Registration Dossier

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Description of key information

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
other: range finding study
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2002
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
No analytical analysis
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
- Purity: 50% test substance in water.
Species:
rat
Sex:
male/female
Route of administration:
oral: feed
Duration of treatment / exposure:
14 d
Remarks:
Doses / Concentrations:
0, 1250, 2500, 5000 ppm of test substance in the diet (i.e., equivalent to achieved dosages of 0, 112, 229 or 436 mg/kg/d for males and 0, 116, 229 and 427 mg/kg/d for females respectively)
Basis: nominal
No. of animals per sex per dose:
6 animals per sex per dose
Control animals:
yes
Details on study design:
Post-exposure period: None
Clinical signs:
no effects observed
Description (incidence and severity):
No relevant clinical signs were observed in any group during the study.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
When compared to controls, a slightly lower mean body weight gain was observed in all treated males and in females given 5000 ppm throughout the study.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Slightly lower mean food consumption was observed in all treated males and in females given 5000 ppm.
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
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
The few differences noted between treated and control animals in the absolute and relative weights of some organs (principally liver, kidneys and thymus) were considered to be of no toxicological importance, as they were minimal and were the contribution of very few individuals.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Macroscopic post mortem examinations: Caecum was distended with faeces in 2/6 males given 2500 ppm; in 4/6 males and all females given 5000 ppm. Ileum was distended with faeces in 1/6 males and 1/6 females given 5000 ppm. The relationship of the distension with faeces to the treatment with the test substance cannot be ruled out.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
> 5 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Key result
Critical effects observed:
not specified
Conclusions:
Under the study conditions, the rat NOAEL for systemic toxicity was > 5,000 ppm (i.e., equivalent to 436 and 427 mg/kg bw/day for males and females respectively).
Executive summary:

A study was conducted to determine the repeated dose oral toxicity of the test substance (50% purity) according to OECD Guideline 407, in compliance with GLP. The experiment performed was a 14 d range finding study in Sprague-Dawley rats at the dietary concentrations of 0, 1250, 2500 and 5000 ppm of test substance (equivalent to achieved dosages of 0, 112, 229 or 436 mg/kg bw/d for males and 0, 116, 229 or 427 mg/kg bw/d for females). Six males and 6 females per dose group were used. Besides lower food intake with related lower increase of body weight gain in all groups, due to the palatability of the test substance, no toxic effects were observed. Under the study conditions, the NOAEL for systemic toxicity was > 5000 ppm (equivalent to 436 and 427 mg/kg bw/d or 218 and 214 mg a.i./kg bw/d for males and females, respectively) (Chevalier, 2002).

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
From November 07, 2001 to October 23, 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
- Purity: 50% test substance in water
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
- Age: approximately 6 weeks at the start of treatment
- Weight at study initiation: mean weight 170 g males, 159 g females
- Number of animals: 10 males and 10 females per dose group (80 in total)
- Housing: 2 animals / cage
Sex:
male/female
Details on test animals and environmental conditions:
10 male, 10 female per dose group
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Remarks:
The test substance consisted of approximately 50% active in water
Details on oral exposure:
continous exposure via oral diet for 13 week (ad libitum)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Before the start of the treatment suitability of proposed preparation procedure was determined by chemical analysis of dietary admixtures. Mixtures were checked for homogeneity, stability and concentration: The concentration in samples (duplicate) taken from each dietary admixture (including the control) prepared for use in weeks 1, 4, 8 and 13 was determined.
- Chemical analysis of test substance in dietary admixtures:
Analysis for homogeneity showed a coefficient of variation of about 9% for levels between 400 and 5.000 ppm. Stability tests showed a deviation from initial values between -2% and +19% after 16d in closed bags, and between -3% and +8% after 9d in open feeders. The concentration in samples (duplicate) taken from each admixture, including the control prepared for use in weeks 1, 4, 8 and 13 was determined. Deviations from nominal concentrations were measured between -12% and +1%.
Duration of treatment / exposure:
13 weeks (93d)
Frequency of treatment:
Continously in feed
Remarks:
Doses / Concentrations:
0, 800, 2000, 5000 ppm of test substance in feed (i.e., equivalent to 0, 28, 68 and 166 mg a.i./kg bw/day for the males, and 0, 30, 74 and 188 mg a.i./kg bw/day for the females, based on food consumption and body weight information).
No. of animals per sex per dose:
10 male, 10 female per dose group
Control animals:
yes
Details on study design:
Post-exposure period: None
Observations and examinations performed and frequency:
- Clinical signs: Each animal was checked twice a d for mortality or signs of morbidity, and at least once a d, at approximately the same time for the recording of clinical signs. Detailed clinical observations were made on all animals outside the home cage, in a standard arena, once before the beginning of the treatment period and once a week until the end of the study. At the end of the treatment period all animals were evaluated 'blind' for functional observation battery (FOB)
- Body weight: Once a week
- Food consumption: Every cage, once a week. Calculated per animal per day.
- Ophthalmoscopic examination: all animals before treatment and control and high dose group animals at end of treatment.
- Haematology: RBC, HB, MCV,PCV, MCHC, MCH, PLAT, WBC with differential cell counts and morphology, PT
- Biochemistry: Na, K, Cl, Ca, Phos, Gluc, Urea, Bil, Prot, Alb, A/G, Chol, Trigl, AP, ASAT, ALAT
Sacrifice and pathology:
- Including organ weight and ratio organ weight to body weight.
- Macroscopic: Complete macroscopic post-mortem examination on all study animals. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues. Adrenals; Aorta; Brain (including medulla/pons; cerebellar and cerebral corte); Cecum; Colon; Duodenum; Epididymides; Esophagus; Heart; Ileum; Jejunum; Kidneys; Liver; Lungs with bronchi; Lymph nodes (mandibular and mesenteric); Mammary glands/area; Ovaries (with oviducts); Pancreas; Pituitary gland; Prostate (dorso-lateral and ventral); Rectum; Salivary glands (sublingual and submandibular); Sciatic nerve; Seminal vesicles (including coagulation gland); Skin; Spinal cord (cervical, thoracic and lumbar); Spleen; Sternum with bone marrow; Stomach with forestomach; Testes; Thymus; Thyroids with parathyroids; Trachea; Urinary bladder; Uterus (horns and cervi); Vagina.
(Preservation of tissue: Eyes with Harderian glands; Femoral bone with articulation; Tongue; Skeletal muscle).
- Microscopic: all tissues for animals of the control and high-dose groups (groups 1 and 4), all macroscopic lesions of all the animals of the low- and intermediate-dose groups.
Other examinations:
No post exposure recovery examinations included.
Statistics:
Depending on normal distribution (Kolmogorov-Lilliefors test, possibly after a logarithmic transformation of the values - except for organ weights) and following testing of homogeneity of variances between groups: Bartlett test (3 or more groups) or Fisher test (2 groups): 
If homogeneous Student test (2 groups) Dunnett test (3 or more groups), else Dunnett test (3 or more groups) Mann-Whitney/Wilcoxon test (2 groups)
If not normal distribution: Dunnett test.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- Body weight gain and food consumption: Normal in 800 and 2000 ppm group. The transient decrease in the first two weeks was caused by the palatability of the test substance in the diet. At 5000 ppm a lower body weight gain was noted (22% lower compared to controls for males and 20% females), which correlated to lower food consumption (12% lower food consumption in males and 7% in females).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- Body weight gain and food consumption: Normal in 800 and 2000 ppm group. The transient decrease in the first two weeks was caused by the palatability of the test substance in the diet. At 5000 ppm a lower body weight gain was noted (22% lower compared to controls for males and 20% females), which correlated to lower food consumption (12% lower food consumption in males and 7% in females).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):




- Gross pathology: No relevant findings.
- Histopathology: No relevant findings.
Haematological findings:
no effects observed
Description (incidence and severity):
No variations in haematological that could be ascribed to the treatment with the test substance (except in the 5000 ppm group females: decreased white blood count (-30%), with lower lymphocytes (-34%) and monocytes counts (-36%)).
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
- Functional observation battery: No signs of neurotoxicity in any group.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
- No differences of toxicological importance in the weights of any organ from the treated animals (except a lower absolute and relative weights for liver in males of the 5000 ppm group).
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
- NOEL: 2000 ppm; NOAEL: 5000 ppm
- No signs indicative of toxicity were observed in any group. At 5000 ppm the only effects was a decrease in body weight gain (statistically significant) correlating with lower food consumption due to the palatability of the test substance.
- Actual dose received by dose level by sex: Average over the whole dosing period 0, 28, 68 and 166 mg a.i./kg bw for the males, and 0, 30, 74 and 188 mg a.i./kg bw for the females, based on food consumption and body weight information.
- Statistical results:
At 5000 ppm (166 mg a.i./kg bw/d in males, 188 mg a.i./kg bw/d in females), the only effects was a decrease in body weight gain (statistically significant) correlating with lower food consumption.
Key result
Dose descriptor:
NOAEL
Effect level:
166 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male
Basis for effect level:
other: see 'Remark'
Key result
Dose descriptor:
NOAEL
Effect level:
188 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
female
Basis for effect level:
other: see 'Remark'
Key result
Critical effects observed:
no
Conclusions:
Under the study conditions, the rat NOAEL for systemic effects was established at the highest tested dose of 5000 ppm (i.e., equivalent to 166 and 188 mg a.i./kg bw/day for males and females, respectively).
Executive summary:

A study was conducted to determine the repeated dose oral toxicity of the test substance (50% purity) according to OECD Guideline 409, in compliance with GLP. This experiment consisted of a 90 d dietary study in Sprague-Dawley rats with test substance concentrations of 0, 800, 2000 and 5000 ppm (equivalent to 0, 28, 68 and 166 mg a.i./kg bw/d for the males and 0, 30, 74 and 188 mg a.i./kg bw/d for the females, based on food consumption and body weight information). Ten males and 10 females per dose group were used. No signs indicative of toxicity were observed in any group. At 5000 ppm, the only effect was a decrease in body weight gain (statistically significant) correlating with lower food consumption due to the low palatability of the test substance. Under the conditions of the study, the rat NOAEL for systemic effects was established at the highest tested dose of 5000 ppm (equivalent to 166 and 188 mg a.i./kg bw/d for males and females, respectively) (Chevalier, 2002).

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
other: range finding study
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2004
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
no
Limit test:
no
Specific details on test material used for the study:
- Batch no.: 667H302 
- Purity: ca. 49.9% test substance (CAS no.: 68424-85-1) in water.
Species:
dog
Strain:
Beagle
Sex:
male/female
Route of administration:
oral: feed
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
Phase I: 4x4 d incremental dose; phase II: 14 d
Remarks:
Doses / Concentrations:
Phase I: 500, 1000, 2000 or 4000 ppm of test substance in the diet for 4d each
Phase II: 2000 ppm of the test substance for 14d
Control animals:
no
Details on study design:
Post-exposure period: none
Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs noted during the two phases.

Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
During phase I, a moderate decrease in body weight was noted in the male especially when given 2000 and 5000 ppm of test substance and a slight decrease was observed in the female when given 5000 ppm test substance.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Phase I: A marked reduction in the food consumption was noted in the male at 2000 ppm and in the female at 5000 ppm.
Phase II: A moderate decrease in the food consumption was noted in the male and the female throughout the phase.
Achieved dosages: 
Phase I:  The respective mean achieved dosages for the dose-levels of 500, 1000, 2000 and 5000 ppm test substance were as follows: males: 12, 23, 8 and 47 mg/kg bw/day, and females: 17, 37, 58 and 42 mg/kg bw/day.
Phase II: The mean achieved dosages at the dose-level of 2000 ppm were as follows: males: 43 mg/kg bw/day (range: 27.2 - 60.3 mg/kg bw/day), and females: 53 mg/kg bw/day (range: 35 - 94.5 mg/kg bw/day).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
No relevant changes were noted in any haematological parameter of the treated animals.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Slight decrease in protein, albumin and triglyceride levels as well as in alkaline phosphatase activity were noted in the male and the female at the end of phase II.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related changes in organ weights.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic post-mortem examination: No relevant necropsy findings were noted.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Details on results:
At 500 and 1000 ppm, no overt signs of toxicity were noted. At 2000 ppm, a moderate to marked decrease in body weight and food consumption was observed in the male. At 5000 ppm, a slight to moderate decrease in body weight and food consumption was observed in the male and the female. No treatment-related laboratory or histopathological changes were noted. Taking into consideration these findings, one male and one female were treated daily with the test substance at the dose-level of 2000 ppm for 2 weeks (phase II). Reduced food consumption was noted throughout the treatment period. A slight decrease in protein, albumin and triglyceride levels and alkaline phosphatase activity was noted. Consequently, the dose-levels of 250, 500 and 1000 ppm of active test substance will be chosen for the 28-d toxicity study (CIT/Study No. 26145 TCC).
Key result
Dose descriptor:
LOAEL
Effect level:
2 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Reduced food consumption throughout the treatment period. A slight decrease in protein, albumin and triglyceride levels and alkaline phosphatase activity; 2000 ppm is equivalent to 43 and 53 mg/kg bw/day in male and female respectively.
Key result
Critical effects observed:
not specified
Conclusions:
Under the study conditions and after a range finding phase, one male and one female dogs were treated daily with the test substance at the dose-level of 2000 ppm (i.e., equivalent to 43 and 53 mg/kg bw/day in male and female respectively) for 2 weeks. Reduced food consumption was noted throughout the treatment period. A slight decrease in protein, albumin and triglyceride levels and alkaline phosphatase activity was noted. Consequently, the dose-levels of 250, 500 and 1000 ppm of active test substance were chosen for the 28-d toxicity study.
Executive summary:

A study was conducted to determine the repeated dose oral toxicity of the test substance (purity 49.9%) according to OECD Guideline 407. This dose range finding study was performed in Beagle dogs. In Phase I, four incremental doses of 500, 1000, 2000 and 5000 ppm were administered to 4 animals (2 males and 2 females). At 500 and 1000 ppm, no overt signs of toxicity were noted. At 2000 ppm, a moderate to marked decrease in body weight and food consumption was seen in the male. At 5000 ppm, a slight to moderate decrease in body weight and food consumption was observed in the male and female. No treatment-related laboratory or histopathological changes were noted. Taking into consideration these findings, one male and one female were then treated daily with the test substance at 2000 ppm (equivalent to 43 and 53 mg/kg bw/d (21.5 and 26.4 mg a.i./kg bw/d) in males and females respectively) for 2 weeks (Phase II). Under the study conditions, reduced food consumption was recorded throughout the treatment period. A slight decrease in protein, albumin and triglyceride levels and alkaline phosphatase activity was noted as well. Consequently, the dose levels of 250, 500 and 1000 ppm of active test substance were chosen for the 28 d toxicity study (Gaou, 2004).

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2006
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
No analytical analysis
Qualifier:
according to
Guideline:
OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity in Non-Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
- ca. 50% test substance (CAS no.: 68424-85-1) in water.
- Test substance (C12: 72.0%; C14: 27.7%; C16: 0.3%)
- Batch No. 667H3002
Species:
dog
Strain:
Beagle
Sex:
male/female
Route of administration:
oral: feed
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
4 weeks
Frequency of treatment:
Continuous
Remarks:
Doses / Concentrations:
500, 1000 and 2000 ppm of test substance in the diet (i.e., equivalent to 250, 500 and 1000 ppm of active test substance).
Control animals:
yes, plain diet
Details on study design:
Post-exposure period: None
Observations and examinations performed and frequency:
A total of 16 Beagle dogs, all less than 9 months old, were randomly allocated to three treated groups and one control group of two males and two females each. The three treated groups received the test substance containing 49.9% of the active substance. The test substance was given by dietary admixture at the concentrations of 500, 1000 and 2000 ppm, corresponding to approximately 250, 500 and 1000 ppm of active test substance, respectively. The control group received untreated diet under the same experimental conditions.
The homogeneity and stability of the active substance under the administration conditions was checked before the start of treatment. Concentrations of the active substance were measured in each dietary admixture in weeks 1 and 4.
The animals were checked daily for mortality and clinical signs. Body weight was recorded weekly. Food consumption was recorded and achieved dosages were calculated daily. Haematological and blood biochemical investigations were performed before the beginning and at the end of the treatment period.
Sacrifice and pathology:
On completion of the treatment period, all animals were killed and submitted to a full macroscopic post-mortem examination. Designated organs were weighed and selected tissue specimens were preserved. A microscopic examination was performed on selected tissues from animals of the control and high-dose groups and on macroscopic lesions from animals of intermediate-dose group.
Clinical signs:
no effects observed
Description (incidence and severity):
At the end of the treatment period, haematological and blood biochemical parameters did not differ between treated and control animals.
Mortality:
no mortality observed
Description (incidence):
No unscheduled death occurred during the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The body weight gain was similar in treated and control animals during the treatment period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The food consumption was not affected by the treatment.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
At the end of the treatment period, haematological parameters did not differ between treated and control animals.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
At the end of the treatment period, blood biochemical parameters did not differ between treated and control animals.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Key result
Dose descriptor:
NOEL
Effect level:
1 000 ppm
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: No treatment related effect up to the highest tested dose; 1000 ppm was corresponding to a mean actual dose level of 36.70 mg a.i./kg bw/day
Dose descriptor:
LOAEL
Effect level:
> 1 000 ppm
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: No treatment related effect up to the highest tested dose; 1000 ppm was corresponding to a mean actual dose level of 36.70 mg a.i./kg bw/day
Key result
Critical effects observed:
no
Conclusions:
Under the study conditions, the 28-d NOEL for systemic effects in Beagle dogs was established at the highest tested dose of 1000 ppm (i.e., corresponding to a mean actual dose level of 36.70 mg a.i./kg bw/day).
Executive summary:

A study was conducted to determine the repeated dose oral toxicity of the test substance (purity 49.9%) according to OECD Guideline 409, in compliance with GLP. The experiment consisted in a 28 d dietary study in Beagle dogs exposed to dose levels of 0, 500, 1000 and 2000 ppm, corresponding to approximately 0, 250, 500 and 1000 ppm a.i., respectively. Two males and 2 females per dose group were used. The homogeneity and stability of the test substance under the administration conditions was checked before treatment start. Concentrations were measured in each dietary admixture in Weeks 1 and 4. No treatment-related effects were observed up to the highest tested dose. Under the conditions of the study, the 28 d NOEL for systemic effects in Beagle dogs was established at the highest tested dose of 1000 ppm (corresponding to a mean actual dose of 36.70 mg a.i./kg bw/d) (Gaou, 2006).

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From June 18, 1987 to June 21, 1988
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPP 82-1 (90-Day Oral Toxicity)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
- Name of test material (as cited in study report): Alkyl dimethyl benzyl ammonium hloride
- Physical state: Pale yellow viscous liquid
- Analytical purity: 80.51 and 79.7% w/w active substance in aqueous/ethanol solution
- Lot/batch No.: Lot # 6158-59-60 (batch 1) and Lot # SC132-65 (batch 2)
- Stability under test conditions: The test substance is hydrolytically and photolytically stable under the conditions of this study and has been shown to be stable in aqueous, alcohol and alcohol/aqueous solutions for extended periods, e.g. at least five years under standard laboratory conditions.
- Storage condition of test material: Room temperature
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
Test animals
- Source: Sprague-Dawley CD rats were obtained from Charles River Breeding Laboratories, Portage, MI, USA.
- Age at study initiation: 7 weeks
- Weight at study initiation: 221.6-250.9 g (males); 147.7-174.4 g (females)
- Housing: Animals were housed one animal/side of divided stainless steel cages (solid sides with wire mesh floors) mounted in a stainless steel Maxi-Rack (Hazleton Systems, Inc., Aberdeen, MD).
- Diet: Ground Purina Certified Rodent Chow # 5002 (Ralstor Purina Co., St. Louis, MO), ad libitum
- Water: Tap water, ad libitum. Water was provided by an automatic watering system with demand control valves mounted on each cage rack
- Acclimation period: 1 week

Environmental conditions:
- Temperature: 22 ± 3°C
- Humidity: 40-70%
- Photoperiod: 12h light/12h dark

In-life dates: From: 18 June 1987 to: 22 September 1987

Route of administration:
oral: feed
Vehicle:
other: Ground Purina Certified Rodent Chow # 5002
Details on oral exposure:
Diet preparation: Test diets were prepared by direct addition of the test substance to ground rodent feed. A concentrated premix was prepared to ensure maximal loss of the ethanol (approximately 12% by weight) from the test substance during the original mixing time of 1h. Test diets were prepared by appropriate dilutions of the concentrated premix or higher diet concentrations.
- Rate of preparation of diet (frequency): Fresh diet was prepared and offered to the animals each week.
- Mixing appropriate amounts with (Type of food): Ground Purina Certified Rodent Chow # 5002
- Storage temperature of food: Diets were stored in polypropylene containers at room temperature.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Experimental diets were analyzed for stability, homogeneity and concentration of test substance in the diet by using liquid chromatography.
- Homogeneity studies performed on samples from all dietary concentrations indicated that test substance was uniformly distributed in the diet.
- Stability studies were conducted on diets (8000 and 100 ppm) stored at room temperature in closed polypropylene containers and in glass jars. These analyses indicated that the test substance was stable in the diets for at least 21 d under both storage conditions for all dose levels.
- Concentration verification analyses revealed that the test substance concentrations were 90.7 to 111.8% of nominal for all 5 concentrations.
Duration of treatment / exposure:
95 and 96 days for males and females, respectively.
Frequency of treatment:
Daily
Remarks:
Doses / Concentrations:
0, 100, 500, 1000, 4000 or 8000 ppm (equivalent to 0, 6, 31 and 62 mg/kg bw/day (males) and 0, 8, 38 and 77 mg/kg bw/day (females). [Due to mortality in the 4000 and 8000 ppm treatment groups, the corresponding daily intakes could not be calculated.]
Basis: nominal in diet
No. of animals per sex per dose:
15
Control animals:
yes, plain diet
Details on study design:
- Rationale for animal assignment: Animals were assigned to test groups, based on body weight, by a computer generated, weight stratified randomization procedure. Only rats with body weights within ± 20% of the population mean for each sex were used in the study.

Assignment of animals: The animals were assigned into following groups in the study:
- Group 1(Diet control): Plain diet
- Group 2: 100 ppm test substance in diet
- Group 3: 500 ppm test substance in diet
- Group 4: 1000 ppm test substance in diet
- Group 5: 4000 ppm test substance in diet
- Group 5: 8000 ppm test substance in diet


Observations and examinations performed and frequency:
MORTALITY and CLINICAL SIGNS: Yes
- Time schedule: Twice daily

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Animals were observed once a week for detailed clinical observations, and six days a week for overt clinical signs.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight data were collected weekly for all animals.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, food consumption data were collected weekly for all animals.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ophthalmic examinations were performed prior to treatment and prior to final sacrifice.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Just prior to sacrifice at study termination
- Anaesthetic used for blood collection: Yes (methoxyflurane)
- Animals fasted: Yes, overnight
- How many animals: Blood samples were collected from 10 animals/sex/group (or from the surviving animals in the 4000 ppm group) for haematology analyses.
- Parameters checked: Hemoglobin, hematocrit, erythrocyte count, erythrocyte indices, platelet count, total leukocyte count, differential leukocyte count, reticulocyte count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Just prior to sacrifice at study termination
- Animals fasted: Yes, overnight
- How many animals: Blood samples were collected from 10 animals/sex/group for clinical chemistry
- Parameters checked: AST (SGPT), ALT (SGOT), creatinine, alkaline phosphatase, gamma glutamyl transpeptidase, glucose, urea nitrogen, total protein, albumin, globulin, A/G ratio, total bilirubin, direct bilirubin, indirect bilirubin, calcium, phosphorous, sodium, potassium, chloride

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
SACRIFICE: Animals were anesthetized with methoxyflurane and exsanguinated by severing the brachial vessel.
GROSS PATHOLOGY: Yes, all animals (surviving) were subject to gross necropsy.
HISTOPATHOLOGY: Yes, histopathologic examinations were performed on selected tissues from 10 randomly selected animals/group from the control and 1000 ppm dose groups. The 1000 ppm group was selected for complete examination because it was the highest dose group without significant mortality.
- Tissues collected for histopathology: Gross lesions, brain, eyes, pituitary, salivary gland, heart, aorta, thymic region, thyroid, lungs, adrenals, trachea, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, spinal cord, pancreas, liver, kidneys, urinary bladder, testes, prostate, epididymis, ovaries, uterus, spleen, lymph nodes, sciatic nerves, sternum
Other examinations:
ORGAN WEIGHT: Prior to sacrifice, body weights were obtained to allow expression of relative organ weights. The liver, spleen, kidney, heart, brain, adrenals, testes and ovaries were weighed for all animals.
Statistics:
- Parametric variables were compared between the dose and control groups using Levene’s test for homogeneity of variances, by analysis of variance and by pooled variance t-tests.
- Non-parametric data were analyzed by the Kruskal-Wallis test or by the Wilcoxon rank sum test as modified by Mann-Whitney.
- Frequency data were compared using Fisher’s exact tests.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
All animals in the 8000 ppm group died. For the 4000 ppm group 12/15 males and 11/15 females died or were sacrificed in a moribund condition.

Mortality:
mortality observed, treatment-related
Description (incidence):
Treatment-related clinical findings were restricted to animals from the 4000 and 8000 ppm groups. The observations were of two types, general cachexia and loose faeces. Findings for the surviving animals were similar to those that died.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Decrease in body weight was observed for the 4000 and 8000 ppm dose group surviving the first week of the study. A slight but significant decrease was also noticed in the males of the 1000 ppm dose group.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Decrease in food consumption was observed for the 4000 and 8000 ppm dose group surviving the first week of the study. A slight decrease in the food consumption was also noticed in the males of the 1000 ppm dose group.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No treatment-related finding at any treatment level was observed.
Haematological findings:
no effects observed
Description (incidence and severity):
No treatment related change was observed for males or females from any treatment group (4000 ppm or lower).
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
No treatment related effect up to 1000 ppm. Significant increases in ALT and phosphorus were observed for 3 surviving males of the 4000 ppm group.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
No treatment related effect up to 1000 ppm was observed.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
No treatment related effect up to 1000 ppm was observed. Gross lesions related to the treatment were restricted to the animals that died in the 8000 and 4000 ppm group and to a lesser degree in the animals that survived the 4000 ppm group. The findings were fecal staining of perineal area, emaciation, colour changes of the organs, ileus consisting of distended fluid and gas-filled viscera.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histopathologic changes were observed for animals in the 4000 and 8000 ppm dose group and consisted of congestion of various organs or tissues, mucosal cell degeneration of the villus tips of small intestine and cecum, submucosal edema of stomach, splenic contraction and hepatocellular atrophy.
Histopathological findings: neoplastic:
not examined
Key result
Dose descriptor:
NOEL
Effect level:
31 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: the dose of 500 ppm is equivalent to 31 mg/kg bw/day for males and 38 mg/kg bw/day for females
Key result
Dose descriptor:
NOEL
Effect level:
38 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: the dose of 500 ppm is equivalent to 31 mg/kg bw/day for males and 38 mg/kg bw/day for females
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
62 mg/kg bw/day (nominal)
System:
other: GI tract, hepatobiliary and spleen (males)
Organ:
stomach
intestine
ileum
colon
liver
spleen
Treatment related:
yes
Dose response relationship:
yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
77 mg/kg bw/day (nominal)
System:
other: GI tract, hepatobiliary and spleen (females)
Organ:
stomach
intestine
ileum
colon
liver
spleen
Treatment related:
yes
Dose response relationship:
yes
Conclusions:
Under the study conditions, the rat NOAEL of the test substance in rats was considered to be 500 ppm in the diet, i.e., equivalent to 31 mg/kg bw/day for males and 38 mg/kg bw/day for females.
Executive summary:

A study was conducted to determine the repeated dose oral toxicity of the test substance (ca. 80% purity) according to OECD Guideline 408 and US EPA OPP 82 -1, in compliance with GLP. This sub-chronic oral toxicity study (90 d) was realised in Sprague-Dawley rats. The rats were administered daily dietary levels of 0, 100, 500, 1000, 4000 and 8000 ppm test substance, equivalent to 0, 6, 31 and 62 mg/kg bw/d (i.e. 0, 4.8, 25 and 50 mg a.i./kg bw/d) (males) and 0, 8, 38 and 77 mg/kg bw/d (i.e. 0, 6.4, 30 and 62 mg a.i./kg bw/d) (females) for 95 and 96 days, respectively. Daily intakes at 4000 and 8000 ppm could not be calculated due to high mortality. The animals were observed for mortality, clinical signs, body weight, food consumption, hematology and clinical chemistry at termination. Gross and histopathological examinations were also performed. A slight trend in reduction of body weight and food consumption in males at 1000 ppm was observed. Besides this, there were no treatment-related findings at 1000 ppm or lower. There was 80 and 100% mortality at 4000 and 8000 ppm, respectively. Clinical signs of toxicity (general cachexia and loose faeces), decreased food consumption and body weights, gross necropsy findings (including increased amounts of liquids or semi-solid material within the stomach, jejunum, ileum and cecum) were also observed at 4000 and 8000 ppm. The cause of morbidity and death was assumed to be shock secondary to fluid and/or ionic shifts in the gastro-intestinal tract. Under the study conditions, the NOAEL was considered to be 500 ppm in the diet, equivalent to 31 mg/kg bw/d (i.e. 25 mg a.i./kg bw/d) for males and 38 mg/kg bw/d (i.e. 30 mg a.i./kg bw/d) for females (Van Miller, 1988).

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity in Non-Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
- Test substance: ca. 50% C12-16-benzyldimethylammonium chloride (CAS no.: 68424-85-1) in water.
- C12-16 BKC (C12: 72.0%; C14: 27.7%; C16: 0.3%)
- Batch No. 667H3002
Species:
dog
Strain:
Beagle
Sex:
male/female
Route of administration:
oral: feed
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Continuous
Remarks:
Doses / Concentrations:
0, 500, 1500 and 3000 ppm of test substance in the diet. The mean achieved dosages of active substance, based on food consumption and body weight information were as follows: 0, 8, 25 and 50 mg/kg bw/day (males); 0, 9, 26 and 45 mg/kg bw/day (females).
No. of animals per sex per dose:
4 males and 4 females per dose group
Control animals:
yes, plain diet
Details on study design:
Post-exposure period: none
Observations and examinations performed and frequency:
Observations included: Clinical signs, mortality, body weight, food consumption, Ophthalmology, haematology, clinical chemistry and urinanalysis.
Sacrifice and pathology:
Pathology of all animals (organ weight, gross pathology) and histopathology on the control and high dose animals.
Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs were attributed to treatment with the test substance.

Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
No treatment related effects on the body weight gain were attributed to the test substance. A mean body weight loss was noted in females from the high-dose group when given 3000 ppm of test substance. This body weight loss was not dose-related and correlated to the decrease of food consumption recorded among the females of that dose group. When the dosing was reduced to 2500 ppm of test substance, a mean body weight gain, similar to that noted in the control females was recorded. Consequently, this effect was associated with the low appetency of the diet at this dose-level.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In the 500 and 1500 ppm dose group, the food consumption was unaffected in males and females. A markedly lower (-27%) food consumption was noted in females at the high dose group of 3000 ppm. After reduction of the dose-level to 2500 ppm test substance, the food consumption was only slightly lower (-6%). No effects seen at ophthalmoscopic examination, haematology, clinical chemistry and urine analysis.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
not examined
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
From week 8, the concentration of test substance was reduced to 2500 ppm (equivalent to 1250 ppm of active substance) in the high-dose female group, due to low food intake. The mean achieved dosages of active substance, based on food consumption and body weight information were as follows: 0, 8, 25 and 50 mg/kg bw/day (males); 0, 9, 26 and 45 mg/kg bw/day (females).
Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male
Basis for effect level:
other: No toxicologically significant effect; 1500 ppm (male) and 1250 ppm (female) were corresponding to 50 and 45 mg a.i./kg/ bwday, respectively.
Key result
Dose descriptor:
NOAEL
Effect level:
45 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
female
Basis for effect level:
other: No toxicologically significant effect; 1500 ppm (male) and 1250 ppm (female) were corresponding to 50 and 45 mg a.i./kg bw/day, respectively.
Key result
Critical effects observed:
no
Conclusions:
Under the study conditions, the 90-d NOAEL for systemic effects in Beagle dogs was established at the highest tested dose of 1500 or 1250 ppm active substance in males and females, respectively (i.e., corresponding to 50 or 45 mg a.i./kg bw/day, respectively).
Executive summary:

A study was conducted to determine the repeated dose oral toxicity of the test substance (ca. 50% purity) according to OECD Guideline 409, in compliance with GLP. This 90 d dietary study in Beagle dogs was performed at doses of 0, 500, 1500 and 3000 ppm (equivalent to 0, 250, 750 or 1500 ppm a.i.). Four males and 4 females per dose group were used. From Week 8, the concentration of test substance was reduced to 2500 ppm (1250 ppm of active substance) in the high dose female group due to low food intake. The mean achieved dosage of active substance, based on food consumption and body weight, were 0, 8, 25 and 50 mg a.i./kg bw/d for males and 0, 9, 26 and 45 mg a.i./kg bw/d for females. No treatment-related toxicologically significant effects were observed up to the highest tested dose. A mean body weight loss observed in females from the high dose group was not dose-related and correlated to the decrease of food consumption recorded among the females of that dose group. When the dosing was reduced to 2500 ppm of test substance, a mean body weight gain, similar to that noted in the control females, was recorded. Consequently, this effect was associated with the reduced palatability of the diet at this dose level. Under the conditions of the study, the 90 d NOAEL for systemic effects in Beagle dogs was established at the highest tested dose of 1500 or 1250 ppm a.i. in males and females, respectively (corresponding to 50 and 45 mg a.i./kg bw/d, respectively) (Guillaumat, 2006).

Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
Deviations:
yes
Remarks:
not considered to have compromised the validity or integrity of the study
Principles of method if other than guideline:
There were some deviations to the protocols:
- the temperature and relative humidity in the animal room were sometimes out of the target ranges
- the analysis of the dietary concentrations was performed in excess in week 104.
- for female D29585, only one sampled of mass was performed instead of two for masses 2110, 2118 and 1205
- for male D29335, because of a technical problem, macroscopic examination was performed after fixation with a pathologist
- rectum was not sampled for female D29626.
These deviations were not considered to have compromised the validity or integrity of the study.
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
- Test substance: ca. 50% C12-16-benzyldimethylammonium chloride (CAS no.: 68424-85-1) in water.
- Batch no 667H3002
- C12-16 BKC (C12: 72.0%; C14: 27.7%; C16: 0.3%)
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Route of administration:
oral: feed
Details on route of administration:
The dietary admixtures were supplied ad libitum. A fourth group of 60 male and 60 female rats received untreated diet under the same experimental conditions, and acted as toxicology (10 males and 10 females) and carcinogenicity (50 males and 50 females) control group.
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
- Three groups of 70 male and 70 female Sprague-Dawley rats were treated with the test substance (containing 49.9% of active substance), by dietary admixtures at constant concentrations of 1000, 2000 and 4000 ppm (corresponding to 499, 998 and 1996 ppm of active substance).
- 20 males and 20 females of each group were used for toxicological investigations and treated for 52 weeks. 50 males and 50 females of each group were used to investigate the carcinogenic potential and were treated for 104 weeks.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
52 weeks
Frequency of treatment:
Ad libitum
Remarks:
Doses / Concentrations:
0, 1000, 2000, 4000 ppm
No. of animals per sex per dose:
- 20 males and 20 females of each group were used for toxicological investigations and were treated for 52 weeks.
- 50 males and 50 females of each group were used to investigate the carcinogenic potential and were treated for 104 weeks.
- A group of 60 male and 60 female rats received untreated diet under the same experimental conditions, and acted as toxicology (10 males and 10 females) and carcinogenicity (50 males and 50 females) control group.
Control animals:
yes, plain diet
Details on study design:
Post-exposure period: none
Observations and examinations performed and frequency:
- Animals were checked at least twice daily for mortality and clinical signs. In addition, detailed clinical observations were made once a week. After 6 months of treatment, all animals were palpated every 2 weeks in order to record the time of onset, location, size, appearance and progression of palpable masses. Body weights were recorded once during the pre-treatment period, on Day 1 and then once a week during the first 13 weeks of the treatment period and then once every 4 weeks until the end of the study. Food consumption was recorded once a week during the first 13 weeks of the study, and then over 7-d periods once every 3 months between weeks 14 and 25, and thereafter once per month until the end of the study.
- Haematological, blood biochemical investigations and urinalysis were performed on all surviving animals of the toxicology sub-groups in weeks 12, 26 and 52. During weeks 52, 78 and 104, differential white blood cell counts were determined for all surviving animals of the control and high-dose carcinogenicity sub-groups.
Sacrifice and pathology:
Surviving animals were killed at the end of the 52-week treatment period for the toxicology sub-groups and at the end of the 104-week treatment period for the carcinogenicity sub-groups. All animals were submitted for a full macroscopic post-mortem examination. Designated organs were weighed and selected tissue specimens were preserved. A microscopic examination was performed on all masses, and on designated tissues of animals from the control sub-groups and from the sub-groups treated at 4000 ppm of test substance sacrificed at the end of the 52 or 104-week treatment periods, and from all animals that died prematurely.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
In the toxicology sub-groups:
- no treatment-related deaths or premature sacrifices occurred during the 52-week treatment period, no treatment-related clinical signs were observed during the 52-week treatment period.
- the mean body weight was significantly lower in the males treated at 4000 ppm than in the controls from week 2 to week 50, due to a lower body weight gain over all the study (from -15% between weeks 0 and 4, to finally -21% between weeks 0 and 50). This lower body weight gain was attributable to a slightly to moderately lower food consumption (ranging from 73.3 to 94.4% of that of controls over the toxicology study), with statistically significant differences occurring during several weeks.
-haematological, blood biochemistry and urinalysis parameters were not affected by the treatment at any dose-level.
-there were no relevant histopathological findings.
In the carcinogenicity sub-groups: -survival rate in animals treated with test substance was not statistically different from to controls. Further, mortality was comparable in terms of time of occurrence and cause. - no test substance-related clinical signs were observed in any treated group.
- the mean body weight was significantly lower in the males and females treated at 4000 ppm than in the controls from week 2 to week 13, due to a lower body weight gain (-14% and -10% for males and females, respectively). This lower body weight gain was attributable to slightly to moderately lower food consumption compared with controls, with statistically significant differences occurring during several weeks. From week 13 to termination, the body weight of the males treated at 4000 ppm was still significantly lower than that of control animals, and this was correlated to a 18% lower body weight gain over the 104 weeks when compared with controls, while the body weight and body weight gain of all other treated animals were in the range of control values.
-there were no differences in the number and localization of palpable masses in test-treated animals and controls.
-there were no significant differences in the differential white blood cell parameters of males and females treated at 4000 ppm, when compared with controls, whatever the sampling time (weeks 52, 78 and 104).
-the administration of the test substance did not induce neoplastic changes under the conditions of this study. Further, no non-neoplastic lesions were found which were considered to represent a treatment-related effect.
Key result
Dose descriptor:
NOEL
Effect level:
1 000 ppm
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
body weight and weight gain
other: see 'Remark'
Dose descriptor:
LOAEL
Effect level:
2 000 ppm
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
2 000 ppm
System:
other: body weight
Organ:
other: general: body weight
Treatment related:
yes
Dose response relationship:
yes
Conclusions:
Under the study conditions, there was a lower body weight gain at 2000 ppm active substance (significant only in males of 52 week chronic study part and in males and females in 104 week carcinogenicity group). Accordingly, the NOEL was established at 1000 ppm active substance (i.e., equivalent to 56 and 65 mg a.i./kg bw/day for males and females in chronic groups or 48 and 58 mg a.i./kg bw/day in males and females for carcinogenicity groups). Further, the test substance was not found to be carcinogenic.
Executive summary:

A study was conducted to determine the repeated dose oral toxicity study of the test substance (ca. 50% purity) according to OECD Guideline 453, in compliance with GLP. This experiment evaluated the chronic toxicity and carcinogenic potential of the test substance in a combined study. The test substance was administered daily to Sprague-Dawley rats by dietary admixture at the concentrations of 1000, 2000 and 4000 ppm (equivalent to 500, 1000 and 2000 ppm a.i.) for 52 weeks (toxicology sub-group) (equivalent to 28, 56 or 109 mg a.i./kg bw/d for males and to 33, 65 or 133 mg a.i./kg bw/d for females, respectively) and for 104 weeks (carcinogenicity sub-group) (corresponding to 24, 48 or 97 mg a.i./kg bw/d for males and 29, 58 or 119 mg a.i./kg bw/d for females). The test substance did not induce any treatment-related mortality or clinical signs when administered daily for 52 or 104 weeks. At 4000 ppm, the mean body weight and body weight gain of the males of the toxicology sub-group and of the males and females of the carcinogenicity sub-group were lower than that of the controls, correlating with slightly lower mean food consumption. There were no significant differences in haematological, biochemical and/or urinalysis parameters at any dose-level for animals of either sub-group, compared with controls. There were no macroscopic or microscopic findings attributable to the test substance at any dose levels. Under the study conditions, due to the observed lower body weight gain at 2000 ppm active substance (significant only in males of 52 week chronic study part; significant in males and females in 104 week carcinogenicity group), the NOEL was established at 1000 ppm active substance (equivalent to 56 and 65 mg a.i./kg bw/d for males and females for chronic groups or 48 and 58 mg a.i./kg bw/d in males and females for carcinogenicity groups) (Appelqvist, 2007).

Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
From March 22, 1988 to July 08, 1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.4300 (Combined Chronic Toxicity / Carcinogenicity)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
- Name of test material (as cited in study report): Alkyl dimethyl benzyl ammonium chloride (ADBAC)
- Physical state: Pale yellow viscous liquid
- Analytical purity: 81.09% active substance in aqueous/ethanol solution.
- Lot/batch No.: 7293K
- Stability under test conditions: The test substance is hydrolytically and photolytically stable under the conditions of this study and has been shown to be stable in aqueous, alcohol and alcohol/aqueous solutions for extended periods, e.g. at least five years under standard laboratory conditions.
- Storage condition of test material: Room temperature
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
Test animals:
- Source: Sprague-Dawley CD rats were obtained from Charles River Breeding Laboratories, Portage MI USA
- Age at study initiation: 8 weeks
- Weight at study initiation: 246.9-308.7 g (males) and 161.8-215.1 g (females)
- Housing: The animals were housed one animal/cage in divided stainless steel cages (solid sides with three mesh floors) mounted in a stainless steel Maxi-Rack (Hazleton Systems, Inc., Aberdeen, MD).
- Diet: Ground Purina Certified Rodent Chow # 5002 (Ralstor Purina Co., St. Louis, MO), ad libitum
- Water: Municipal water, ad libitum. Water was provided by an automatic watering system with demand control valves mounted on each rack.
- Acclimation period: One week

Environmental conditions:
- Temperature: 66-77 °F
- Humidity: 40-70%
- Air changes: 8/h
- Photoperiod: Fluorescent lighting was provided 12 h/d using an automatic timer.

In-life dates: From: 22 March 1988 to: 27 March 1990
Route of administration:
oral: feed
Vehicle:
ethanol
Details on oral exposure:
Diet preparation:
Test diets were prepared by direct addition of the test substance to ground rodent feed. A concentrated premix was prepared to ensure maximal loss of the ethanol (approximately 12% by weight) from the test substance during the original mixing time of 1h. Test diets were prepared by appropriate dilutions of the concentrated premix or higher diet concentrations.
- Rate of preparation of diet (frequency): Fresh diet was prepared and offered to the animals each week.
- Mixing appropriate amounts with (Type of food): Ground Purina Certified Rodent Chow # 5002
- Storage temperature of food: Diets were stored in polypropylene containers at room temperature.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Experimental diets were analyzed for stability, homogeneity and concentration of test substance by using liquid chromatography.
- Homogeneity studies performed on samples from all dietary concentrations indicated that test substance was uniformly distributed in the diet.
- Stability studies were conducted on diets (300 and 200 ppm) stored at room temperature in closed polypropylene containers and in glass jars. These analyses indicated that the test substance was stable in the diets for at least 14d in glass jars and stable for at least 21d in closed polyethylene containers.
- Concentration verification analyses of 87 samples (mean of duplicate assays) for the diets revealed that the test substance concentration ranged from 92.4 to 110.0% of nominal for all 3 concentrations.
Duration of treatment / exposure:
24 months (104 weeks)

Frequency of treatment:
Daily
Remarks:
Doses / Concentrations:
0, 300, 1000 or 2000 ppm test substance (i.e., equivalent to mean intake levels of 0, 13, 44, and 88 mg/kg bw/day (males) and 0, 17, 57 and 116 mg/kg bw/day (females).
Basis: nominal in diet
No. of animals per sex per dose:
60
Control animals:
yes, plain diet
Details on study design:
Rationale for animal assignment:
Animals considered suitable for study on the basis of pretest health screen. Evaluations for fecal parasites, clinical pathology, gross pathology, histology and serology conducted during the pretest period indicated that the animals were free of infectious disease and parasites. Animals were assigned to test groups, based on body weight, by a computer generated, weight stratified randomization procedure. Only rats with body weights within ± 20% of the population mean for each sex were used in the study.

Assignment of animals: The animals were assigned into following groups in the study:
- Group 1(Diet control): Plain diet
- Group 2 (Low dose): 300 ppm test substance in diet
- Group 3 (Mid dose): 1000 ppm test substance in diet
- Group 4(High dose): 2000 ppm test substance in diet
- Group 5 (Diet control): Plain diet


Observations and examinations performed and frequency:
MORTALITY/MORBIDITY: Yes
- Time schedule: Twice daily

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were performed once each week. Observations for overt clinical signs were made once daily on days when detailed observations were not conducted.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight data were collected weekly for the first 14 weeks and every other week thereafter.

FOOD CONSUMPTION: Yes
- Time schedule: Food consumption data were collected weekly for the first 14 weeks and every other week thereafter.

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ophthalmologic examinations were performed prior to study initiation and prior to final sacrifice.
- Dose groups that were examined: All animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At 26, 52, 78, and 104 weeks of study
- Animals fasted: Yes (overnight)
- How many animals: 15 animals/sex/group at 26, 52, 78, and 104 weeks of study and on all animals prior to necropsy.
- Parameters checked: Hemoglobin, hematocrit, erythrocyte count, erythrocyte indices, platelet count, total leukocyte count, differential leukocyte count, reticulocyte count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At 26, 52, 78, and 104 weeks of study
- Animals fasted: Yes (overnight)
- How many animals: 15 animals/sex/group at 26, 52, 78, and 104 weeks of study and on all animals prior to necropsy.
- Parameters checked: Glucose, urea nitrogen, creatinine, AST (SGPT), ALT (SGOT), creatine kinase, gamma glutamyl transpeptidase, alkaline phosphatase, total protein, total cholesterol, albumin, globulin, A/G ratio, total bilirubin, direct bilirubin, indirect bilirubin, calcium, phosphorous, sodium, potassium, chloride.

URINALYSIS: Yes
- Time schedule for collection of urine: At 25, 51, 77, and 103 weeks of study.
- How many animals: 15 animals/sex/group at 25, 51, 77, and 103 weeks of study.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: Color, appearance, specific gravity, total volume, pH, protein, glucose, ketone, bilirubin, blood, urobilirubinogen, microscopic elements.

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:
SACRIFICE: Animals were anesthetized with methoxyflurane and exsanguinated by severing the brachial vessel. 

GROSS PATHOLOGY: Yes, complete gross postmortem examination was performed on all animals.

HISTOPATHOLOGY: Yes, microscopic examination was performed on all tissues from the two controls and high dose animals as well as lungs, liver, kidneys, and gross lesions in the mid and low dose groups. In addition to gross lesions and tissue masses, the following tissues were examined: spinal cord, brain, pituitary, thyroid, thymic region, trachea, lungs, heart, salivary gland, liver, spleen, kidneys, adrenals, pancreas, testes, epididymis, prostate, seminal vesicles, ovaries, uterus, vagina, mammary gland, skin, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, urinary bladder, representative lymph nodes, peripheral nerve, sternum, femur, thigh musculature, eyes and aorta.
Other examinations:
Organ weight: The liver, kidneys, adrenals, spleen, brain with stem, heart, testes and ovaries were weighed for all animals sacrificed at termination.
Statistics:
Parametric variables were compared between the test and control groups using Leven’s test for homogeneity of variances, by analysis of variance and by pooled variance t-tests. Non-parametric data were analyzed by the Kruskal-Wallis test or by the Wilcoxon rank sum test as modified by Mann-Whitney. Frequency data were compared using Fisher’s exact tests. Mortality and mean days to first palpable mass were analysed using life-table analyses. The two control groups were treated as independent entities for statistical or other interpretative purposes.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
An increased incidence of loose faeces was noted in the male rats in all groups treated with the test substance. Based upon previous 14-day and 90-day dietary studies with the test substance, the increased incidence of loose faeces in this study was considered potentially treatment-related; however, the lack of a dose response relationship in incidence and the sporadic nature of the observation throughout all dose groups limited the importance of this finding. There were no other clinical signs observed in male rats considered to be treatment-related. No clinical signs observed in the female rats were considered to be related to treatment with test substance.
Mortality:
no mortality observed
Description (incidence):
No treatment-related findings were observed at any treatment level.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The mean absolute body weights of the 2000 ppm group male and female rats were statistically significantly decreased at most measurement periods from Week 1 to Week 26 (male) and Week 1 to 60 (female) and, while not consistently statistically significant, remained decreased throughout the study. No treatment related changes were observed in the other groups.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
A transient food consumption decrease was reported in the male rats in the 1000 ppm treatment group during the first few months of the study. Mean food consumption values for the 2000 ppm dose group were generally depressed 2 to 12% throughout the study.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
35.64 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male
Basis for effect level:
other: NOAEL of 44 mg/kg bw/day (i.e., equivalent to 35.64 mg a.i./kg bw/day). Based on the treatment related decrease in body weight and food consumption at the highest dose of 88 mg/kg bw/day.
Key result
Dose descriptor:
NOAEL
Effect level:
46.17 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
female
Basis for effect level:
other: NOAEL of 57 mg/kg bw/day (i.e., equivalent to 46.17 mg a.i./kg bw/day). Based on the treatment related decrease in body weight and food consumption at the highest dose of 116 mg/kg bw/day in females.
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
2 000 ppm
System:
other: body weight and food consumption
Treatment related:
yes
Dose response relationship:
yes
Conclusions:
Under the study conditions, the rat NOAEL for chronic toxicity was determined to be 1000 ppm in diet (44 mg/kg bw/day for males and 57 mg/kg bw/day for females i.e., equivalent to 35.64 and 46.17 mg a.i./kg bw/day respectively).
Executive summary:

A study was conducted to determine the repeated dose oral toxicity of the test substance (ca. 81% purity) according to OECD Guideline 453 and US EPA OPPTS 870.4300, in compliance with GLP. This two year combined dietary toxicity and carcinogenicity study was conducted in Sprague-Dawley CD rats. The test substance was administered rats (60/sex/group) at dose levels of 0, 300, 1000 or 2000 ppm test substance (equivalent to mean intake levels of 0, 13, 44, and 88 mg/kg bw/d in males and 0, 17, 57 and 116 mg/kg bw/d in females) in the diet daily for 104 weeks. There were two control groups of 60/sex/group each. The animals were observed twice daily and body weights and clinical findings were recorded periodically. Clinical pathology measurements (haematology, clinical chemistry and urinalysis) were made at 6, 12, 18 and 24 months. At termination, a thorough post-mortem examination was conducted on all animals. Histopathology was conducted on a full set of tissues and organs from all animals in the control and high dose groups as well as on selected organs from animals in the low and mid dose groups. An increased incidence of loose faeces in male rats was observed which was considered to be potentially treatment-related, however, was not of biological significance. A reduction in mean absolute bodyweights and food consumption was observed in males and females in the high dose group. No other treatment related effects were observed for clinical pathology, organ weights, gross and histopathology or ophthalmology. Based on the results of the study, the NOAEL for chronic toxicity was determined to be 1000 ppm in diet (44 mg/kg bw/d for males and 57 mg/kg bw/d for females, equivalent to 35.64 and 46.17 mg a.i./kg bw/d respectively)(Gill, 1991).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
47.5 mg/kg bw/day
Study duration:
subchronic
Species:
dog
Quality of whole database:
The information requirement for this tonnage band is sufficiently met with the available data.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Data waiving:
exposure considerations
Justification for data waiving:
other:
Reason / purpose:
data waiving: supporting information
Reason / purpose:
data waiving: supporting information
Critical effects observed:
not specified

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Data waiving:
exposure considerations
Justification for data waiving:
other:
Reason / purpose:
data waiving: supporting information
Reason / purpose:
data waiving: supporting information
Critical effects observed:
not specified

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Data waiving:
exposure considerations
Justification for data waiving:
other:
Reason / purpose:
data waiving: supporting information
Reason / purpose:
data waiving: supporting information
Critical effects observed:
not specified

Repeated dose toxicity: dermal - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Data waiving:
exposure considerations
Justification for data waiving:
other:
Reason / purpose:
data waiving: supporting information
Reason / purpose:
data waiving: supporting information
Critical effects observed:
not specified

Additional information

Oral

Study 1. A study was conducted to determine the repeated dose oral toxicity of the test substance (50% purity) according to OECD Guideline 407, in compliance with GLP. The experiment performed was a 14 d range finding study in Sprague-Dawley rats at the dietary concentrations of 0, 1250, 2500 and 5000 ppm of test substance (equivalent to achieved dosages of 0, 112, 229 or 436 mg/kg bw/d for males and 0, 116, 229 or 427 mg/kg bw/d for females). Six males and 6 females per dose group were used. Besides lower food intake with related lower increase of body weight gain in all groups, due to the palatability of the test substance, no toxic effects were observed. Under the study conditions, the NOAEL for systemic toxicity was > 5000 ppm (equivalent to 436 and 427 mg/kg bw/d or 218 and 214 mg a.i./kg bw/d for males and females, respectively) (Chevalier, 2002).

Study 2. A study was conducted to determine the repeated dose oral toxicity of the test substance (50% purity) according to OECD Guideline 409, in compliance with GLP. This experiment consisted of a 90 d dietary study in Sprague-Dawley rats with test substance concentrations of 0, 800, 2000 and 5000 ppm (equivalent to 0, 28, 68 and 166 mg a.i./kg bw/d for the males and 0, 30, 74 and 188 mg a.i./kg bw/d for the females, based on food consumption and body weight information). Ten males and 10 females per dose group were used. No signs indicative of toxicity were observed in any group. At 5000 ppm, the only effect was a decrease in body weight gain (statistically significant) correlating with lower food consumption due to the low palatability of the test substance. Under the conditions of the study, the rat NOAEL for systemic effects was established at the highest tested dose of 5000 ppm (equivalent to 166 and 188 mg a.i./kg bw/d for males and females, respectively) (Chevalier, 2002).

Study 3. A study was conducted to determine the repeated dose oral toxicity of the test substance (purity 49.9%) according to OECD Guideline 407. This dose range finding study was performed in Beagle dogs. In Phase I, four incremental doses of 500, 1000, 2000 and 5000 ppm were administered to 4 animals (2 males and 2 females). At 500 and 1000 ppm, no overt signs of toxicity were noted. At 2000 ppm, a moderate to marked decrease in body weight and food consumption was seen in the male. At 5000 ppm, a slight to moderate decrease in body weight and food consumption was observed in the male and female. No treatment-related laboratory or histopathological changes were noted. Taking into consideration these findings, one male and one female were then treated daily with the test substance at 2000 ppm (equivalent to 43 and 53 mg/kg bw/d (21.5 and 26.4 mg a.i./kg bw/d) in males and females respectively) for 2 weeks (Phase II). Under the study conditions, reduced food consumption was recorded throughout the treatment period. A slight decrease in protein, albumin and triglyceride levels and alkaline phosphatase activity was noted as well. Consequently, the dose levels of 250, 500 and 1000 ppm of active test substance were chosen for the 28 d toxicity study (Gaou, 2004).

Study 4. A study was conducted to determine the repeated dose oral toxicity of the test substance (purity 49.9%) according to OECD Guideline 409, in compliance with GLP. The experiment consisted in a 28 d dietary study in Beagle dogs exposed to dose levels of 0, 500, 1000 and 2000 ppm, corresponding to approximately 0, 250, 500 and 1000 ppm a.i., respectively. Two males and 2 females per dose group were used. The homogeneity and stability of the test substance under the administration conditions was checked before treatment start. Concentrations were measured in each dietary admixture in Weeks 1 and 4. No treatment-related effects were observed up to the highest tested dose. Under the conditions of the study, the 28 d NOEL for systemic effects in Beagle dogs was established at the highest tested dose of 1000 ppm (corresponding to a mean actual dose of 36.70 mg a.i./kg bw/d) (Gaou, 2006).

Study 5. A study was conducted to determine the repeated dose oral toxicity of the test substance (ca. 80% purity) according to OECD Guideline 408 and US EPA OPP 82 -1, in compliance with GLP. This sub-chronic oral toxicity study (90 d) was realised in Sprague-Dawley rats. The rats were administered daily dietary levels of 0, 100, 500, 1000, 4000 and 8000 ppm test substance, equivalent to 0, 6, 31 and 62 mg/kg bw/d (i.e. 0, 4.8, 25 and 50 mg a.i./kg bw/d) (males) and 0, 8, 38 and 77 mg/kg bw/d (i.e. 0, 6.4, 30 and 62 mg a.i./kg bw/d) (females) for 95 and 96 days, respectively. Daily intakes at 4000 and 8000 ppm could not be calculated due to high mortality. The animals were observed for mortality, clinical signs, body weight, food consumption, hematology and clinical chemistry at termination. Gross and histopathological examinations were also performed. A slight trend in reduction of body weight and food consumption in males at 1000 ppm was observed. Besides this, there were no treatment-related findings at 1000 ppm or lower. There was 80 and 100% mortality at 4000 and 8000 ppm, respectively. Clinical signs of toxicity (general cachexia and loose faeces), decreased food consumption and body weights, gross necropsy findings (including increased amounts of liquids or semi-solid material within the stomach, jejunum, ileum and cecum) were also observed at 4000 and 8000 ppm. The cause of morbidity and death was assumed to be shock secondary to fluid and/or ionic shifts in the gastro-intestinal tract. Under the study conditions, the NOAEL was considered to be 500 ppm in the diet, equivalent to 31 mg/kg bw/d (i.e. 25 mg a.i./kg bw/d) for males and 38 mg/kg bw/d (i.e. 30 mg a.i./kg bw/d) for females (Van Miller, 1988).

Study 6. A study was conducted to determine the repeated dose oral toxicity of the test substance (ca. 50% purity) according to OECD Guideline 409, in compliance with GLP. This 90 d dietary study in Beagle dogs was performed at doses of 0, 500, 1500 and 3000 ppm (equivalent to 0, 250, 750 or 1500 ppm a.i.). Four males and 4 females per dose group were used. From Week 8, the concentration of test substance was reduced to 2500 ppm (1250 ppm of active substance) in the high dose female group due to low food intake. The mean achieved dosage of active substance, based on food consumption and body weight, were 0, 8, 25 and 50 mg a.i./kg bw/d for males and 0, 9, 26 and 45 mg a.i./kg bw/d for females. No treatment-related toxicologically significant effects were observed up to the highest tested dose. A mean body weight loss observed in females from the high dose group was not dose-related and correlated to the decrease of food consumption recorded among the females of that dose group. When the dosing was reduced to 2500 ppm of test substance, a mean body weight gain, similar to that noted in the control females, was recorded. Consequently, this effect was associated with the reduced palatability of the diet at this dose level. Under the conditions of the study, the 90 d NOAEL for systemic effects in Beagle dogs was established at the highest tested dose of 1500 or 1250 ppm a.i. in males and females, respectively (corresponding to 50 and 45 mg a.i./kg bw/d, respectively) (Guillaumat, 2006).

Study 7. A study was conducted to determine the repeated dose oral toxicity study of the test substance (ca. 50% purity) according to OECD Guideline 453, in compliance with GLP. This experiment evaluated the chronic toxicity and carcinogenic potential of the test substance in a combined study. The test substance was administered daily to Sprague-Dawley rats by dietary admixture at the concentrations of 1000, 2000 and 4000 ppm (equivalent to 500, 1000 and 2000 ppm a.i.) for 52 weeks (toxicology sub-group) (equivalent to 28, 56 or 109 mg a.i./kg bw/d for males and to 33, 65 or 133 mg a.i./kg bw/d for females, respectively) and for 104 weeks (carcinogenicity sub-group) (corresponding to 24, 48 or 97 mg a.i./kg bw/d for males and 29, 58 or 119 mg a.i./kg bw/d for females). The test substance did not induce any treatment-related mortality or clinical signs when administered daily for 52 or 104 weeks. At 4000 ppm, the mean body weight and body weight gain of the males of the toxicology sub-group and of the males and females of the carcinogenicity sub-group were lower than that of the controls, correlating with slightly lower mean food consumption. There were no significant differences in haematological, biochemical and/or urinalysis parameters at any dose-level for animals of either sub-group, compared with controls. There were no macroscopic or microscopic findings attributable to the test substance at any dose levels. Under the study conditions, due to the observed lower body weight gain at 2000 ppm active substance (significant only in males of 52 week chronic study part; significant in males and females in 104 week carcinogenicity group), the NOEL was established at 1000 ppm active substance (equivalent to 56 and 65 mg a.i./kg bw/d for males and females for chronic groups or 48 and 58 mg a.i./kg bw/d in males and females for carcinogenicity groups) (Appelqvist, 2007).

Study 8. A study was conducted to determine the repeated dose oral toxicity of the test substance (ca. 81% purity) according to OECD Guideline 453 and US EPA OPPTS 870.4300, in compliance with GLP. This two year combined dietary toxicity and carcinogenicity study was conducted in Sprague-Dawley CD rats. The test substance was administered rats (60/sex/group) at dose levels of 0, 300, 1000 or 2000 ppm test substance (equivalent to mean intake levels of 0, 13, 44, and 88 mg/kg bw/d in males and 0, 17, 57 and 116 mg/kg bw/d in females) in the diet daily for 104 weeks. There were two control groups of 60/sex/group each. The animals were observed twice daily and body weights and clinical findings were recorded periodically. Clinical pathology measurements (haematology, clinical chemistry and urinalysis) were made at 6, 12, 18 and 24 months. At termination, a thorough post-mortem examination was conducted on all animals. Histopathology was conducted on a full set of tissues and organs from all animals in the control and high dose groups as well as on selected organs from animals in the low and mid dose groups. An increased incidence of loose faeces in male rats was observed which was considered to be potentially treatment-related, however, was not of biological significance. A reduction in mean absolute bodyweights and food consumption was observed in males and females in the high dose group. No other treatment related effects were observed for clinical pathology, organ weights, gross and histopathology or ophthalmology. Based on the results of the study, the NOAEL for chronic toxicity was determined to be 1000 ppm in diet (44 mg/kg bw/d for males and 57 mg/kg bw/d for females, equivalent to 35.64 and 46.17 mg a.i./kg bw/d respectively) (Gill, 1991).

 

Justification for classification or non-classification

Based on the observed effects and the available NOAELs and LOAELs from the repeated dose studies, it can be concluded that the test substance does not require classification for this endpoint according to CLP (EC 1272/2008) criteria.