

DETAILED CLINICAL OBSERVATIONS:
- Time schedule:
1.) At least once daily: morbidity, pertinent behavioral changes, signs of overt toxicity, littering and lactation behavior of the dams
2.) On weekdays: the parturition behavior of the dams
3.) The day of littering was considered the 24-hour period from about 15.00 h of one day until about 15.00 h of the following day.

BODY WEIGHT:
Body weight was determined before the start of the administration period in order to randomize the animals. During the administration period body weight was determined on study day 0 (start of the administration period) and thereafter once a week at the same time of the day (in the morning). The body weight change of the animals was calculated from these results.

The following exceptions are notable for the female animals:

- During the mating period the parental females were weighed on the day of positive evidence of sperm on gestational day(GD) 0 and on GD 7, 14 and 20.
- Females with litter were weighed on the day of parturition on postnatal day(PND) 0 and on PND 4.
- Females without a litter and without positive evidence of sperm in the vaginal smear were weighed weekly. These body weight data were solely used for the calculations of the dose volume.

FOOD CONSUMPTION:
Generally, food consumption was determined once a week for male and female parental animals, with the following exceptions:
- Food consumption was not determined during the mating period (male and female F0 animals).
- Food consumption of the F0 females with evidence of sperm was determined on GD 0-7, 7-14, 14-20.
- Food consumption of F0 females, which gave birth to a litter, was determined for PND 1-4.

Food consumption was not determined in females without positive evidence of sperm (during the mating period of dams used in parallel) and females without litter (during the lactation period of dams used in parallel).

WATER CONSUMPTION:
Drinking water consumption was monitored by daily visual inspection of the water bottles for any changes in volume.

FUNCTIONAL OBSERVATION BATTERY:
A functional observational battery (FOB) was performed in five animals per sex and group at the end of the administration period. The FOB started with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable. The observations were performed at random.

MOTOR ACTIVITY ASSESSMENT:
The motor activity assessment (MA) was carried out in five animals per sex and group at the end of the administration period. Motor activity was measured on the same day as FOB was performed.


FEMALE REPRODUCTION DATA:
The pairing partners, the number of mating days until vaginal sperm were detected and gestational status were recorded for F0 females. For the females, mating, fertility and gestation indices were calculated for F1 litters. The total number of pups delivered and the number of liveborn and stillborn pups were noted, and the live birth index was calculated for F1 litters.

Fetal examinations:

PUP NUMBER AND STATUS AT DELIVERY:
All pups delivered from the F0 parents (F1 litter) were examined as soon as possible on the day of birth to determine the total number of pups, the sex and the number of liveborn and stillborn pups in each litter. At the same time, the pups were also examined for macroscopically evident changes. Pups, which died before this initial examination, were defined as stillborn pups.

PUP VIABILITY/MORTALITY:
In general, a check was made for any dead or moribund pups twice daily on workdays (once in the morning and once in the afternoon) or as a rule, only in the morning on Saturdays, Sundays or public holidays. The number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PND 1-4 (lactation period) were determined. Pups which died accidentally or were sacrificed due to maternal death were not included in these calculations. The number of live pups/litter was calculated on the day after birth, and on lactation day 4. The viability index was calculated.

SEX RATIO:
On the day of birth (PND 0) the sex of the pups was determined by observing the distance between the anus and the base of the genital tubercle; normally, the anogenital distance is considerably greater in male than in female pups. The sex of the pups was finally confirmed at necropsy.

PUP CLINICAL OBSERVATION:
The live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams.

PUP BODY WEIGHT DATA:
The pups were weighed on the day after birth (PND 1) and on PND 4. Pups' body weight change was calculated from these results. The individual weights were always determined at about the same time of the day (in the morning). Pup body weights and pup body weight change were listed for males, females and males + females. “Runts” were defined on the basis of the body weights on PND 1. "Runts" are pups that weigh less than 75% of the mean weight of the respective control pups.

Statistics:

Please refer to any other information on material and methods incl. tables

Indices:

Female: female mating index, female fertility index, gestation index
Live birth index, viability index, sex ratio

Historical control data:

Historical reproduction toxicity data and clinical pathology data were included in the report.

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
CLINICAL SIGNS AND MORTALITY:
No parental animal died prematurely in the present study. Brightly yellowish discolored urine and light yellow discolored feces were observed during the administration period at detailed clinical observations (DCO) in all dosed animals starting at DCO on study days 7 (urine) and 14 (feces).
The effects were related to the test substance but assessed as being non-adverse.

FOOD CONSUMPTION:
During the premating period food consumption was significantly decreased in female animals of test group 3 (1000 mg/kg bw/d), i.e. on study day 7 (-15%) and in mean of means (-11%). However, as the body weight gain of these animals did not show any impairment during the first week of treatment the finding was assessed as being spontaneous.

WATER CONSUMPTION:
No test substance-related, adverse findings were noted.

BODY WEIGHT DATA:
No test substance-related changes in body weight or body weight gain were observed for female animals of test groups 1-3 (100, 300 and 1000 mg/kg bw/d) when compared to control groups.

FUNCTIONAL OBSERVATION BATTERY:
Deviations from "zero values" were obtained in several rats. However, as most findings were equally distributed between test-substance treated groups and controls, were without a dose-response relationship or occurred in single rats only, these observations were considered to have been incidental.

MOTOR ACTIVITY MEASUREMENT:
There were no significant deviations concerning the overall motor activity (summation of all intervals) and regarding the single intervals in female animals of all test groups in comparison to the concurrent control group.

FEMALE REPRODUCTION AND DELIVERY DATA:
The female mating index calculated after the mating period for F1 litter was 100% for all test groups (0, 100, 300, 1000 mg/kg bw/d). The mean duration until sperm was detected in vaginal smears (GD 0) was 2.3, 2.3, 3.9 and 3.3 days in test groups 0-3 (0, 100, 300, 1000 mg/kg bw/d, respectively).

All sperm positive rats delivered pups with the exception of two females of test group 1 (100 mg/kg bw/d), two females of test group 2 (300 mg/kg bw/d) and one female of test group 3 (1000 mg/kg bw/d), which were mated with males but did not become pregnant. The female fertility index varied between 80% and 100%. The mean duration of gestation was between 22.0 and 22.2 days and did not show significant differences. The gestation index reached 100% in all test groups including the control group. The rate of liveborn pups was not affected by the test substance, as indicated by live birth indices of 99.2% (control group), 100.0% (test groups 1 and 2) and 99.0% (test group 3). Single stillborn pups were seen in control group 0 and test group 3 (1000 mg/kg bw/d). A relation to treatment was excluded.


HEMATOLOGY:
No treatment-related changes among hematological parameters were observed.

CLINICAL CHEMISTRY:
In rats of test group 3 (1000 mg/kg bw/d) creatinine values and, urea levels were increased. Potassium levels were higher in females of test groups 1 and 3 (100 and 1000 mg/kg bw/d) but the potassium mean in test group 1 was within the historical control range. Therefore, the potassium level alteration in females of test group 1 (100 mg/kg bw/d) was regarded as incidental and not treatment-related.

URINALYSES:
No treatment-related changes among urinalysis parameters were observed.

ABSOLUTE WEIGHTS:
When compared to the control group 0 (set to 100%), none of the mean absolute weight parameters showed significant differences.

RELATIVE ORGAN WEIGHTS:
When compared to the control group 0 (set to 100%), the mean relative weight of the kidneys was significantly increased in females of test group 3 (1000 mg/kg bw/d). All other mean relative weight parameters in females and all weight parameters in males did not show significant differences when compared to the control group 0. For the increased relative kidney weight in female animals of test group 3 a treatment-related effect could not be ruled out.

GROSS LESIONS:
A treatment-related yellow discoloration of contents was observed in the glandular stomach (all dose groups) and the cecum (1000 mg/kg bw/day). All findings occurred individually. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

FERTILITY:
The female animals, which were not pregnant as well as the male mating partners, did not show relevant gross lesions. In male mating partners of test group 3 (1000 mg/kg bw/d) the size of testes and of epididymides were reduced. One male animal of test group 3 (1000 mg/kg bw/d) showed an extreme (grade 5) diffuse degeneration of the testes that caused an aspermia in the epididymides. Therefore, the corresponding female animal was not pregnant. The occurrence of these findings was regarded to be incidental. All other females that were not pregnant as well as their male mating partners were not investigated histopathologically.

HISTOPATHOLOGY:

LIVER
In the centrilobular regions of the liver, the occurrence of single cell necrosis or apoptosis was minimally increased in female animals of test group 3 (1000 mg/kg bw/d). The increased number of single cell necroses or apoptotic bodies in animals of test group 3 (1000 mg/kg bw/d) was considered to be treatment-related.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
PUPS NUMBER AND STATUS AT DELIVERY:
The mean number of delivered pups per dam and the rate of liveborn and stillborn pups reflect the normal range of biological variation inherent in the strain used in this study.

PUPS VIABILITY/MORTALITY:
The viability index as indicator for pup mortality between postnatal day (PND) 0 and 4 was 100% for test groups 1, 2 and 3 (100, 300 and 1000 mg/kg bw/d). In the control group the viability index was decreased down to 99.2% because 1 pup was found dead. This decreased viability index was within the historical control data and reflected the normal range of biological variation inherent in the strain used in this study.

SEX RATIO:
The sex distribution and sex ratios of live F1 pups on the day of birth and on PND 4 did not show biologically relevant differences between test groups.

PUP CLINICAL OBSERVATION:
The surviving F1 pups of any test group did not show adverse clinical signs up to scheduled sacrifice on PND 4.

PUP BODY WEIGHT DATA:
No test substance-related significant changes in body weight or body weight change were observed for male and female pups of test groups 1-3 (100, 300 and 1000 mg/kg bw/d) when compared to control groups.
Two male runts were seen in 1 litter of the control group 0 on PND 1. Also, 1 male runt was observed in test group 2 (300 mg/kg bw/d) on PND 1 and each 2 female runts were detected in test groups 1 (100 mg/kg bw/d) and 3 (1000 mg/kg bw/d). All values were within the range of the biological variation inherent in the strain of rats used for this study.

PUPS NECROPSY OBSERVATIONS:
No test substance-related effects were observed.

Abnormalities:

not specified

Developmental effects observed:

not specified

STABILITY ANALYSES:

The stability of the test substance indrinking waterwas demonstrated over a period of 4 hours at room temperature. As the mixtures were stored no longer than this time period, the stability was guaranteed.

HOMOGENEITY CONTROL ANALYSES:

Considering the low relative standard deviation in the homogeneity analysis, it can be concluded that the test item was distributed homogeneously in drinking water.

CONCENTRATION CONTROL ANALYSES:

The concentration control analyses of all concentrations revealed that the values were in the expected range of the target concentrations, i.e. were always in a range of about 98-100% of the nominal concentrations. Taken together, the results demonstrate the correctness of the concentrations of the test item.

FOOD ANALYSES:

On the basis of duration of use and the analytical findings with respect to chemical and microbiological contaminants the diet was found to be suitable.

DRINKING WATER ANALYSES:

On the basis of the analytical findings the drinking water was found to be suitable.

BEDDING AND ENRICHMENT ANALYSES:

On the basis of the analytical findings the bedding and the enrichment are found to be suitable.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

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

Please refer to the discussion of "Effects on fertility" (see above).

Justification for selection of Effect on developmental toxicity: via oral route:
only available reliable study

Justification for classification or non-classification

Based on the results obtained from reproduction/developmental testing, the test substance is not considered to be subject to classification and labelling for toxicity to reproduction/development according to Directive 67/548/EEC (DSD) and Regulation (EC) No 1272/2008 (CLP).