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Toxicological information

Repeated dose toxicity: dermal

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Administrative data

Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1989-04-18 to 1990-12-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report Date:
1990

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EPA OPP 82-3 (Subchronic Dermal Toxicity 90 Days)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
Chemical name: N-Alkyl (C12-C18)-N,N-dimethyl-N-benzylammonium chloride (ADBAC C12-18)
Molecular formula: C9H13NRCl where R = C12H25, C14H29, C16H33 or C18H37
CAS: 68391-01-5
Appearance: very viscous, gel-like liquid
Specific details on test material used for the study:
- Appearance: Pale yellow, viscous liquid
- Percent active material: 81.09% (w/w)
- Storage: Room temperature
- Stability of test substance: stable at room temperature

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles Rivers Breeding Laboratories (Portage, MI)
- Age at study initiation: approximately 8 weeks old
- Females (if applicable) nulliparous and non-pregnant: Yes
- Housing: Upon arrival, the animals were housed in two animals per side of divided stainless steel cages (solid sides with wire mesh floors) mounted in a stainless steel Maxi-Rack. The purpose of the double housing was to help acclimate the rats to the automatic watering system. Approximately one week later, the animals were individually housed in similar caging and remained housed one animal per cage side throughout the study. A layer of Deotized Animal Cage Board was kept under each cage and changed at least three times per week
- Diet (e.g. ad libitum): Ad libitum, Ground Purina Certified Rodent Chow #5002 (Ralston Purina Co., St. Louis, MO)
- Water (e.g. ad libitum): Ad libitum, tap water

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 66 - 750 °F
- Humidity (%): 40 - 70 %
- Photoperiod (hrs dark / hrs light): 12/12
- Air changes (per hr): 15

Administration / exposure

Type of coverage:
occlusive
Vehicle:
water
Details on exposure:
TEST SITE
- Area of exposure: Dorsal area of the trunk
- Type of wrap used: Vetrap Bandaging Tape (3M Personal Care Products, St. Paul, MN) and secured with Elastikon Elastic Tape

REMOVAL OF TEST SUBSTANCE
- Washing (if done): After the exposure, wrapping was removed and the application site was rinsed. Water was liberally applied to the treatment area and the area was blotted using a 4" x 4" gauze pad.
- Time after start of exposure: 6 h

OBSERVATION TIME POINTS (indicate if minutes, hours or days): Observations for mortality were made twice daily. Detailed clinical observations, with special attention to the skin of the dose site, were performed once each week and observations for overt clinical signs were made on all other days. Ophthalmic observations were performed for all animals, using an indirect ophthalmoscope, prior to the start of the dosing and prior to final sacrifice.
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 alkyl dimethyl benzyl ammonium chloride 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 alkyl dimethyl benzyl ammonium chloride 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 8 and 13 was analyzed.
Duration of treatment / exposure:
6 h
Frequency of treatment:
All animals were dosed 5 days/week, Monday through Friday, for 13 weeks. 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.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control
Dose / conc.:
2 mg/kg bw/day (nominal)
Remarks:
Low
Dose / conc.:
6 mg/kg bw/day (nominal)
Remarks:
Mid
Dose / conc.:
20 mg/kg bw/day (nominal)
Remarks:
High
No. of animals per sex per dose:
15
Control animals:
yes, concurrent vehicle
Details on study design:
- Rationale for animal assignment (if not random): 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.
- Dose selection rationale: Doses were selected on the basis of findings from a preliminary skin irritation screen at a range of ADBAC concentrations of 0.03% to 10% active ingredient. The highest concentration of the test substance which could be applied according to the dose regimen scheduled to be used in this study without producing more than slight skin irritation was 1.0%.
Positive control:
n.a.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: Not specified

BODY WEIGHT: Yes
- Time schedule for examinations: Prior to sacrifice

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once each week and observations for overt clinical signs were made on all other days.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to the start of dosing and prior to final sacrifice.
- Dose groups that were examined: All animals were examined

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to sacrifice
- Anaesthetic used for blood collection: Yes, methoxyflurane
- Animals fasted: Yes, animals were fasted overnight prior to the final procedures
- How many animals: all animals (90 males and 90 females)
- Parameters checked in Table 1 in box “Any other information on materials & methods incl. tables” were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to sacrifice
- Animals fasted: Yes, animals were fasted overnight prior to the final procedures
- How many animals: all animals (90 males and 90 females)
- Parameters checked in Table 2 in box “Any other information on materials & methods incl. tables” were examined.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes. On the 15th day of the study, all animals were killed by methoxyflurane overdose. The skin at the application site of all sacrificed animals was harvested and fixed in 10 % neutral buffered formalin for possible future histopathological examination.
GROSS PATHOLOGY: Yes (see table 3 under "Any other information on materials and methods incl. tables"). Tissues were harvested and saved in 102 neutral buffered formalin.

HISTOPATHOLOGY: Yes, all harvested tissues 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:
Organ weights: The liver, kidneys, heart, spleen, brain with stem, adrenals, testes (males), and ovaries (females) were weighed for all sacrificed animals.
Statistics:
Data for continuous, parametric variables were intercompared for the dose and control groups by use of Levene's test for homogeneity of variances, by analysis of variance, and by pooled variance t-tests. The t-tests were used, if the analysis of variance was significant, to delineate which groups differed from the control group. If Levene's test indicated heterogeneous variances, the groups were compared by an analysis of variance for unequal variances followed, if necessary, by separate 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 where appropriate. All statistical tests, except the frequency comparisons were performed using BMDP Statistical Software (Dixon, 1985). The frequency data tests are described in Biometry (Sokal, R. R. and Rohlf, F. J., W. H. Freeman and Company: San Francisco, 1969). The fiducial limit of 0.05 was used as the critical level of significance for all tests.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical signs were limited to exfoliation at the treatment area for one female in the high dose treatment group observed on the last day of dosing only, excoriation on the abdomen of one female in the mid dose treatment group, and crust and ulceration on the skin adjacent to the treatment area for one male in the high dose treatment group. Based on the few numbers of observations, the location of the lesions, and/or the time of observation, these findings were considered unrelated to test material treatment.
Dermal irritation:
effects observed, non-treatment-related
Description (incidence and severity):
Hyperkeratosis was observed in the treatment area of females in the ADBAC treatment groups and males in all treatment groups (including control). These lesions were of minimal to mild severity for females and minimal to moderate severity for males. The relationship of these lesions to test article administration was questionable based on the high incidence in the control male animals and the potential for the development of hyperkeratosis as a result of clipping and repeated periods of dermal occlusion for all animals in this study.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female from the high dose treatment group died during the first day of treatment. This death was not considered to be related to treatment with the test material but rather a reaction to the stress of the wrapping procedure.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no treatment-related alterations in body weight or body weight gain for animals in this study.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no treatment-related alterations in food consumption for animals in this study. Occasional statistically significant increases in mean food consumption observed for females in the ADBAC treated groups compared to the mean for the control group were considered to be chance occurrences.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Corneal crystals were observed for both males and females (distributed across all groups including control) at both the pre-study examination (97% of the males and 85% of the females) and the examination prior to final sacrifice (97% of the males and 77% of the females). The lesion was considered minimal and there was no evidence that the lesions were related to treatment with the test material. Other lesions were infrequent and were not considered related to treatment with ADBAC.
Haematological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related biologically significant alterations in hematology for animals in this study
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were no treatment-related biologically significant alterations in clinical chemistry parameters for animals in this study
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):
There were no treatment-related findings observed.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no gross treatment-related findings in tissues other than the treated skin.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no microscopic treatment-related findings in tissues other than the treated skin.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Details on results:
The occluded cutaneous treatment of male and female Sprague-Dawley CD® rats with alkyl dimethyl benzyl ammonium chloride for five days per week for 13 weeks at dosage levels of 2.0, 6.0, or 20.0 mg/kg body weight/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. Slight local irritation (hyperkeratosis) was observed for males in all treatment groups (including controls) and for females in each of the ADBAC treatment groups. Since the 1.0 % concentration represented the maximum concentration which could be applied without producing more than slight skin irritation, and the 2.0 mg/kg body weight volume represented the maximum volume which could be applied, without run-off, the 20 mg/kg body weight/day dosage level represented the maximum dosage level that could be evaluated in this test system.

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
20 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed
Key result
Dose descriptor:
NOAEL
Remarks:
local
Effect level:
20 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed
Remarks on result:
other: equal to 51 µg/cm² for males and 33 µg/cm² for females

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Pre-study information

Evaluations for fecal parasites, histology, serology, hematology, and clinical chemistry evaluations which were conducted during the pretest period indicated that the animals were free of infectious disease and parasites. The animals were, therefore, considered to be acceptable for use on the study.

Analytical results

Homogeneity studies performed on samples from all dosing solutions indicated that the test material was uniformly distributed. Stability studies were conducted on solutions (1.0% and 0.1%) 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 96.8 to 98.6 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 94.4 to 109.0 percent of nominal for all 3 concentrations.

Converting dermal NOAEL in mg/kg bw/day to NOAEL in mg/cm²/day:

NOAEL (mg/cm²/day) = NOAEL (mg/kg bw/day) x body weight (in kg)/total application area (in cm²)

- the average weight of the rats are 265.95 g (males) and 171.8 g (females) based on the weight at study initiation

- total application area: 4" x 4" = 10.16 cm x 10.16 cm = 103.23 cm ²

NOAEL (mg/cm²/day) = 20 mg/kg bw/day x 265.95 g (males) or 171.8 g (females)/103.23 cm² = 0.051 mg/cm² (males) and 0.033 mg/cm² (females)

Applicant's summary and conclusion

Conclusions:
In this study, dermal treatment with the test item, alkyl dimethyl benzyl ammonium chloride on male and female Sprague Dawley rats for 90 days resulted in no systemic toxicity at dose levels of 2.0, 6,0 and 20.0 mg/kg bw. Hence, the systemic and local NOAEL for the test item under the conditions of this study was 20 mg/kg/day which is equal to 51 µg/cm² for males and 33 µg/cm² for females.
Executive summary:

In a repeated dose dermal toxicity study conducted according to EPA guideline OPP 82-3, the test item ADBAC C12 -C18 (81.09% active ingredient) was applied to the shaved skin of male and female Sprague Dawley rats (15/sex/dose) at dose levels of 0, 2.0, 6.0 and 20.0 mg/kg body weight/day for five days per week for 90 days. 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 were observed. Slight local irritation(hyperkeratosis) was observed for males in all treatment groups (including controls) and for females in each of the ADBAC C12 -C18 treatment groups. Based on the results obtained in this study, the systemic and local NOAEL for ADBAC C12 -C18 under the conditions of this study is 20 mg/kg which is equal to 51 µg/cm² for males and 33 µg/cm² for females.