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Description of key information

K-Salt is considered negative for dermal sensitization potential in the LLNA. The validity of the study was confirmed via a positive response with 25% (HCA), a weak sensitiser.

The vehicle control and positive control in the definitive LLNA were within the acceptable ranges and fulfilled the requirements for a valid assay.

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:
29 November 2017 to 21 December 2017
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:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source of test material: The Dow Chemical Company, Midland, Michigan (United States)
- Source and lot No.of test material: YY00G7P285
- Expiration date of the lot: December 17, 2017
- Purity: 99.8% min.
- Puriity test date: Not specified

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient temperature
- Stability under test conditions: Not determined, assumed stable for the duration of the study.
- Solubility and stability of the test substance in the solvent/vehicle: Not determined, assumed stable for the durtion of the study
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: None

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Solublilised in Dimethylformamide (DMF) up to 50% w/v.

FORM AS APPLIED IN THE TEST (if different from that of starting material) . The test susbstance, a crystalline powder, was solublised in Dimethylformamide (DMF) up to 50% w/v.
Species:
mouse
Strain:
CBA:J
Remarks:
Mus musculus
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Animal Breeding Facility, Jai Research Foundation
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 10 to 11 weeks old at the initiation of treatment
- Weight at study initiation: 19.7 to 25.6g
- Housing: Mice were housed individually in solid floor polypropylene (size: ca. 290 mm x 220 mm x 140 mm) solid bottom cages which conform to the size recommendations in the most recent Guide for the Care and Use of Laboratory Animals (Natl. Res. Council, 2011). These cages had stainless steel top grills through which pellet feed and drinking water were provided; steam sterilized corn cob bedding was used and changed along with the cage at least twice a week. On test day 6, animals were housed in metabolic cages.
- Diet (e.g. ad libitum): Teklad Certified Global High Fiber Rat/Mice feed manufactured by Envigo, USA.
- Water (e.g. ad libitum): UV sterilised water (Reverse Osmosis water filtration system)
- Acclimation period: 6 days prior to dosing
- Indication of any skin lesions: No, all mice were received into the experimental procedure room after veterinary examination for health condition and allowed to acclimatise to the laboratory conditions for a period of 6 days prior to commencement of dosing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 23 °C
- Humidity (%): 57 to 66%
- Air changes (per hr): 16 air changes/hour
- Photoperiod (hrs dark / hrs light): The photoperiod was 12 h artificial light and 12 h darkness, light hours being 06:00 – 18:00 h (photoperiod maintained through an automatic timer).
- IN-LIFE DATES: From: To: 1 December 2017 to 21 December 2017
Vehicle:
dimethylformamide
Concentration:
In the main test, K-Salt was tested at concentrations of 2%, 10% and 50% (w/v) in DMF
No. of animals per dose:
5 mice per group
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: Prior to the screening test, a solubility test was conducted to identify an appropriate vehicle for testing K-Salt. The test item was insoluble in guideline recommended LLNA vehicle, acetone/olive oil (AOO) (4:1 v/v) at 25%. It was however soluble in dimethylformamide (DMF) up to 50% w/v, therefore, DMF was selected as the vehicle.

Doses selected for the irritation screening study were based on maximum solubility of the test item in DMF. Selected dose concentrations for the screening study included: 1%, 2.5%, 5%, 10%, 25% and 50% (w/v) in DMF.

An irritation screening test was conducted to identify the highest dose that does not cause irritation or overt systemic toxicity. Six groups of mice (2 mice per group) were treated with K-Salt at concentrations of: 1%, 2.5%, 5%, 10%, 25% and or 50% (w/v) in DMF (25 µL/ear) for three consecutive days (days 1, 2 and 3). The dose was spread evenly over the dorsal surface of the ear. Clinical observations were recorded daily during the experiment. Ear thickness of each animal was measured (apex of the pinna) using a micrometer (digital micrometer; serial N° 293.821) on days 1 (pre-dose), 3 and 6. Increases in ear swelling on days 3 and 6 were calculated for each animal relative to the thickness measurement taken on day 1.

Body weight was recorded on days 1 and 6 (prior to termination). The ears were evaluated daily for erythema (except test item treated group animals due to the color of the test material). Ear swelling and erythema were was used to identify concentrations of the test material that produced irritation to the ears of mice. Previous studies have determined that chemicals with irritancy potential, but without sensitization potential, can produce a detectable proliferative response when administered at concentrations that induce excessive local irritation (Kimber et al., 1994 and ICCVAM, 1999). Excessive local skin irritation is indicated by an erythema score ≥3 on any day of measurement and/or an increase in ear thickness of ≥25% on day 3 or 6 relative to the measurement on day 1 (OECD Test Guideline 429, 24 July 2010). These data along with expert judgment are used in the selection of the final doses to be used in the LLNA.

- Irritation: None observed
- Systemic toxicity: None observed
- Ear thickness measurements: yes, no treatment related effects observed
- Erythema scores: None observed

All mice appeared active and healthy, and there were no consistent treatment-related effects on body weight. There was no increase in ear swelling of 25% or greater observed in the 1%, 2.5%, 5%, 10%, 25% orand 50% (w/v) K-Salt in DMF test concentration groups.

MAIN STUDY
Dose selection for the main study was based on toxicity data generated in the screening study. K-Salt did not demonstrated greater than 5% reduction in body weight and no ≥ 25% increase in ear thickness was observed in any tested concentrations in the screening study. Therefore, in the main study K-Salt was tested at 2%, 10% and 50% (w/v) in DMF concentrations. Fresh dose solutions were prepared daily prior to application. The concentrations of the dose solutions were not verified analytically.

Prior to treatment, the animals were weighed and the ears were checked for any abnormalities or clinical signs of diseases or injury. Twenty five healthy naive female mice without pre-existing ear irritation were selected and distributed into treatment groups (5 mice per group). Three treatment groups (G22 to G24) were treated topically once daily for three consecutive days (days 1, 2 and 3) on the dorsal surface of both ears (25 L/ear) using a calibrated micropipette with K-Salt at concentrations of 2%, 10% and or 50% (w/v) in DMF, respectively. The dose was gently spread evenly over the dorsal surface of the ear using the tip of the pipette.

Mice from the vehicle control group (G21) and positive control group (G25) were handled in the same manner but received 25 µL/ear of vehicle (DMF) and or 25% a-Hexylcinnamaldehyde (v/v) in vehicle (DMF), respectively. No treatment was applied on days 4 and 5 for any group. All dosage preparations were freshly prepared on the day of application.

Group mean body weights of treated animals were comparable with the vehicle control group. There were no indications of clinical or systemic toxicity in K-Salt treated animals.

ANIMAL ASSIGNMENT AND TREATMENT
After acclimatisation animals were randomized into different groups using in-house developed, validated computer software.

In the screening study, K-Salt did not demonstrated greater than 5% reduction in body weight and no ≥ 25% increase in ear thickness was observed in any tested concentrations in the screening study. Therefore, in the main study K-Salt was tested at 2%, 10% and 50% (w/v) in DMF concentrations. Fresh dose solutions were prepared daily prior to application. The concentrations of the dose solutions were not verified analytically.

- Name of test method: Local Lymph Node Assay (LLNA) in accordence with:
OECD, 2010: The Organisation for Economic Co-operation and Development (OECD) Guidelines for the Testing of Chemicals, OECD 429, Skin Sensitisation: Local Lymph Node Assay, adopted by the Council on July 22, 2010.
U.S. EPA, 2003: The United States Environmental Protection Agency (EPA), Health Effects Test Guidelines, OPPTS 870.2600, “Skin Sensitization” (EPA 712-C-03-197), March 2003.
EC, 2012: The Official Journal of the European Community (L142): Part B: Methods for the Determination of Toxicity and other Health Effects, B.42. Skin Sensitization: Local Lymph Node Assay EC 440/2008 adopted July 06, 2012.

- Criteria used to consider a positive response:
The proliferate response of lymph nodes from each mouse was expressed as the number of radioactive DPM/ per mouse, calculated by subtracting out background DPM (measured in 1 mL of 5% TCA aliquot).

Stimulation index (SI) = mean DPM of test group divided by mean DPM of solvent/vehicle control group

The DPM/mouse, along with an appropriate measure of inter-animal variability (i.e., mean ± standard deviation), were calculated for each test group and vehicle and positive control groups. Final results were expressed as the (SI) which is calculated as a ratio of the mean DPM of test group divided by mean DPM of vehicle control group. Any test item that produces a SI > 3 in the LLNA is considered “positive” for dermal sensitization potential (Kimber et al., 1994).

TREATMENT PREPARATION AND ADMINISTRATION:
K-Salt did not demonstrated greater than 5% reduction in body weight and no ≥ 25% increase in ear thickness was observed in any tested concentrations in the screening study. Therefore, in the main study K-Salt was tested at 2%, 10% and 50% (w/v) in DMF concentrations. Fresh dose solutions were prepared daily prior to application. The concentrations of the dose solutions were not verified analytically.

Prior to treatment, animals were weighed and the ears were checked for any abnormalities, clinical signs of diseases or injury. Twenty five healthy naive female mice without pre-existing ear irritation were selected and distributed into treatment groups (5 mice per group). Three treatment groups (G22 to G24) were treated topically once daily for three consecutive days (days 1, 2 and 3) on the dorsal surface of both ears (25 µL/ear) with K-Salt at concentrations of 2%, 10% and or 50% (w/v) in DMF, respectively. The dose was gently spread evenly over the dorsal surface of the ear using the tip of the pipette.

Mice from the vehicle control group (G21) and positive control group (G25) were handled in the same manner but received 25 µL/ear of vehicle (DMF) and 25% a-Hexylcinnamaldehyde (v/v) in vehicle (DMF), respectively. No treatment was applied on days 4 and 5 for any group. All dosage preparations were freshly prepared on the day of application. All procedures including, administration of 3H-methyl thymidine (on day 6), individual animal observations, individual and group animal body weights (prior to dosing on day 1, and prior to of 3H-TdR administration on day 6), collection of lymph nodes, cell suspension preparation and determination of cellular proliferation were inaccordence with regulatory requirements.


Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
In addition to an assessment of the magnitude of the SI, statistical analysis was carried out for the assessment of the dose response relationship and pair-wise comparison made between the treatment and the solvent/vehicle control group. All the parameters characterised by continuous data such as body weight and radioactive DPM were subjected to Bartlett’s test to meet the homogeneity of variance before conducting Analysis of Variance (ANOVA). To compare vehicle and positive control data, Student's t-test was performed to calculate significance.
Positive control results:
The SI of 5.45 obtained for the concurrent positive control, α-Hexylcinnamaldehyde, showed greater than a three-fold increase over the vehicle control value indicating a clear positive response for this known weak sensitizer that confirmed the reliability of this test procedure.
Key result
Parameter:
SI
Value:
1
Test group / Remarks:
5 mice
Key result
Parameter:
SI
Value:
0.86
Test group / Remarks:
5 mice
Key result
Parameter:
SI
Value:
1.25
Test group / Remarks:
5 mice
Key result
Parameter:
SI
Value:
2.04
Test group / Remarks:
5 mice
Key result
Parameter:
SI
Value:
5.45
Test group / Remarks:
5 mice
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA

Group N° Dose Concentration No° of Mice Group Mean (DPM) Standard Deviation Stimulation Index (SI)

G21 Vehicle control 5 793.80 319.35 1.00
G22 2% K-Salt 5 679.00 288.71 0.86
G23 10% K-Salt 5 992.50 566.59 1.25
G24 50% K-Salt 5 1622.40* 477.10 2.04
G25 Positive control 5 4326.60** 1001.39 5.45

* Significantly higher than vehicle control (p≤0.05)
** Significantly higher than vehicle control (p≤0.01)

Positive control (HCA) = α-Hexylcinnamaldehyde
DPM = Disintegrations per minute
DMF = Dimethylformamide

DETAILS ON STIMULATION INDEX CALCULATION
Stimulation Index = Mean DPM of test group divided by mean DPM of solvent/vehicle control group

The SI obtained for 2%, 10% and 50% (w/v) K-Salt showed less than three-fold increase over the vehicle control value; therefore, an EC3 calculation was not possible.


CLINICAL OBSERVATIONS: None observed

BODY WEIGHTS: The mean body weight of positive control as well as K-Salt treated mice was comparable to that of the vehicle control group. All animals gained bodyweight by the end of the study.
Interpretation of results:
GHS criteria not met
Conclusions:
K-Salt is considered negative for dermal sensitization potential in the LLNA. The validity of the study was confirmed via a positive response with 25% (HCA), a weak sensitiser.
The vehicle control and positive control in the definitive LLNA were within the acceptable ranges and fulfilled the requirements for a valid assay.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

This substance is not classified. K-Salt is considered negative for dermal sensitization potential in the LLNA. The validity of the study was confirmed via a positive response with 25% (HCA), a weak sensitiser.

The vehicle control and positive control in the definitive LLNA were within the acceptable ranges and fulfilled the requirements for a valid assay.