Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
4 May - 12 July 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP/Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
not specified
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
not specified
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Benzyltrimethylammonium Chloride, Lot #: ZMG-184366 was used. Purity was 99.9%

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/J
Sex:
female
Details on test animals and environmental conditions:
Housing: The animals were individually housed in plastic solid bottom cages with bedding during the dosing and resting phases of the study. After final weighing until sacrifice, animals were housed in their respective dose groups in plastic cages with bedding. Bedding in the plastic, solid bottom cages was changed at least once per week. All caging conformed to the size recommendations in the most recent Guide for the Care and Use of Laboratory Animals (Natl. Res. Council of the National Academies, 2011).

Animal Room Temperature and Relative Humidity Ranges: 19-23ºC and 59-66%, respectively.

Animal Room Air Changes/Hour: 12. Airflow measurements are evaluated regularly and the records are kept on file at Eurofins PSL

Photoperiod: 12-hour light/dark cycle

Acclimation Period: 7-14 days

Food: Harlan Teklad Certified Global 16% Protein Rodent Diet® #2016C. The diet was available ad libitum.

Water: Filtered tap water was supplied ad libitum by an automatic water dispensing system.

Contaminants: There were no known contaminants reasonably expected to be found in the food or water at levels which would have interfered with the results of this study. Analyses of the food and water are conducted regularly and the records are kept on file at Eurofins PSL. The most recent water analysis was conducted in July 2012. Harlan Teklad Certified Global 16% Protein Rodent Diet® #2016C, Lot Number: 2016C-032112MA, was analyzed in April 2012.

Identification
Cage: Each cage was identified with a cage card indicating at least the study number, identification, and sex of the animal.

Animal: Each animal was marked with a color code and given a sequential animal number assigned to study 34440, which constituted unique identification.

Study design: in vivo (LLNA)

Vehicle:
other: 1% Pluronic L92 surfactant in distilled water
Concentration:
Concentrations of 10%, 25% and 50% were selected for the main test based on results of the preliminary screening test. An additional dose level of 35% was also prepared based on the results (mortality) at the 50% concentration.
No. of animals per dose:
5 mice/group
Details on study design:
Preliminary Toxicity Testing
Three test substance concentrations and the vehicle control were used for preliminary toxicity testing. Test substance concentrations of 50%, 25% and 10% in 1% Pluronic L92 surfactant in distilled water were tested to determine the highest achievable level that avoids overt systemic toxicity and excessive local irritation. The top dose of 50% was selected based on maximum solubility, compatibility, and viscosity of the test substance with the vehicle while maintaining a pipettable solution that could be applied to the mouse ear (documented in the raw data). Each group consisted of two mice. The ears of each mouse were scored for erythema and edema prior to dosing on Days 1, 2, 3, and prior to termination on Day 6 according to the scoring system.

Because aqueous materials may not be readily compatible with the standard solvents, topical administration can be problematic. Guidelines indicate that hydrophilic materials are incorporated into a vehicle system that wets the skin and does not immediately run off, in this regard, L92 provides good skin wetting properties for prolonged dermal contact, and has been shown to yield positive LLNA results using a number of water soluble dermal sensitizers (Ryan et al., 2002). In addition, consistent LLNA results were obtained across laboratories for various water soluble dermal sensitizers and formulations in an interlaboratory study that examined L92 as a vehicle (Boverhof et al., 2008).

Furthermore, L92 surfactant should demonstrate improved compatibility with the test material when considering formulation properties, thus resulting in more realistic test conditions when compared with human exposure.

Twenty-five microliters (25 uL) of the appropriate dilution of the test substance concentration or the vehicle alone was applied to the dorsum of both ears of each mouse (50 uL total) for three consecutive days (Days 1, 2 and 3). Application was done using an appropriate size micropipette to accurately deliver 25 uL. The dose was gently spread as evenly as possible over the dorsal surface of the ear using the tip of the pipette. No treatment was made on Days 4 and 5. On Days 1, 2, 3 and 6, each site was evaluated for local irritation (erythema & edema) according to the scoring system.

Animals were observed daily for signs of toxicity. The Study Director and Sponsor used this data in conjunction with any pre-existing data to select the maximum concentration to be tested in the main LLNA study. Test substance concentrations of 10%, 25% and 50% w/w mixtures 1% Pluronic L92 surfactant in distilled water were selected for testing. Due to mortality in four animals at the 50% concentration in the main test, an additional dose level of 35% and an additional vehicle control group were added to the study to aid in the interpretation of the sensitization potential of the test substance.

Sample Preparation
Dilutions of the test substance were prepared as w/w mixtures in 1% Pluronic L92 surfactant in distilled water. Concentrations of 10%, 25% and 50% were selected for the main test based on results of the preliminary screening test. An additional dose level of 35% was also prepared based on the results (mortality) at the 50% concentration. A single concentration of a 25% w/w mixture of HCA in 1% Pluronic L92 surfactant in distilled water was prepared as a positive control. All dosage preparations were freshly prepared on the day of administration.

Test Substance Application
Beginning on Day 1, a volume of 25 uL of the vehicle, the appropriate test substance concentration, or the positive control substance was applied to the dorsum of both ears of each surviving mouse once per day for three consecutive days (Days 1, 2, and/or 3) using a micropipette. During application, the material was gently spread as evenly as possible over the dorsal surface of the ear using the tip of the pipette.

Dermal Scoring
Prior to each application (Days 1, 2, and/or 3), the ears were evaluated for erythema and edema according to the modified Draize scoring system (Draize et al., 1944). All ears of surviving animals were also evaluated on Day 6.

3H-methyl Thymidine Injections
On Day 6 of the study (three days after the final topical application) 250 uL of sterile phosphate buffered saline (PBS, Lot #: 051M8212) containing 20 uCi of 3H-methyl thymidine (Lot #201205 was injected intravenously via the tail vein of each mouse.

Lymph node assessment
Approximately five hours after the injection, the draining auricular lymph nodes from all surviving animals were excised. The lymph nodes were pooled for each individual mouse. A single cell suspension of lymph node cells (LNC) was prepared in PBS by gently massaging the lymph nodes between the frosted ends of two microscope slides over a collection vessel. The slides were then rinsed briefly with PBS into the vessel. The contents of the vessel were transferred to a centrifuge tube and washed with an excess of PBS and centrifuged for approximately 10 minutes at 1800 rpm, with a RCF5 of 489G. This process was carried out twice. In both cases, the supernatant was decanted and discarded following each centrifugation. After the second wash, approximately 5 mL of 5% trichloroacetic acid in distilled water (TCA, Lot #: 112549) was then added to the sediment and the tube was vortexed briefly. The DNA was then precipitated in the 5% TCA at approximately 5.1-6.3C overnight (approximately 18 hours).

Following the overnight precipitation of the DNA, the tubes were centrifuged again for approximately 10 minutes and the supernatant was discarded. The resulting precipitate was resuspended using 1 mL of the 5% TCA and transferred to 10 mL of scintillation fluid. Incorporation of 3H-methyl thymidine was measured by beta-scintillation counting and expressed as disintegrations per minute, minus background dpm.

Clinical Observations
All test and preliminary screening mice were observed for signs of mortality, gross toxicity, and/or behavioral changes daily or until death occurred. All surviving test mice were euthanized via overdose of inhaled Isoflurane anesthetic on Day 6.

Body Weights
All test mice were weighed on Day 1 (prior to the first application) and prior to sacrifice on test Day 6 or after death.

EVALUATION
The mean and standard deviation of the body weight, body weight gain and dpm values were calculated for surviving animals from Groups 1 through 4 in the main test. A stimulation index (SI) was derived for selected experimental groups by dividing the mean dpm of these experimental groups by the mean dpm of the vehicle control group. Any test material that produces a SI > 3 in the LLNA is normally considered “positive” for dermal sensitization potential (Kimber et al., 1994). While a SI > 3 was originally developed empirically, a robust statistical evaluation indicated that it is an acceptable practical value for hazard identification (Basketter et al., 1999). If applicable, the EC3 value will be calculated to determine the concentration of test substance that induces a stimulation index of 3.0. It is determined by linear interpolation between two points of the stimulation indices axis, one above (a,b) and one below (c,d) the stimulation index of 3.0. Since the two resulting dose levels induced a stimulation index less than 3.0, the EC3 could not be calculated.

References:
Basketter, D. A., Lea, L. J., Cooper, K., Stocks, J., Dickens, A., Pate, I., Dearman R. J., and Kimber, I. (1999). Threshold for classification as a skin sensitizer in the local lymph node assay: a statistical evaluation. Food and Chemical Toxicology 37(12), 1167-1174.

Boverhof, D. R., Wiescinski, C. M., Botham, P., Lees, D., Debruyne, E., Repetto-Larsay, M., Ladics, G., Hoban, D., Gamer, A., Remmele, M., Wang-Fan, W., Ullmann, L. G., Mehta, J., Billington, R. and Woolhiser, M. R. (2008). Interlaboratory validation of 25% Pluronic L92 surfactant as a suitable, aqueous vehicle for testing pesticide formulations using the murine local lymph node assay. Toxicol. Sci. 105(1), 79-85.

Draize, J.H., Woodward, G., and Calvery, H.O. (1944). Methods for the study of irritation and toxicity of substances applied topically to the skin and mucous membranes. J. Pharmacol. Exp. Ther.; 82:377-390.

Kimber, I., Dearman, R. J., Scholes, E. W., and Basketter, D. A. (1994). The local lymph node assay: developments and applications. Toxicology 93, 13-31.

Ryan, C. A., Cruse, L. W., Skinner, R. A., Dearman, R. J., Kimber, I., and Gerberick, G. F. (2002). Examination of a vehicle for use with water soluble materials in the murine local lymph node assay. Fd. Chem. Toxicol., 40, 1719-1725.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Statistical analysis was performed on the body weight, body weight gain and dpm values. Significance was judged at p <0.05. The treated group and vehicle control group were compared using a One-Way Analysis of Variance (ANOVA), followed by comparison of the treated groups to the vehicle control group by Dunnett’s t-test for multiple comparisons. Where variances are considered significantly different by Bartlett’s test, groups were compared using a non-parametric method (Kruskal-Wallis non parametric analysis of variance followed by Dunn’s test) (INSTAT Biostatistics, Graph Pad Software, San Diego, CA). Outlier analysis was conducted using Grubbs (1969).

Reference:
Grubbs, Frank E., Procedures for Detecting Outlying Observations in Samples, Technometrics, Vol. 11, No. 1, 1969, pp. 1-21.

Results and discussion

Positive control results:
HCA resulted in a positive response in the LLNA. See Table 1.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: Treatment of mice with 10% and 25% of Benzyltrimethylammonium Chloride (BTMAC) resulted in stimulation index values of 1.06 and 1.32, respectively, relative to vehicle control mice.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: See Table 1

Any other information on results incl. tables

A preliminary screening study was conducted with concentrations of 10%, 25% and 50% w/w in 1% Pluronic L92 surfactant in distilled water and the vehicle alone. The top dose of 50% was selected based on maximum solubility, compatibility, and viscosity of the test substance with the vehicle while maintaining a pipettable solution that could be applied to the mouse ear (documented in the raw data). On Days 1, 2, 3 and/or 6, each dose site was evaluated for local irritation. One mouse from Group 4P (50% concentration) was found dead on Day 6. There were no clinical signs of toxicity prior to death. Gross necropsy revealed discoloration of the lungs and intestines. All other mice appeared active and healthy. There was no dermal irritation observed in the vehicle control group or 10% test concentration group. Very slight erythema was seen at the 25% and 50% concentrations.

Based on the results of the screening assay, 10%, 25% and 50% w/w concentrations in 1% Pluronic L92 surfactant in distilled water and the vehicle alone were evaluated in the main study. Four out of five mice from the 50% concentration were found dead by Day 6. All surviving mice appeared active and healthy, and there were no consistent treatment-related effects on body weight for the survivors over the course of the study. There was no dermal irritation noted for the treatment group at the 10% concentration and very slight erythema observed at various time points at the 25% and 50% concentrations. Due to the mortality of the four animals at the 50% concentration, an additional dose level of 35% was evaluated, along with an additional vehicle control group. However, due to the death of two of the five mice by Day 1 at the 35% concentration, all animals from the test and vehicle control groups were euthanized for humane reasons on Day 2. There was no dermal irritation and no clinical signs of toxicity observed in the mice from either of these groups prior to death.

Gross necropsy of two of the decedents from the 50% treatment group revealed discoloration of the intestines. No gross abnormalities were noted in the two other decedents at 50%, the decedents at 35% or for the euthanized animals from the additional vehicle control and 35% groups (Groups 6 and 7).

No dermal irritation was observed at any site in the original vehicle control group (Group 1). Very slight to well-defined erythema and slight edema was noted in the positive control group (Group 2).

Treatment of mice with 10% and 25% of Benzyltrimethylammonium Chloride (BTMAC) resulted in stimulation index values of 1.06 and 1.32, respectively, relative to vehicle control mice. As a stimulation index (SI) of greater than 3.0 was not observed, Benzyltrimethylammonium Chloride (BTMAC) was considered negative for dermal sensitization potential in the LLNA at concentrations of less than or equal to 25%. Proper conduct of the LLNA was confirmed via a positive response with 25% HCA, a moderate contact sensitizer, which elicited a proliferative response with a SI value of 4.16 relative to vehicle controls.

Table 1 DPM values for each treatment group

 Dose Level  DPM1  SI
 Vehicle Control (1% Pluronic L92 surfactant in distilled water)  730.74 + 181.07  -
 Positive Control (25% HCA in 1% Pluronic L92 surfactant in distilled water)  3038.88 + 437.32  4.16
 10% Test Substance in 1% Pluronic L92 surfactant in distilled water  776.10 + 227.07  1.06
 25% Test Substance in 1% Pluronic L92 surfactant in distilled water  966.43 + 203.22  1.32

1 Mean + Standard Deviation

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: expert judgment
Conclusions:
As a stimulation index (SI) of greater than 3.0 was not observed, Benzyltrimethylammonium Chloride (BTMAC) was considered negative for dermal sensitization potential in the LLNA at concentrations of less than or equal to 25%. Proper conduct of the LLNA was confirmed via a positive response with 25% HCA, a moderate contact sensitizer.
Executive summary:

This study was conducted at Eurofins Product Safety Labs (EPSL) under contract of REACHCentrum, “RC”, representing the member companies of the BTMAC SIEF Leadership Team, “SLT”: Polystar Europe SLU, SACHEM Europe B.V., TANATEX Chemicals B.V. and Dow Duetschland Anlagengesellschaft mbH. Dow is a paying and participating member of REACHCentrum and as such is a co-owner of this study.

A dermal sensitization test was conducted with female mice to determine the potential for Benzyltrimethylammonium Chloride (BTMAC) to produce sensitization after repeated topical applications. Three concentrations (10%, 25% and 50%) of the test substance in 1% Pluronic L92 surfactant in distilled water or the vehicle alone were initially topically applied to twenty healthy female test mice (5 mice/group) for three consecutive days. Due to mortality observed at the 50% concentration, an additional dose level of 35% of the test substance and an additional vehicle control group were added to the study to aid in the interpretation of the sensitization potential of the test substance. There were five female mice in each of these groups. The 35% concentration was also terminated due to mortality; therefore, 25% was the maximum tolerated dose. Three days after the last application, the surviving mice were given a 20 μCi IV injection of 3H-methyl thymidine. Five hours later, the draining (auricular) lymph nodes were harvested and prepared for analysis in a scintillation counter. The results are presented in disintegrations per minute per mouse (dpm/mouse). Each animal’s ears were also evaluated for erythema and edema prior to each application and again on Day 6, prior to the IV injection. A positive control group (five female mice) was maintained under the same environmental conditions and treated with a 25% w/w mixture of alpha-Hexylcinnamaldehyde Technical (HCA) in 1% Pluronic L92 surfactant in distilled water in the same manner as the test animals. A table summarizing the results of the LLNA is found below:

   Mean DPM  Stimulation Index
 Control  730.74  -
 Positive Control  3038.88  4.16
 10% Test Substance  776.10  1.06
 25% Test Substance  966.43  1.32

As a stimulation index (SI) of greater than 3.0 was not observed, Benzyltrimethylammonium Chloride (BTMAC) was considered negative for dermal sensitization potential in the LLNA at concentrations of less than or equal to 25%. Proper conduct of the LLNA was confirmed via a positive response with 25% HCA, a moderate contact sensitizer.