Quaternary ammonium compounds, bis(hydrogenated tallow alkyl)dimethyl, chlorides, reaction products with polyethylenepolyamines and tall-oil fatty acids, humates hydrochlorides

Endpoint summary

Description of key information

This study was based on Guideline 429, Skin Sensitization: Local Lymph Node Assay, the OECD Guideline for Testing of Chemicals, revised July 2010. Organolignite was determined to not be a sensitizer when tested at concentrations of 15%, 30% or 61%, the highest achievable concentration (solvent used was Dimethyl Sulphoxide), in the Murine Local Lymph Node Assay. No adverse effects were observed during the course of this study.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-07-08 to 2013-07-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

yes

Remarks:

A protocol deviation was noted because lymph node samples were centrifuged at 1-6 degrees C instead of 4 degrees C. The Study Director commented that this deviation did not affect the quality or integrity of the study.

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: The Jackson Laboratory, Bar Harbor, Maine, USA
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 18-23.5 grams
- Housing: Upon arrival, the animals were housed in groups of 4 in suspended, stainless steel, wire mesh cages to allow time to adapt to the automatic
watering system. For the study, animals were individually housed in the same type of caging.
- Diet (e.g., ad libitum): Block Lab Diet® (Certified Rodent Diet #5002, PMI Nutrition International, Inc.) was available ad libitum.
- Water (e.g., ad libitum): Tap water was available ad libitum via an automatic watering system.
- Acclimation period: 6-8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-26 °C
- Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light): 12 hrs of light and dark

Vehicle:

dimethyl sulphoxide

Concentration:

Treated groups were administered the test article at 15%, 30%, or 61% (maximum achievable dose in vehicle).

No. of animals per dose:

Five

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: DMSO was determined to be the most effective vehicle for the test article, organolignite.
- Irritation: The test article, organolignite, was administered once on Day 1 via dermal application to the surface of both ears using a 1 mL syringe.
The test article was administered at dose concentrations of 4, 8, 15, 30, or 61% at a dose volume of 25 µL/ear. The formulations were stirred
continuously throughout dosing using a magnetic stir bar and stir plate. On Day 6, surviving Phase 1 animals were euthanized.


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: OECD Guideline 429, Skin Sensitization: Local Lymph Node Assay.
- Criteria used to consider a positive response: A sensitization index equal to or greater than 3.0.
TREATMENT PREPARATION AND ADMINISTRATION: The test article vehicle, dimethyl sulphoxide (DMSO); the vehicle for the positive control,
Acetone/Olive Oil (4:1 v/v); the positive control, α Hexylcinnamaldehyde (HCA); or the test article, organolignite; were administered daily for three
consecutive days (Days 1 to 3) via dermal application to the surface of both ears at approximately the same time each day (±1 hour) using a 1 mL
syringe. The treated groups were administered the test article at dose concentrations of 15%, 30%, or 61%, and the positive control article was
administered as a 35% solution of HCA in vehicle. Both treated and control groups were administered the vehicle, positive control, or test article at a dose volume of 25 µL/ear. On Day 6, the cell proliferation marker article, [3H] methyl thymidine, was administered to all groups via bolus intravenous injection via the tail vein at a target dose level of 20 µCi/animal and a target dose volume of 0.25 mL/animal.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

Body Weights: Analyzed by Group Pair-wise Comparisons (Levene’s/ANOVA Dunnett’s/Welch’s)
Disintegrations per minute (DPM): Analyzed by Log Transformation/Group Pair-wise Comparisons
Group Pair-Wise Comparisons (Levene’s/ANOVA-Dunnett’s/Welch’s):
If sample sizes for all groups were three or greater, Levene’s test was used to assess homogeneity of group variances for each specified endpoint
and for all collection intervals. If Levene’s test was not significant (p>=0.01), a pooled estimate of the variance (Mean Square Error or MSE) was
computed from a one-way analysis of variance (ANOVA) and utilized by a Dunnett’s comparison of each treatment group with the control group. If
Levene’s test was significant (p<0.01), comparisons with the control group were made using Welch’s t-test with a Bonferroni correction. Results of all pair-wise comparisons are reported at the 0.05 and 0.01 significance levels. All endpoints were analyzed using two-tailed tests.

Log Transformation/Group Pair-wise Comparisons:
Historical data indicate that DPMs are not normally distributed; therefore, a log transformation was performed on these data. The transformed data
were analyzed as described in the Group Pair-wise Comparisons section.

Positive control results:

In the LLNA, the positive control group (35% HCA) resulted in a sensitization index (SI) of 5.33, which is indicative of a sensitizer (SI ≥3).
In addition, the SI correlated well with the mean dpm values, which were significantly increased in the 35% HCA group relative to the vehicle group.

Parameter:

SI

Value:

0.92

Test group / Remarks:

15% test concentration

Parameter:

SI

Value:

0.75

Test group / Remarks:

30% test concentration

Parameter:

SI

Value:

1.07

Test group / Remarks:

61% test concentration

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

The raw data were expressed as disintegrations per minute (dpm) per animal and background values were determined. Individual values that fell within 3 times the background value were assigned a value of 0 (zero). Table 1 below presents the mean back-transformed least squares (BTLS) dpm and mean dpm for each group. Animals treated with organolignite show similar values when compared to the control group. The positive control responded appropriately and a significant increase in mean BTLS was observed when compared to the control.

Table 1.

Summary of Disintegrations per minute (dpm)
	DMSO (5)	15% (5)	30% (5)	61% (5)	Acetone/Olive Oil	35% HCA
Mean	1190.4	989.6	939.8	1347.4	1004.0	5074.6
Mean BTLS	1003.0	919.0	749.0	1075.0	897.0	4781.0*

* Significantly different from Vehicle (Acetone/Olive Oil); (p<0.01)

Interpretation of results:

GHS criteria not met

Conclusions:

Organolignite was determined to not be a sensitizer when tested at concentrations of 15%, 30% or 61% in Dimethyl Sulphoxide (DMSO), the highest
achievable concentration, in the Murine Local Lymph Node Assay. No adverse effects were observed during the course of this study.

Executive summary:

This study was conducted to evaluate whether the substance, organolignite, elicits skin sensitization reactions (allergic contact dermatitis) in the Murine Local Lymph Node Assay (MLLNA). This study was conducted in accordance with MPI Research Standard Operating Procedures (SOPs) and the protocol and amendments as approved by the Sponsor. A protocol deviation was acknowledged by the Study Director and documented in the raw data. In the opinion of the Study Director, this deviation did not affect the quality or integrity of the study. This study was based on Guideline 429, Skin Sensitization: Local Lymph Node Assay, the Organization for Economic Cooperation and Development (OECD) Guideline for Testing of Chemicals, revised July 2010. Organolignite was determined to not be a sensitizer when tested at concentrations of 15%, 30% or 61% (the highest achievable concentration; solvent used was dimethyl sulphoxide) in the Murine Local Lymph Node Assay. No adverse effects were observed during the course of this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

This study was conducted to evaluate whether the substance, organolignite, elicits skin sensitization reactions (allergic contact dermatitis) in the Murine Local Lymph Node Assay (MLLNA). This study was based on Guideline 429, Skin Sensitization: Local Lymph Node Assay, the OECD Guideline for Testing of Chemicals, revised July 2010. Organolignite was determined to not be a sensitizer when tested at concentrations of 15%, 30% or 61%, the highest achievable concentration (solvent used was Dimethyl Sulphoxide), in the Murine Local Lymph Node Assay. No adverse effects were observed during the course of this study.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Organolignite does not meet the criteria for classification and labeling as a skin sensitizer under CLP EU Regulation 1272/2008 based on the result of the mouse Local Lymph Node Assay (LLNA).