Registration Dossier

Administrative data

Description of key information

The NOAEL for systemic effects after repeated oral exposure was found to be 10 mg/kg bw/day in dogs (chronic study).
In the same study, a NOAEL of 3 mg/kg bw/day for local effects was determined.
The NOAEL for local dermal effects was determined to be 6 mg/kg bw/day (0.045 mg/cm2) for the read across substance.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
chronic toxicity: oral
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP and OECD Guideline study For justification of read-across please refer to section 13.
Qualifier:
according to
Guideline:
OECD Guideline 452 (Chronic Toxicity Studies)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPP 83-1 (Chronic Toxicity)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
dog
Strain:
Beagle
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 8.5 to 9.5 months old
- Weight at study initiation: body weights ranged from 7.5 to 13.5 kg for the males and 6.8 to 9.7 kg for the females.
- Housing: individually housed in elevated stainless steel cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 3 weeks prior to initiation of treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 59 to 88
- Humidity (%): 11 to 96
- Photoperiod (hrs dark / hrs light): 12

IN-LIFE DATES:
From: 14 June 1989
To: 14 June 1990
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): A mixture of test material and canine diet was prepared weekly, portions of the diet mixture were diluted 9:1 (water:feed) daily for dose
administration.
- Mixing appropriate amounts with (Type of food): Purina® Certified Canine Diet Meal
- Storage temperature of food: Room temperature
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of dosing slurries that were prepared prior to the initiation of the study were analyzed for test article homogeneity and for Day 0 stability analyses. These samples were obtained from the top, middle, and bottom of dosing slurries prepared for each treatment group.
Samples of the dosing slurries prepared from dietary mixes 7 and 14 days after preparation of the dietary mixes from the pretreatment diet mix were evaluated for stability.
Samples of the test article/diet mix prepared pretreatment and weekly during the study were retained frozen. Samples of the dosing slurries for each group for Days 1, 8, 15, 22, 50, 78, 106, 134, 162, 190, 218, 246, 274, 302, 330, and 358 were analyzed for verification of concentration. Samples of the dosing slurries from Groups 1-3 for Days 2 and 3 were analyzed for verification of concentration because the values obtained on Day 1 were more than 10 % below target.
The analytical method used for the test material in a 9:1 water:feed slurry was UV-VIS spectrophotometry.
Results from the homogeneity analyses revealed that the dosing slurries were homogeneous having a % relative standard deviation (RSD) less than 5 %. Stability data also indicate that the test material was stable in feed for at least 14 days. Analyses of the prepared dosing slurries prepared on Day 1 revealed low concentrations of the test material for Groups 2 and 3 which were 66.5 and 86.2 % of target, respectively. These levels were then remixed on Day 2 and the resultant dosing slurries were found to still be low (80.6 and 81.6 % of target for Groups 2 and 3, respectively). These levels were again remixed on Day 3 and concentrations were then within acceptable limits. Verification of concentrations results for the remainder of the study indicated that the dosing slurries were within acceptable limits (+/- 10 % of target) with the exception of the Day 15 and Day 50 Group 3 mixes which were slightly lower (88.1 and 89.8 % of target, respectively).
Duration of treatment / exposure:
52 weeks
Frequency of treatment:
Two daily doses, 7 days/week
Remarks:
Doses / Concentrations:
0, 3, 10 and 30 (changed to 20 on Day 36) mg/kg bw/day
Basis:
nominal in diet
No. of animals per sex per dose:
4/sex
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: It was anticipated that at the higher dosage level(s), some toxicological or pharmacological effect(s) would be observed and that at the lower dosage level(s) no treatment-related effects would be seen
- Rationale for animal assignment: Random
- Rationale for selecting satellite groups: None
- Post-exposure recovery period in satellite groups: None
- Section schedule rationale: Random
Positive control:
Not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations and physical examinations were performed once each week. Physical examinations were performed weekly by laboratory personnel and at least once every 3 months by a staff veterinarian.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights data were collected weekly for all animals for the first 14 weeks and then every 2 weeks thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Time schedule: Food consumption data were collected weekly for all animals for the first 14 weeks and then every 2 weeks thereafter

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule: Ophthalmic examinations were performed prior to treatment and prior final sacrifice
- How many animals: Ophthalmoscopic examinations were performed on all animals prior to treatment and prior to termination using indirect ophthalmoscopy on all animals. A 1 % Mydriacyl solution was used for pupil dilation.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Prior to treatment and at termination. Blood samples were collected via the jugular vein.
- Anaesthetic used for blood collection: Yes (sodium thiamylal)
- Animals fasted: Yes (overnight)
- How many animals: All animals
- Parameters checked:
cell morphology
corrected leukocyte count
erythrocyte count
hematocrit
hemoglobin
platelet
mean cell volume
leukocyte count
leukocyte differential
mean cell hemoglobin
mean cell hemoglobin concentration
reticulocyte count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Prior to initiation of dosing and at Weeks 13, 26, and 52
- Animals fasted: Yes (overnight)
- How many animals: All animals
- Parameters checked:
alanine aminotransferase
albumin (ALBUMIN)
aspartate aminotransferase
blood urea nitrogen
calcium
chloride
creatine kinase
creatinine
gamma glutamyltransferase
globulin
glucose
inorganic phosphorus
potassium
sodium
total bilirubin
total cholesterol
total protein

URINALYSIS: Yes
- Time schedule for collection of urine: Prior to initiation of dosing and at Weeks 13, 26, and 52
- Collection of urine: Samples were collected from cagepans overnight
- Animals fasted: Yes (overnight)
- How many animals: All animals
- Parameters checked:
appearance
urine volume
specific gravity
occult blood
bilirubin
urobilinogen
microscopic examination of
sediment
fecal flotation (O & P; performed once prior to the initiation of dosing)
protein
pH
glucose
reducing substances
ketones
nitrites (
Sacrifice and pathology:
SACRIFICE AND GROSS PATHOLOGY
After 52 weeks of treatment, all surviving animals were weighed, anesthetized with sodium thiamylal, and exsanguinated. Necropsies were performed on each animal (including Group 2 male No. 26729 which was found dead) by trained personnel under the direct supervision of a pathologist.
Findings were recorded.
The necropsy included examination of the following:
external surface
all orifices
cranial cavity
carcass
external surface of the brain and spinal cord and the cut surfaces of the spinal cord (at necropsy); the cut surfaces of the brain were examined at the time of tissue trimming
nasal cavity and paranasal sinuses
thoracic, abdominal, and pelvic cavities and their viscera
cervical tissues and organs

ORGAN WEIGHTS
For each terminally sacrificed animal, the following organs (when present) were weighed following careful dissection and trimming to remove fat and other contiguous tissue in a uniform manner:
adrenals
brain with brainstem
heart
kidneys
liver with drained gallbladder
ovaries
pituitary
spleen
testes with epididymides
thyroid/parathyroids
Using these values, the organ-to-body-weight ratios were calculated.

TISSUE PRESERVATION
The following tissues (when present) from each animal were preserved in 10 % neutral-buffered formalin.
adrenals
aorta
brain with brainstem (medulla/pons, cerebellar cortex, and cerebral cortex)
mandibular and mesenteric lymph nodes
mid-thoracic spinal cord
ovaries
pancreas
cervical spinal cord
colon, cecum, rectum, duodenum, jejunum, ileum
esophagus
eyes (including optic nerve, eyes were fixed in a glutaraldehyde fixative)
femur including articular surface
gall bladder
heart
kidneys
lesions
liver (representative section from each lobe)
lumbar spinal cord
lung with mainstem bronchi (Lungs were inflated with formalin via the trachea)
mammary gland (female only)
pituitary
prostate
mandibular salivary glands
sciatic nerve
skin
skeletal muscle (thigh)
spleen
sternum with bone marrow
stomach
testes with epididymides
thymus
thyroid with parathyroids
trachea
urinary bladder (urinary bladders were inflated with formalin for examination after fixation)
uterus

HISTOPATHOLOGY
The aforementioned tissues were embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined microscopically from all animals.
Other examinations:
None.
Statistics:
Parametric variables were intercompared for the dose and control groups using Levene’s test for homogeneity of variance and by analysis of variance. Non-parametric data were transformed by log10, square, square root, reciprocal, angular (arcsine), or rank transformation. Dunnett’s test was used to compare significant results from the analysis of variance.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
There were generally higher incidences of emesis, salivation, and soft/mucoid/liquid faeces in the two higher dose groups than in the low dose group and control. The incidence of these clinical signs was high at 30 mg/kg bw/day but decreased to tolerable levels when dosage was lowered to 20 mg/kg bw/day. One animal in the 3 mg/kg bw/day died on Day 42 due to gavage error. All other animals survived to study termination.
Mortality:
mortality observed, treatment-related
Description (incidence):
There were generally higher incidences of emesis, salivation, and soft/mucoid/liquid faeces in the two higher dose groups than in the low dose group and control. The incidence of these clinical signs was high at 30 mg/kg bw/day but decreased to tolerable levels when dosage was lowered to 20 mg/kg bw/day. One animal in the 3 mg/kg bw/day died on Day 42 due to gavage error. All other animals survived to study termination.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 30 mg/kg bw/day, mean bw changes for Weeks 0-4 were significantly decreased for males and females compared to control groups. When the dose level was decreased, the body weight changes generally became comparable to or greater than the control values.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Mean total food consumption was significantly decreased in Week 1 for the 3 mg/kg bw/day males and females and in Weeks 1-4 for females only.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Slight, non-significant decreases in erythrocyte count, haemoglobin and haematocrit were observed in high dose males and females at 13, 26 and 52 weeks. No other differences from control were considered to be treatment-related.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Total cholesterol was significantly decreased in high dose group females at 13 weeks. Total protein was significantly decreased in high dose males at Week 52 and albumin was significantly decreased in this group.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: systemic effects
Dose descriptor:
NOAEL
Effect level:
3 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: local effects
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
10 mg/kg bw/day
Study duration:
chronic
Species:
dog
Quality of whole database:
GLP and OECD Guideline study

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

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: reliable study report, no guideline available For justification of read-across please refer to section 13.
Qualifier:
no guideline available
Principles of method if other than guideline:
The test material was applied dermally to the clipped skin of rats daily for a period of 14 days, 5 days/week. During this period and after the last application, skin reactions as signs of irritancy were recorded according to the Draize scale.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Sprague-Dawley, Inc., Indianapolis, IN, USA
- Age at study initiation: Approximately 7 weeks
- Weight at study initiation: 206.2-234.1 g
- Housing: 6/group
Type of coverage:
other: The treatment area was uncovered following the first 6 applications and covered following the last 4 applications.
Vehicle:
water
Details on exposure:
TEST SITE
- Area of exposure: dorsal area from the scapular region to just above the rump, approx. 5 cm wide

TEST MATERIAL
- Amount applied: 2.0 mL per kg body weight
- Concentration: 0, 0.03/0.6, 0.1, 0.3, 1.0 and 3.0 % (irritation was not observed during the first week of the study, animals which received a 0.03 % solution of test substance for the first five applications received a 0.6 % solution of test substance for the last five applications)
- Constant volume or concentration used: yes

DURATIN OF EXPOSURE
- Two weeks (10 applications), with the exception of the high-dose (3.0%) animals which were only dosed for 5 days due to severe skin irritation.

Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
no data
Duration of treatment / exposure:
10 applications except for the highest dose group: 5 applications, 6 hours/day
Frequency of treatment:
5 days/week
Remarks:
Doses / Concentrations:
0, 0.03/0.6, 0.1, 0.3, 1.0 and 3.0 % (animals which received a 0.03% solution of test substance for the first five applications received a 0.6% solution of test substance for the last five applications)
Basis:
other: active substance
No. of animals per sex per dose:
6
Control animals:
yes, concurrent vehicle
Positive control:
not required
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily

DERMAL IRRITATION: Yes
- Time schedule for examinations: The skin of the application site was evaluated for erythema and edema six hours after the 5th application, prior to the 6th application, six hours after the 10th application, and prior to sacrifice.
Sacrifice and pathology:
HISTOPATHOLOGY: The skin at the application site was collected and fixed for possible future histopathological examination.
Other examinations:
not applicable
Statistics:
not applicable
Clinical signs:
effects observed, treatment-related
Dermal irritation:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
not examined
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
No mortality occured. Perinasal and periocular encrustation were observed in all high-dose (3.0%) animals during the first week of the study and in one animal at the 0.1% level on Day 12. Hyperactivity lasting several minutes was observed in animals in the 1.0% treatment group immediately following dose application on Day 8.

DERMAL IRRITATION
Erythema and edema were found from the 1 % dose group onwards. No erythema or edema were present in the lower dose groups.
Dose descriptor:
NOAEL
Effect level:
6 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male
Basis for effect level:
other: local skin effects after 2 week application calculated from the concentration of 0.3 % a.s. in water at 2 mL/kg bw/day (3 mg/mL x 2 mL/kg bw/day = 6 mg/kg bw/d) corresponding to 0.045 mg/cm2
Critical effects observed:
not specified

It was assumed that the treated body surface was 10 %. The total surface body of rat (male and female) is 400 cm2 and the mean body weight is 300 g. Assuming that 10 % of the body surface has been exposed to the test substance, the resulting exposed area is of 40 cm2.

The NOAEC in mg/cm2 was calculated according to the following formula:

NOAEC in mg /cm2 =( Total dose applied in mg)/( Treated surface in cm2) =

(average animal weight in kg * dose in mg kg bw)/ (treated surface area in cm2)

NOAEC = (0.3 kg x 6 mg/kg bw/day) / 40cm2 = 0.045 mg/cm2

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
0.045 mg/cm²
Study duration:
subacute
Species:
rat
Quality of whole database:
reliable study, sufficient for assessment

Additional information

Systemic and local effects - oral

A study (key study) was carried out according to OECD Guideline 452 (Chronic Toxicity Studies) and EPA OPP 83-1 (Chronic Toxicity) using the read-across substance Didecyldimethylammonium chloride (DDAC). 32 purebred beagle dogs were divided into four groups (four/sex/group) and used to evaluate the chronic oral toxicity of test material over 52 weeks of treatment. The compound was administered by oral gavage in a 9:1 water: feed slurry given in two equally divided a.m and p.m doses. Initially dose levels of 0, 3, 10, and 30 mg/kg bw/day were administered. Daily observations, periodic body weights, food consumption, hematology, clinical chemistry, ophthalmoscopic examinations, gross pathologic examinations, selected organ weights and histopathologic examinations of selected organs were utilized to detect treatment-related effects. During the first 4.5 weeks of the study, several of the dogs in the 30 mg/kg bw/day dose group demonstrated potentially life threatening body weight and food consumption depressions and displayed an increased incidence of soft feces. Based upon these observations, the dosage level in this group was reduced from 30 mg/kg bw/day to 20 mg/kg bw/day. In addition, because in some cases, the body weight and/or food consumption depressions were rather severe, the dogs in this group were removed from treatment completely during study days 31-36 and then reinstated at the 20 mg/kg bw/day dose level. Thereafter, body weights, food consumption and body weight changes were comparable to controls. No treatment-related clinical signs were noted in the 3.0 mg/kg bw/day Group. One male in the 3.0 mg/kg bw/day group was found dead on day 42 due to an error in test item administration. In the 10 mg/kg bw/day and 20 mg/kg bw/day group an increased incidence of emesis, salivation and soft/mucoid/liquid feces was observed as compared to controls. Treatment-related changes in clinical pathology parameters were mild and limited to the high dose group. These changes consisted of slight decreases in mean erythrocyte counts, hemoglobin and hematocrit values and decreases in mean total cholesterol values (significant in females) and decreases in total protein and albumin values (males). No treatment-related changes were noted in ophthalmology findings, gross pathological examinations, organ weight data, or from histopathological examination of selected organs and tissues. Under the conditions of this study, 10.0 mg/kg bw/day was considered to be the no observed adverse effect level (NOAEL) for systemic toxicity.

However the clinical signs, on which the NOAEL derivation is based, are consistent with the irritation/corrosive properties of the test item and are due to local effects. The NOAEL for local effects was determined to be 3 mg/kg bw/d. The read across substance reacts immediately with the gut mucosa and only a limited amount becomes systemically available. At 10 mg/kg bw/d effects were limited to local effects, the occurrence of systemic effects (body weight decrease), is reported at 30 mg/kg bw/d.

In a 90-day repeated dose oral toxicity study the test item was administered to 10 rats/sex/dose group via feed. Dose groups were 0, 100, 500, 2000 ppm (active ingredient) per day, but the highest dose group was lowered on day 29 to 1000 ppm due to strong clinical effects. These values are corresponding to 0, 22, 113, 273 mg/kg bw/day (active ingredient). Animals in the mid-dose group were erroneously treated with the low dose and vice-versa starting from day 35; the exchanged allocation persisted till study termination. This deviation was not considered to impact the result of the study. During the study, no mortality occurred and no treatment related effects were observed concerning water consumption, ophthalmoscopic findings, haematology, clinical chemistry, organ weights. In addition no gross findings were made at necropsy. In the highest dose group hunched posture, fur staining and incidents of tiptoe gait were observed and weight gains were reduced. In the 500 ppm dose group, weight gains were found to be reduced during the first two weeks and weights remained lower when compared to the control group during the study. Females of the highest dose group exhibited an increase in startle reflex. In males of the highest dose group as well as of the 500 ppm dose group a higher incidence of haemosiderin accumulation in the kidneys was observed. Based on these findings, the NOAEL of the active ingredient was determined to be 100 ppm, which is equivalent to 22 mg/kg bw/day. This study was used as supporting study.

 

local effects - dermal

To assess the local dermal effects of the read across substance DDAC a 14 days skin irritation study was conducted with male Sprague Dawley rats. 6 male animals per dose group were treated with the test material for a period of 14 days, 5/days/week. The applied doses were 0, 0.03/0.6, 0.1, 0.3, 1.0 and 3.0 % active substance at a dose volume of 2mL/ kg body weight. Because irritation was not observed during the first week of the study, animals which received a 0.03% solution of test substance for the first five applications received a 0.6% solution of test substance for the last five applications. Duration of exposure per day was 6 hours. As vehicle, water was used. Dermal scores were recorded according to the Draize scale six hours after the 5th application, prior to the 6th application, six hours after the 10th application, and prior to sacrifice. Erythema and edema were found from the 1 % dose group onwards. No erythema or edema were present in the lower dose groups. No mortality occurred. Perinasal and periocular encrustation were observed in all high-dose (3.0%) animals during the first week of the study and in one animal at the 0.1% level on Day 12. Hyperactivity lasting several minutes was observed in animals in the 1.0% treatment group immediately following dose application on Day 8. As a result of the study, the NOAEL for local skin effects was determined to be 0.3 % active substance in water at a dose volume of 2 mL/kg bw/day, which corresponds to 6 mg/kg bw/day (0.045 mg/cm2).

 

 

 


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
reliable chronic repeated dose study, most appropriate for assessment

Justification for selection of repeated dose toxicity dermal - local effects endpoint:
only one study available

Justification for classification or non-classification

Based on the results of the available data, the test item is not classified and labelled for long-term toxicity according to Regulation (EC) No 1272/2008 (CLP).