Reaction mass of bis(N-decyl-N,N-dimethyldecan-1-aminium) carbonate and N-decyl-N,N-dimethyldecan-1-aminium hydrogen carbonate

Endpoint summary

Description of key information

Standard repeated dose studies have been conducted using the structurally-related substance Didecyldimethylammonium chloride (DDAC) in rats, mice and dogs via the oral route and in rats via the dermal route. As both source and target chemicals have identical organic cations with hydrophobic side chains - the only difference is the inorganic anion carbonate or chloride with negligible contribution to the hazard properties - both substances can be predicted to have similar movement and fate characteristics. In addition effects of repeated dermal exposure to the test item Didecyldimethylammonium carbonate (DDA carbonate) have been investigated in a non-guideline GLP dermal study (21-Day Repeated Dose Dermal Irritation Study in Female Rats). A summary on the studies presented and results obtained is shown in the discussion section below. 

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:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

- Reliability: GLP study - Read-across justification: As both source (Didecyldimethylammonium chloride, DDAC) and target chemicals (Didecyldimethylammonium carbonate, DDA carbonate) have identical organic cations with hydrophobic side chains - the only difference is the inorganic anion carbonate or chloride with negligible contribution to the hazard properties- both substances can be predicted to have similar movement and fate characteristics.

Qualifier:

according to guideline

Guideline:

OECD Guideline 452 (Chronic Toxicity Studies)

Qualifier:

according to guideline

Guideline:

EPA OPP 83-1 (Chronic Toxicity)

GLP compliance:

yes

Limit test:

no

Species:

dog

Strain:

Beagle

Sex:

male/female

Details on test animals or test system 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: feed

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 (if not random): Random
- Rationale for selecting satellite groups: None
- Post-exposure recovery period in satellite groups: None
- Section schedule rationale (if not random): 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 (if feeding study):
- 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 (COR WBC)
erythrocyte count (RBC)
hematocrit (HCT)
hemoglobin (HGB)
platelet (PLATELET)
mean cell volume (MCV)
leukocyte count (WBC)
leukocyte differential
mean cell hemoglobin (MCH)
mean cell hemoglobin concentration (MCHC)
reticulocyte count (RETIC)

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 (ALT)
albumin (ALBUMIN)
aspartate aminotransferase (AST)
blood urea nitrogen (BUN)
calcium (CALCIUM)
chloride (CHLORIDE)
creatine kinase (CK)
creatinine (CREAT)
gamma glutamyltransferase (GGT)
globulin (GLOBULIN)
glucose (GLUCOSE)
inorganic phosphorus (IN PHOS)
potassium (POTAS)
sodium (SODIUM)
total bilirubin (T BILl)
total cholesterol (T CHOL)
total protein (T PROT)

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 (UTRANS, UCOLOR)
urine volume (U VOL)
specific gravity (SP GR)
occult blood (OC BLD-U)
bilirubin (BILIRUB)
urobilinogen (UROBIL)
microscopic examination of
sediment
fecal flotation (O & P; performed once prior to the initiation of dosing)
protein (PROTEIN)
pH (PH)
glucose (GLUCOSE)
reducing substances (RED SUBS)
ketones (KETONES)
nitrites (NITRITE)

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 (uinary 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.

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):

see details below

Urinalysis findings:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Details on results:

MORTALITY AND CLINICAL OBSERVATIONS
One animal, Group 2 male No. 26729, was found dead on Day 42 of study. The cause of death was considered to be gavage error. All other animals survived to study termination.
There were generally higher incidences of emesis, salivation, and soft/mucoid/liquid feces in Groups 3 and 4 than in Groups 1 and 2. The incidence of salivation, emesis, and soft/mucoid/liquid feces was high in the Group 4 animals when they were receiving 30 mg/kg bw/day and decreased to tolerable levels after the dosage was lowered to 20 mg/kg bw/day. Other observations that were noted and did not appear to be related to treatment included (but were not limited to): lacrimation, rough haircoat, injected sclera, and thin appearance. Observations made during the quarterly veterinary physical examinations were generally comparable to those noted weekly.

BODY WEIGHTS AND FOOD CONSUMPTION
Group 4 mean body weight change values for Weeks 0-4 were significantly decreased for both males and females compared to the control values. However, when the dose level was decreased, the Group 4 mean body weight change values generally became comparable to or greater than control values. The Group 4 female value for Weeks 5-13 was significantly increased when compared to the female control value for the same interval.
Group mean total food consumption at Week 1 was significantly decreased for Groups 2 and 4 females and also Group 2 females at Week 3. The statistical analysis for Weeks 1-4 also yielded significant decreases for the Groups 2 and 4 females.

OPHTHALMOLOGY
The ophthalmoscopic observations noted at the termination of the study were also observed at the initial examination. There were not any ophthalmoscopic abnormalities related to the compound or dose level.

CLINICAL PATHOLOGY
Evaluation of the hematology data revealed slight, nonsignificant decreases in red cell mass (erythrocyte count, hemoglobin, and hematocrit) in the Group 4 males and females at Weeks 13, 26, and 52.
The apparent significant decreases in absolute eosinophil count and absolute lymphocyte count in the Groups 2 through 4 females at Week 13 appear to be due primarily to a slightly elevated control group value at Week 13, as well as generally lower values in Groups 2 through 4 at the pretreatment interval. Incidental significant decreases occurred in the Group 4 male leukocyte count and corrected leukocyte count at Week -2.
The significantly decreased platelet count in the Group 3 females at Week 26 was considered to be spurious, based on the low magnitude of the change, the inconsistent occurrence across intervals, presence of similar differences at the pretreatment interval, and the lack of a dose response.
Evaluation of the clinical chemistry data revealed significantly decreased total cholesterol in the Group 4 females at Week 13, and non-significantly decreased mean values in the Group 4 animals at the remaining intervals of analysis during the treatment period. Total protein was significantly decreased in the Group 4 males at Week 52, while albumin was significantly decreased in the Group 4 males at all intervals of analysis during the treatment period. In the females, total protein and albumin were significantly decreased in Group 3 at Week 26, apparently due to reduced levels at the pretreatment interval. Sporadic increases in the mean aspartate aminotransferase and alanine aminotransferase values occurred in control and treated animals at several intervals, without any clear relationship to treatment.
Considerable variation was seen in individual creatine kinase values, however, most creatine kinase values were generally comparable to the range of values noted at the pretreatment interval and/or the range of control group values at the various intervals.
The significant increases in potassium in the groups 2 and 4 females at Week 52 were considered to be incidental, based on the low magnitude of the change, the inconsistent occurrence across intervals, and the lack of a dose response.
Urinalyses were generally comparable between control and treated groups. The fecal parasite exam performed at Pretest -1 for Group 3 female 26741 revealed the presence of coccidia. All other animals had negative findings for fecal ova and parasites.

GROSS PATHOLOGY AND ORGAN WEIGHTS
All gross pathology findings were considered to be incidental and commonly occurring spontaneous changes in the dog. There were no significances noted for organ weight data.

HISTOPATHOLOGY
Microscopic changes observed in treated animals were considered consistent with commonly occurring spontaneous and/or technique related processes in the dog. No test compound related changes were observed in animals which received up to 20 (reduced from 30) mg/kg bw/day.

Dose descriptor:

LOAEL

Effect level:

20 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Critical effects observed:

not specified

Conclusions:

The purpose of this study was to evaluate the chronic toxicity of the test material when administered by gastric gavage to dogs at doses ranging from 3.0 - 30.0 mg/kg bw/day. Previous dose range finding studies in which the test material had been administered in the diet or by oral gavage had shown that 30 mg/kg bw/day was the maximum dose which could be administered orally to dogs. In this study test material given to dogs, at a dose of 30 mg/kg bw/day (administered twice daily) produced salivation, frequent emesis, weight loss, and decreased food consumption in the males and females. Because these effects were rather severe, the dogs in this group were removed from treatment completely during study days 31-36 and then reinstated at a slightly reduced dose level of 20 mg/kg bw/day. At the 20 mg/kg bw/day level, these signs subsided to a tolerable level and all dogs in this group survived to terminal sacrifice. At the 10 mg/kg bw/day dose levels, an increased incidence of emesis, salivation and soft/mucoid/liquid feces was observed as compared to controls. No consistent treatment-related effects
were observed at the 3.0 mg/kg bw/day dose level.
In conclusion, chronic administration of test material at doses ranging from 3.0 - 20 mg/kg bw/day was not associated with mortality, changes in organ weights, gross pathological findings, ophthalmoscopic changes, or microscopic changes in selected organs and tissues. The 20 mg/kg bw/day dose level was associated with slight decreases in mean erythrocyte counts, and hemoglobin and hematocrit values, and decreases in mean total cholesterol, total protein and albumin values. In addition, the 20 and 10 mg/kg bw/day dose levels were associated with an increased incidence of emesis, salivation and soft/mucoid/liquid feces as compared to controls. The 3.0 mg/kg bw/day dose level for 52-weeks did not elicit any consistent or apparent treatment-related changes.
Therefore, under the conditions of this study, 10.0 mg/kg bw/day was considered to be the no-observable effect level for systemic toxicity.

Executive summary:

A study was carried out according to OECD Guideline 452 (Chronic Toxicity Studies) and EPA OPP 83-1 (Chronic Toxicity) using the structural analog Didecyldimethylammonium chloride (DDAC). In view of the chemical and structural similarities (the relevant chemical part of both, DDAC and DDACarbonate, under the conditions of this test is the common quaternary ammonium cation Didecyldimethylammonium+), it is considered that the data are adequate for DDACarbonate.

Thirty two 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. However, during the first four and onehalf 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. 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.

The dosage levels evaluated in this study were selected on the basis of two previous dose range finding studies. In one of the previous dose range finding studies, the test material was administered in the diet at dosage levels of 30, 60, and 90 mg/kg bw/day. In the other study, test material was administered by oral gavage at dosage levels between 7.5 and 60.0 mg/kg bw/day. These two preliminary studies were considered to be necessary since it was known that diet rejection and/or emesis rather than systemic toxicity were the limiting factors regarding the quantity of test material which could be administered to dogs orally. The purpose of these preliminary studies, therefore, was to determine the method of oral administration that would allow for the maximum amount of test material to be administered as well as selecting dose levels for this chronic study. The results of the preliminary studies indicated that administration by oral gavage using a daily divided dose regimen allowed for the largest quantity of test material to be administered orally.

No treatment-related clinical signs were noted in the 3.0 mg/kg bw/day Group. One male (26729) in the 3.0 mg/kg bw/day group was found dead on day 42 of study as the result of aspirating the water:feed slurry into the lungs. A transient decrease in mean total food consumption values (approximately 82% of control values) were noted for females receiving 3.0 mg/kg bw/day during weeks 1-4 probably a reflection of adapting to the dosing procedure. 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. During weeks 1-4, animals which were receiving 30 mg/kg bw/day exhibited weight loss as evidenced by significantly lower mean body weight change values. This weight loss was accompanied by a significant decrease in mean total food consumption values in the Group 4 females. When dosing was discontinued in these dogs, weight loss subsided, and when dosing was reinstated at 20 mg/kg bw/day a significant weight gain was noted for females during weeks 5-13. Thereafter, body weights, food consumption and body weight changes were comparable 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-observable effect level (NOEL) for systemic toxicity.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

10 mg/kg bw/day

Study duration:

chronic

Species:

dog

Quality of whole database:

Good quality database.

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

Link to relevant study records

Reference

Endpoint:

sub-chronic 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: see 'Remark'

Remarks:

- Reliability: GLP study according to international guidelines - Read-across justification: As both source (Didecyldimethylammonium chloride, DDAC) and target chemicals (Didecyldimethylammonium carbonate, DDA carbonate) have identical organic cations with hydrophobic side chains - the only difference is the inorganic anion carbonate or chloride with negligible contribution to the hazard properties- both substances can be predicted to have similar movement and fate characteristics.

Qualifier:

according to guideline

Guideline:

EPA OPP 82-3 (Subchronic Dermal Toxicity 90 Days)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 8 weeks
- Weight at study initiation: males: 221.3-264.5 g; females: 166.7-219.11 g
- Fasting period before study: none
- Housing: individually housed in stainless steel cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 3 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 66 to 75
- Humidity (%): 40 and 70
- Photoperiod (hrs dark / hrs light): 12

IN-LIFE DATES:
From: 11 January 1987
To: 12 April 1987

Type of coverage:

occlusive

Vehicle:

water

Details on exposure:

TEST SITE
- Area of exposure: 4" x 4"
- % coverage: 100 %
- Type of wrap if used: Vetrap® Bandaging Tape
- Time intervals for shavings or clippings: Weekly and as needed

REMOVAL OF TEST SUBSTANCE
- Washing: After removal of the wraps, the application site was rinsed. Water was liberally applied to the treatment area and the area was blotted using a 4" x 4" (8-ply) gauze pad.
- Time after start of exposure: 6 hours

TEST MATERIAL
- Amount(s) applied: 2.0 ml/kg bw
- Concentration (if solution): 0, 2, 6 and 12 mg/kg bw
- Constant volume or concentration used: Yes

VEHICLE
- Justification for use and choice of vehicle: Water
- Amount(s) applied: 2 mg/kg bw
- Concentration: 0, 0.1, 0.3 and 0.6% (w/w)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Before initiation of the study, a trial batch of the dose solutions was prepared to assess the homogeneity and stability of the test material in water. For homogeneity, 3 samples each from the top, middle, and bottom of the mixing vessel were analyzed. Stability was evaluated 7 and 14 days after preparation by determining the test material concentration in triplicate samples in solutions prepared at the high and low concentrations and stored at room temperature.
Samples of the actual dosing solutions were taken and analyzed for test material concentration (prior to being used for dosing) during study weeks 1 through 4. In subsequent weeks, the samples were stored refrigerated, and a sample from the preparations made in weeks a and 13 were
analyzed.
Homogeneity studies performed on samples from all dosing solutions indicated that the test material was uniformly distributed. Stability studies were conducted on solutions (0.1 % and 0.6 %) stored at room temperature. These analyses indicated that the test material was stable in solution for at least 14 days. The values of analyses for the stability tests for both solutions ranged from 95.0 to 102.2 percent of nominal when assayed at 0, 7, and 14 days.
Concentration verification analyses (mean of duplicate assays) for the solutions prepared for the study ranged from 94.2 to 108.0 percent of nominal for all 3 concentrations.

Duration of treatment / exposure:

All animals were dosed for 13 weeks.

Frequency of treatment:

5 days/week, Monday through Friday. The females were also dosed on Saturday of the 13th week so that only 2 days elapsed between the administration of the last dose and the final sacrifice. This was done so that the elapsed time from dosing to sacrifice was the same as for the males.

Remarks:

Doses / Concentrations:
0, 2, 6 and 12 mg/kg bw/day
Basis:
nominal per unit body weight

No. of animals per sex per dose:

15/sex

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Three graduated dosage levels of the test material were evaluated in three groups of rats, anticipating that at the higher dosage level(s) some toxicological or pharmacological effect(s) will be observed and that at the lower dosage level(s) no treatment-related effects will be seen.
- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: no satellite groups selected
- Post-exposure recovery period in satellite groups: none
- Section schedule rationale (if not random): none

Positive control:

Not applicable

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes / No / No data
- Time schedule:

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: Animals were observed once a week for detailed clinical observations, and six days a week for overt clinical signs. Skin reactions were scored according to a modified Draize procedure when signs were observed on Friday after dosing and on Monday before dosing.

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

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Food consumption data were collected weekly for all animals.

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Prior to initiation and prior to sacrifice.
- Dose groups that were examined:

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood was obtained just prior to sacrifice from all surviving animals via the retroorbital sinus for clinical chemistry and hematology analyses.
- Anaesthetic used for blood collection: Yes (methoxyflurane)
- Animals fasted: Yes (overnight)
- How many animals: All animals
- Parameters checked:
total leukocyte count
erythrocyte count
hemoglobin
hematocrit
erythrocyte indices
platelet count
differential leukocyte count
reticulocyte count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood was obtained just prior to sacrifice from all surviving animals via the retroorbital sinus for clinical chemistry and hematology analyses.
- Animals fasted: Yes (overnight)
- How many animals: All animals
- Parameters checked:
AST (SGPT)
ALT (SGOT)
creatinine
alkaline phosphatase
gamma glutamyl transpeptidase
glucose
urea nitrogen
total protein
albumin
globulin (calculated)
A/G ratio (calculated)
total bilirubin
direct bilirubin
indirect bilirubin (calculated)
calcium
phosphorus
sodium
potassium
chloride

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Gross lesions
Brain, incl. cerebral cortex, cerebellar cortex, medulla/ pons
Eyes
Pituitary
Salivary gland (mandibular with submandibular lymph node)
Heart
Aorta
Thymic region
Thyroid - parathyroid complex
Lungs with mainstem bronchi
Adrenals
Spinal cord
Cervical, Thoracic, Lumbar
Pancreas
Liver (3 lobes)
Kidneys
Urinary bladder
Testes
Prostate
Epididymis
Ovaries
Vagina
Uterus (corpus and cervix)
Spleen
Lymph nodes (mesenteric & mediastinal)Trachea
Esophagus
Stomach
Duodenum, Jejunum, Ileum,
Cecum, Colon, Rectum (entire organs)
Exorbital lacrymal gland
Femur (including articular surface)
Skeletal muscle
Sciatic nerve
Mammary gland (female)
Skin (application site & non-application site)
Sternum (including marrow)
Feet and ears were saved for identification purposes.

HISTOPATHOLOGY: Yes
All harvested tissues (see list above) from all male and female rats in the control and high dose groups were processed histologically and examined microscopically. In addition, the skin, lungs, liver, kidneys, and all gross lesions were processed and examined histologically from all animals in the mid and low dose groups.

Other examinations:

None.

Statistics:

Parametric variables were intercompared for 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:

no effects observed

Dermal irritation:

effects observed, treatment-related

Description (incidence and severity):

Skin irritation (erythema, oedema) was observed at 6 and 12 mg/kg bw/day primarily early in the study (Days 5-8).

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Details on results:

CLINICAL OBSERVATIONS AND MORTALITY
No treatment-related clinical signs of toxicity were observed in either sex during the 90-day treatment period. Gradable skin irritation (erythema and/or edema) was observed principally on Days 5 and 8 for several males and during the first 5 weeks for several females from some groups. Therefore, the skin of the treatment area was evaluated using the Draize scoring system for erythema and edema for all animals after dosing on study Days 5, 12, 19, 26,and 33, and prior to dosing on study Days 8, 15, 22, 29, and 36.
Signs of skin irritation included erythema (scored as barely perceptible or well defined) at the application site of animals from the low dose group (1 female), mid dose group (2 males and 4 females), and high dose group (6 males and 14 females). Edema (scored as barely perceptible or well defined) was also observed in animals from the mid dose group (1 male and 2 females) and high dose group (1 male and 9 females). Erythema and edema were observed only for females in the high dose treatment group after study Week 2 with the exception of isolated observations of erythema for one male in the high dose group and one female in each of the low and mid dose groups. Erythema and edema were observed for females in the high dose group through study Weeks 5 and 4, respectively. Mild exfoliation in the treatment area was also observed for approximately half of the males and most of the females in the high dose groups throughout the dosing period.
No male rats died during the study. One female from the low dose group and one female from the high dose group were found dead on study Days 77 and 63, respectively, and one female from the mid dose group was sacrificed in a moribund condition on Day 75. None of the deaths were attributed to treatment with the test substance.

FOOD CONSUMPTION
The data for the animals with significant food spillage are removed from each test period. No treatment-related differences were observed for either sex at any dose level.

BODY WEIGHTS
No treatment-related effects on body weight or weight gain were observed for either sex from any dose group. The occasional statistically significant increases in weight gain observed for females from the low and mid dose groups compared to controls were considered a result of random variation.

OPHTHALMIC EXAMINATIONS
No treatment-related findings were made for either sex at any dose level.

CLINICAL PATHOLOGY
No treatment-related effects on hematology or serum chemistry measurements occurred in any group of either sex.

ORGAN WEIGHTS AND FINAL BODY WEIGHTS
No treatment-related effect on organ weights (absolute or relative) was observed for male or female rats administered test material.
Statistically significant differences (adrenal glands relative to body weight in males from the mid dose group and spleen weight relative to body weight in females from the mid dose group) in treated animals compared to controls were considered spurious.

GROSS PATHOLOGY AND HISTOPATHOLOGY
Treatment-related gross findings indicative of minimal to mild skin irritation were observed at the two highest dosage levels used in this study. Microscopically, lesions were observed in the treated skin for some animals from each treatment level. The greatest incidence of animals with clear histologic evidence of dermal and/or epidermal inflammation (epidermitis, dermatitis, vacuolar degeneration, hemorrhage, ulceration) occurred for females at the high dose. No other gross or microscopic pathologic findings were attributed to treatment with the test material.

Dose descriptor:

NOAEL

Effect level:

12 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

not specified

Conclusions:

Other than mild skin irritation, the occluded cutaneous treatment of male and female Sprague-Dawley rats with test material five days per week for 13 weeks at dosage levels of 2.0, 6.0, or 12.0 mg/kg bw/day resulted in no systemic toxicity as measured by clinical signs, food consumption, body weights or weight gain, ophthalmic changes, hematology or clinical chemistry measurements, gross pathology or histopathology. Since the 0.6 % concentration represented the maximum concentration which could be applied without producing more than slight skin irritation, and the 2.0 mL/kg bw volume represented the maximum volume which could be applied without run-off, the 12 mg/kg bw/day dosage level represented the maximum dosage level that could be evaluated in this test system. In addition, a local effect LOAEL of 2 mg/kg bw/day can be identified from this study.

Executive summary:

A study was carried out according to EPA OPP 82-3 (Subchronic Dermal Toxicity 90 Days) using the structural analog Didecyldimethylammonium chloride (DDAC). In view of the chemical and structural similarities (the relevant chemical part of both, DDAC and DDACarbonate, under the conditions of this test is the common quaternary ammonium cation Didecyldimethylammonium+), it is considered that the data are adequate for DDACarbonate. Solutions of test material were applied to the clipped backs of Sprague-Dawley CD® rats. The solutions were applied daily, five days per week, for 13 weeks. The dosing sites were occluded for at least 6 hours each day of dosing after which time the dressings were removed and the application site rinsed with tap water and blotted dry. Concentrations of 0, 0.1, 0.3 and 0.6 % (w/w) were evaluated in groups consisting of 15 male and 15 female rats. All solutions were administered at a constant volume of 2.0 mL/kg bw/day. The three concentrations, therefore, corresponded to dosage levels of 2, 6, and 12 mg/kg bw/day. Since the 0.6 % concentration represented the maximum concentration which could be applied without producing more than slight skin irritation, and the 2.0 mL/kg bw volume represented the maximum volume which could be applied without run-off, the 12 mg/kg bw/day dosage level represented the maximum dosage level that could be evaluated in this test system.

No treatment-related systemic toxicity as measured by clinical signs, food consumption, body weights or weight gain, ophthalmic changes, hematology or clinical chemistry measurements, gross pathology or histopathology was observed. Mild skin irritation at the treatment site was observed grossly in most animals treated with test material at a concentration of 0.6 %. Mild skin irritation was also observed grossly in a few animals at the 0.3 % concentration and in one female at the lowest concentration (0.1 %).

Changes in skin at the application site, indicative of irritation, were also observed microscopically for some animals at all dose levels. These microscopic changes were most evident in the female rats from the mid and high dose groups.

The systemic NOAEL was determined to be 12 mg/kg bw/day for males and females. In addition, a local effect NOAEL of 2 mg/kg bw/day could be identified from this study.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

12 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Good quality database.

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

sub-chronic 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: see 'Remark'

Remarks:

- Reliability: GLP study according to international guidelines - Read-across justification: As both source (Didecyldimethylammonium chloride, DDAC) and target chemicals (Didecyldimethylammonium carbonate, DDA carbonate) have identical organic cations with hydrophobic side chains - the only difference is the inorganic anion carbonate or chloride with negligible contribution to the hazard properties- both substances can be predicted to have similar movement and fate characteristics.

Qualifier:

according to guideline

Guideline:

EPA OPP 82-3 (Subchronic Dermal Toxicity 90 Days)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 8 weeks
- Weight at study initiation: males: 221.3-264.5 g; females: 166.7-219.11 g
- Fasting period before study: none
- Housing: individually housed in stainless steel cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 3 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 66 to 75
- Humidity (%): 40 and 70
- Photoperiod (hrs dark / hrs light): 12

IN-LIFE DATES:
From: 11 January 1987
To: 12 April 1987

Type of coverage:

occlusive

Vehicle:

water

Details on exposure:

TEST SITE
- Area of exposure: 4" x 4"
- % coverage: 100 %
- Type of wrap if used: Vetrap® Bandaging Tape
- Time intervals for shavings or clippings: Weekly and as needed

REMOVAL OF TEST SUBSTANCE
- Washing: After removal of the wraps, the application site was rinsed. Water was liberally applied to the treatment area and the area was blotted using a 4" x 4" (8-ply) gauze pad.
- Time after start of exposure: 6 hours

TEST MATERIAL
- Amount(s) applied: 2.0 ml/kg bw
- Concentration (if solution): 0, 2, 6 and 12 mg/kg bw
- Constant volume or concentration used: Yes

VEHICLE
- Justification for use and choice of vehicle: Water
- Amount(s) applied: 2 mg/kg bw
- Concentration: 0, 0.1, 0.3 and 0.6% (w/w)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Before initiation of the study, a trial batch of the dose solutions was prepared to assess the homogeneity and stability of the test material in water. For homogeneity, 3 samples each from the top, middle, and bottom of the mixing vessel were analyzed. Stability was evaluated 7 and 14 days after preparation by determining the test material concentration in triplicate samples in solutions prepared at the high and low concentrations and stored at room temperature.
Samples of the actual dosing solutions were taken and analyzed for test material concentration (prior to being used for dosing) during study weeks 1 through 4. In subsequent weeks, the samples were stored refrigerated, and a sample from the preparations made in weeks a and 13 were
analyzed.
Homogeneity studies performed on samples from all dosing solutions indicated that the test material was uniformly distributed. Stability studies were conducted on solutions (0.1 % and 0.6 %) stored at room temperature. These analyses indicated that the test material was stable in solution for at least 14 days. The values of analyses for the stability tests for both solutions ranged from 95.0 to 102.2 percent of nominal when assayed at 0, 7, and 14 days.
Concentration verification analyses (mean of duplicate assays) for the solutions prepared for the study ranged from 94.2 to 108.0 percent of nominal for all 3 concentrations.

Duration of treatment / exposure:

All animals were dosed for 13 weeks.

Frequency of treatment:

5 days/week, Monday through Friday. The females were also dosed on Saturday of the 13th week so that only 2 days elapsed between the administration of the last dose and the final sacrifice. This was done so that the elapsed time from dosing to sacrifice was the same as for the males.

Remarks:

Doses / Concentrations:
0, 2, 6 and 12 mg/kg bw/day
Basis:
nominal per unit body weight

No. of animals per sex per dose:

15/sex

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Three graduated dosage levels of the test material were evaluated in three groups of rats, anticipating that at the higher dosage level(s) some toxicological or pharmacological effect(s) will be observed and that at the lower dosage level(s) no treatment-related effects will be seen.
- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: no satellite groups selected
- Post-exposure recovery period in satellite groups: none
- Section schedule rationale (if not random): none

Positive control:

Not applicable

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes / No / No data
- Time schedule:

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: Animals were observed once a week for detailed clinical observations, and six days a week for overt clinical signs. Skin reactions were scored according to a modified Draize procedure when signs were observed on Friday after dosing and on Monday before dosing.

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

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Food consumption data were collected weekly for all animals.

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Prior to initiation and prior to sacrifice.
- Dose groups that were examined:

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood was obtained just prior to sacrifice from all surviving animals via the retroorbital sinus for clinical chemistry and hematology analyses.
- Anaesthetic used for blood collection: Yes (methoxyflurane)
- Animals fasted: Yes (overnight)
- How many animals: All animals
- Parameters checked:
total leukocyte count
erythrocyte count
hemoglobin
hematocrit
erythrocyte indices
platelet count
differential leukocyte count
reticulocyte count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood was obtained just prior to sacrifice from all surviving animals via the retroorbital sinus for clinical chemistry and hematology analyses.
- Animals fasted: Yes (overnight)
- How many animals: All animals
- Parameters checked:
AST (SGPT)
ALT (SGOT)
creatinine
alkaline phosphatase
gamma glutamyl transpeptidase
glucose
urea nitrogen
total protein
albumin
globulin (calculated)
A/G ratio (calculated)
total bilirubin
direct bilirubin
indirect bilirubin (calculated)
calcium
phosphorus
sodium
potassium
chloride

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Gross lesions
Brain, incl. cerebral cortex, cerebellar cortex, medulla/ pons
Eyes
Pituitary
Salivary gland (mandibular with submandibular lymph node)
Heart
Aorta
Thymic region
Thyroid - parathyroid complex
Lungs with mainstem bronchi
Adrenals
Spinal cord
Cervical, Thoracic, Lumbar
Pancreas
Liver (3 lobes)
Kidneys
Urinary bladder
Testes
Prostate
Epididymis
Ovaries
Vagina
Uterus (corpus and cervix)
Spleen
Lymph nodes (mesenteric & mediastinal)Trachea
Esophagus
Stomach
Duodenum, Jejunum, Ileum,
Cecum, Colon, Rectum (entire organs)
Exorbital lacrymal gland
Femur (including articular surface)
Skeletal muscle
Sciatic nerve
Mammary gland (female)
Skin (application site & non-application site)
Sternum (including marrow)
Feet and ears were saved for identification purposes.

HISTOPATHOLOGY: Yes
All harvested tissues (see list above) from all male and female rats in the control and high dose groups were processed histologically and examined microscopically. In addition, the skin, lungs, liver, kidneys, and all gross lesions were processed and examined histologically from all animals in the mid and low dose groups.

Other examinations:

None.

Statistics:

Parametric variables were intercompared for 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:

no effects observed

Dermal irritation:

effects observed, treatment-related

Description (incidence and severity):

Skin irritation (erythema, oedema) was observed at 6 and 12 mg/kg bw/day primarily early in the study (Days 5-8).

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Details on results:

CLINICAL OBSERVATIONS AND MORTALITY
No treatment-related clinical signs of toxicity were observed in either sex during the 90-day treatment period. Gradable skin irritation (erythema and/or edema) was observed principally on Days 5 and 8 for several males and during the first 5 weeks for several females from some groups. Therefore, the skin of the treatment area was evaluated using the Draize scoring system for erythema and edema for all animals after dosing on study Days 5, 12, 19, 26,and 33, and prior to dosing on study Days 8, 15, 22, 29, and 36.
Signs of skin irritation included erythema (scored as barely perceptible or well defined) at the application site of animals from the low dose group (1 female), mid dose group (2 males and 4 females), and high dose group (6 males and 14 females). Edema (scored as barely perceptible or well defined) was also observed in animals from the mid dose group (1 male and 2 females) and high dose group (1 male and 9 females). Erythema and edema were observed only for females in the high dose treatment group after study Week 2 with the exception of isolated observations of erythema for one male in the high dose group and one female in each of the low and mid dose groups. Erythema and edema were observed for females in the high dose group through study Weeks 5 and 4, respectively. Mild exfoliation in the treatment area was also observed for approximately half of the males and most of the females in the high dose groups throughout the dosing period.
No male rats died during the study. One female from the low dose group and one female from the high dose group were found dead on study Days 77 and 63, respectively, and one female from the mid dose group was sacrificed in a moribund condition on Day 75. None of the deaths were attributed to treatment with the test substance.

FOOD CONSUMPTION
The data for the animals with significant food spillage are removed from each test period. No treatment-related differences were observed for either sex at any dose level.

BODY WEIGHTS
No treatment-related effects on body weight or weight gain were observed for either sex from any dose group. The occasional statistically significant increases in weight gain observed for females from the low and mid dose groups compared to controls were considered a result of random variation.

OPHTHALMIC EXAMINATIONS
No treatment-related findings were made for either sex at any dose level.

CLINICAL PATHOLOGY
No treatment-related effects on hematology or serum chemistry measurements occurred in any group of either sex.

ORGAN WEIGHTS AND FINAL BODY WEIGHTS
No treatment-related effect on organ weights (absolute or relative) was observed for male or female rats administered test material.
Statistically significant differences (adrenal glands relative to body weight in males from the mid dose group and spleen weight relative to body weight in females from the mid dose group) in treated animals compared to controls were considered spurious.

GROSS PATHOLOGY AND HISTOPATHOLOGY
Treatment-related gross findings indicative of minimal to mild skin irritation were observed at the two highest dosage levels used in this study. Microscopically, lesions were observed in the treated skin for some animals from each treatment level. The greatest incidence of animals with clear histologic evidence of dermal and/or epidermal inflammation (epidermitis, dermatitis, vacuolar degeneration, hemorrhage, ulceration) occurred for females at the high dose. No other gross or microscopic pathologic findings were attributed to treatment with the test material.

Dose descriptor:

NOAEL

Effect level:

12 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

not specified

Conclusions:

Other than mild skin irritation, the occluded cutaneous treatment of male and female Sprague-Dawley rats with test material five days per week for 13 weeks at dosage levels of 2.0, 6.0, or 12.0 mg/kg bw/day resulted in no systemic toxicity as measured by clinical signs, food consumption, body weights or weight gain, ophthalmic changes, hematology or clinical chemistry measurements, gross pathology or histopathology. Since the 0.6 % concentration represented the maximum concentration which could be applied without producing more than slight skin irritation, and the 2.0 mL/kg bw volume represented the maximum volume which could be applied without run-off, the 12 mg/kg bw/day dosage level represented the maximum dosage level that could be evaluated in this test system. In addition, a local effect LOAEL of 2 mg/kg bw/day can be identified from this study.

Executive summary:

A study was carried out according to EPA OPP 82-3 (Subchronic Dermal Toxicity 90 Days) using the structural analog Didecyldimethylammonium chloride (DDAC). In view of the chemical and structural similarities (the relevant chemical part of both, DDAC and DDACarbonate, under the conditions of this test is the common quaternary ammonium cation Didecyldimethylammonium+), it is considered that the data are adequate for DDACarbonate. Solutions of test material were applied to the clipped backs of Sprague-Dawley CD® rats. The solutions were applied daily, five days per week, for 13 weeks. The dosing sites were occluded for at least 6 hours each day of dosing after which time the dressings were removed and the application site rinsed with tap water and blotted dry. Concentrations of 0, 0.1, 0.3 and 0.6 % (w/w) were evaluated in groups consisting of 15 male and 15 female rats. All solutions were administered at a constant volume of 2.0 mL/kg bw/day. The three concentrations, therefore, corresponded to dosage levels of 2, 6, and 12 mg/kg bw/day. Since the 0.6 % concentration represented the maximum concentration which could be applied without producing more than slight skin irritation, and the 2.0 mL/kg bw volume represented the maximum volume which could be applied without run-off, the 12 mg/kg bw/day dosage level represented the maximum dosage level that could be evaluated in this test system.

No treatment-related systemic toxicity as measured by clinical signs, food consumption, body weights or weight gain, ophthalmic changes, hematology or clinical chemistry measurements, gross pathology or histopathology was observed. Mild skin irritation at the treatment site was observed grossly in most animals treated with test material at a concentration of 0.6 %. Mild skin irritation was also observed grossly in a few animals at the 0.3 % concentration and in one female at the lowest concentration (0.1 %).

Changes in skin at the application site, indicative of irritation, were also observed microscopically for some animals at all dose levels. These microscopic changes were most evident in the female rats from the mid and high dose groups.

The systemic NOAEL was determined to be 12 mg/kg bw/day for males and females. In addition, a local effect NOAEL of 2 mg/kg bw/day could be identified from this study.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

0.01 mg/cm²

Study duration:

subacute

Species:

rat

Quality of whole database:

Good quality database.

Additional information

Available repeated dose toxicity studies were previously summarized and evaluated by the UK MSCA (2012). Standard repeated dose studies have been conducted using the structurally-related substance Didecyldimethylammonium chloride (DDAC) in rats, mice and dogs via the oral route and in rats via the dermal route. As both source and target chemicals have identical organic cations with hydrophobic side chains - the only difference is the inorganic anion carbonate or chloride with negligible contribution to the hazard properties - both substances can be predicted to have similar movement and fate characteristics. In addition effects of repeated exposure to the test item Didecyldimethylammonium carbonate (DDACarbonate) have been investigated in a non-guideline GLP dermal study (21-Day Repeated Dose Dermal Irritation Study in Female Rats).

The results from the oral studies reveal a pattern of response (local corrosion rapidly followed by signs of generalised toxicity and death) that is consistent with the mode of action of a corrosive substance. Therefore, the systemic effects observed in these studies are regarded as secondary to the local irritation/corrosion caused by the test substance and as a result the NOAELs and LOAELs identified are for the underlying local toxicity. The doses presented have been adjusted to represent 100 % active ingredient.

In the oral studies, exposure to 3000 ppm DDAC (daily intakes not calculated but assumed to be approximately 3 x the 1000 ppm doses) for 90 days via the diet resulted in 80 % and 97 % mortality in rats (183 (m) and 222 (f) mg/kg bw/day). Fifty percent mortality was observed in dogs exposed to a gavage dose of 60 mg/kg bw/day DDAC for 8 weeks. Other key adverse effects identified in these studies included severe retardation of body weight gain and reduced body weight and food consumption. In this key chronic oral toxicity study in dogs the no-observable effect level for systemic toxicity (NOAEL) was considered to be 10.0 mg/kg bw/day.

Limitations were identified in both of these studies; in the 8-week study the dosing procedure was changed from a single daily dose to 2 half doses per day after 2 weeks exposure which may have affected the extent of the observed toxicity and in the 52-week study sickness was observed in the control animals. Despite these uncertainties these LOAELs can be used in the risk characterisation because similar adverse effects were observed in each study reducing concern over their reliability. Chronic dietary studies in rats and mice also revealed signs of generalised toxicity, mainly reductions in body weight and body weight gain. However these effects occurred at higher doses than those observed in the sub-acute and sub-chronic studies in dogs. Overall, dogs appear to be more sensitive to the adverse effects of repeated oral exposure to DDAC than rats and mice and toxicity occurs at lower doses in gavage studies compared to dietary studies. Given the underlying mode of action of DDAC/DDACarbonate toxicity (i.e. irritation/corrosion) and the anticipated pattern of oral exposure in humans, gavage administration is considered a relevant exposure route for use in the risk characterisation of DDACarbonate.

In addition to the above mentioned repeated dose toxicity studies two studies were carried out according to OECD Guideline 414 and EPA OPP 83-3 (Prenatal Developmental Toxicity Study) using the structural analog Didecyldimethylammonium chloride (DDAC). The NOAEL for maternal toxicity revealed in the key developmental study was 1.0 mg/kg bw/day. The observed adverse effects at higher concentrations were considered to be either the result of or a consequence of local irritation. The NOAEL is used for oral DNEL derivation.

In a standard 90-day dermal study, no systemic toxicity was observed up to the highest dose tested of 12 mg/kg bw/day DDAC (100 % AI). Signs of skin irritation were observed at all doses and the incidence of these lesions increased with increasing dose. At the lowest dose tested, reversible, well defined erythema in 1 female (day 15 only) and epidermitis in 1 female and 1 male were observed. As the erythema was observed in 1 female at 1 time-point only and because the microscopic changes were very mild in nature these effects were not considered adverse and a local toxicity NOAEL of 2 mg/kg bw/day (0.1 % solution) was identified. As local effects are concentration-dependent rather than dose-dependent the NOAEL of 2 mg/kg bw/day has been converted to a concentration-based NOAEC of 0.0165 mg/cm2/day (see below). The calculated NOAEC is similar to the LOAEC revealed in the non-standard repeated dose irritation study conducted with DDACarbonate (100 % AI).

Conversion of dermal dose to dermal concentration:

2 mg/kg bw/day X 0.35 kg (bw default – TGD) X 1000) / 42.5*= 0.0165 mg/cm2/day

Treated area (cm2) based on the assumption that 10 % of the body surface area was exposed to DDAC. Default value of 350 g body weight (males in dermal toxicity studies) and 425 cm2 for total body surface area was used (ECB TGD, 2003).

Reference: UK MSCA (2012) Didecyldimethylammonium Carbonate / Bicarbonate (DDACarbonate) Product-type 08 (Wood Preservative). Assessment Report for the Inclusion of Active Substances in Annex I to Directive 98/8/EC.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
GLP study carried out using the structural analog Didecyldimethylammonium chloride (DDAC). As both source (DDAC) and target chemicals (Didecyldimethylammonium carbonate, DDA carbonate) have identical organic cations with hydrophobic side chains - the only difference is the inorganic anion carbonate or chloride with negligible contribution to the hazard properties - both substances can be predicted to have similar movement and fate characteristics.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
According to REACH Regulation (EC) No 1907/2006 Annex VIII, column 1 and 2, section 8.6.1 repeated dose toxicity testing via the inhalation route was waived, as inhalation of the substance is unlikely. The vapor pressure of Didecyldimethylammonium carbonate was determined to be 7.7E-3 Pa at 25 °C. Further, Didecyldimethylammonium carbonate is marketed as an aqueous solution and thus, exposure to aerosols, particles or droplets of an inhalable size is highly unlikely. In addition, the results of a dermal toxicity study and skin irritation studies indicate that the substance is corrosive. Further data are not required as appropriate risk mitigation measures can be employed to prevent inhalation exposure.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
According to REACH Regulation (EC) No 1907/2006 Annex VIII, column 1 and 2, section 8.6.1 repeated dose toxicity testing via the inhalation route was waived, as inhalation of the substance is unlikely. The vapor pressure of Didecyldimethylammonium carbonate was determined to be 7.7E-3 Pa at 25 °C. Further, Didecyldimethylammonium carbonate is marketed as an aqueous solution and thus, exposure to aerosols, particles or droplets of an inhalable size is highly unlikely. In addition, the results of a dermal toxicity study and skin irritation studies indicate that the substance is corrosive. Further data are not required as appropriate risk mitigation measures can be employed to prevent inhalation exposure.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
GLP study according to international guidelines carried out using the structural analog Didecyldimethylammonium chloride (DDAC). As both source (DDAC) and target chemicals (Didecyldimethylammonium carbonate, DDA carbonate) have identical organic cations with hydrophobic side chains - the only difference is the inorganic anion carbonate or chloride with negligible contribution to the hazard properties - both substances can be predicted to have similar movement and fate characteristics.

Justification for selection of repeated dose toxicity dermal - local effects endpoint:
GLP study according to international guidelines carried out using the structural analog Didecyldimethylammonium chloride (DDAC). As both source (DDAC) and target chemicals (Didecyldimethylammonium carbonate, DDA carbonate) have identical organic cations with hydrophobic side chains - the only difference is the inorganic anion carbonate or chloride with negligible contribution to the hazard properties - both substances can be predicted to have similar movement and fate characteristics. The no observed adverse effect concentration (NOAEC) in this study was determined to be 10 µg AI/cm2/day.
In addition a GLP study was carried out to evaluate the dermal irritation of Didecyldimethylammonium carbonate and to provide results in the same units as those used to report human exposure, i.e. µg test substance per cm2 skin. The low observed adverse effect concentration (LOAEC) in this study was determined to be 10 µg AI/cm2/day.

Justification for classification or non-classification

Based on the information available the substance is not classified and labeled according to Regulation (EC) No 1272/2008 (CLP) and according to Directive 67/548/EEC (DSD).