[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

Description of key information

In a reliable combined repeated dose and reproductive/developmental toxicity screening test (OECD TG 422) with the registered dye by oral gavage in rats systemic toxicity occurred at a dose level of 1000 mg/kg bw/day. The NOAEL was determined to be 300 mg/kg bw/day. As specific target organs the kidney and the liver were affected at the highest dose tested (1000 mg/kg bw/day), however, without influence on classification and labelling.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

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 oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test material 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.

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

Observations and examinations performed and frequency:

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 gestation 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.

Sacrifice and pathology:

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 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:
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/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.

Statistics:

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

Details on results:

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.

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. 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.

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

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: There were signs of systemic toxicity at a dose level of 1000 mg/kg bw/day in animals of both sexes. Thus, the NOAEL for general systemic toxicity was 300 mg/kg bw/day in males and females.

Critical effects observed:

not specified

STABILITY ANALYSES:

The stability of the test substance in drinking 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

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Repeated dose toxicity: oral route

In a repeated dose oral toxicity study (BASF SE, 2013, OECD 422) the test item 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. 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 behaviour. 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 (passive effect). 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. 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 present study indicated the kidney and the liver as target organs triggered at the limit 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 and was without histopathological correlation. The effect in the liver was considered to be test item related and adverse. However, due to the high concentrations at which the effect was seen, a serious health risk for humans is not expected. In conclusion, based on the systemic effects at 1000 mg/kg bw/day the No Observed Adverse Effect Level (NOAEL) for males and females was established at 300 mg/kg bw/day.

 

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
only available reliable study

Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: liver; urogenital: kidneys

Justification for classification or non-classification

Based on the results of the repeated dose toxicity testing the test item does not need to be subjected to classification and labelling for danger of serious damage to health by prolonged exposure (R48) according to Directive 67/548/EEC (DSD) or for specific target organ toxicity after repeated exposure (STOT RE) according to Regulation (EC) No 1272/2008 (CLP).