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Description of key information

Mouse Local Lymphnode Assay was conducted to determine the allergenic potential of the test chemical. The assay was performed according to OECD 429 Guidelines.

Therefore, based on the criteria of this study, treatment with test chemical at concentrations of 25%, 50% and 100% did not result in a stimulation index of 3.0 or greater and hence the substance at concentrations up to 100% did not have skin sensitizing activity.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Remarks:
The study was done in 2011, before the changes in legislation concerning animal testing entered into force.
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May 2011 - December 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
data is from experimental reports
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
24 April 2022
Deviations:
not specified
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
March 2003
Deviations:
not specified
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2004/73/EC L 152, 2004
Deviations:
not specified
Principles of method if other than guideline:
Mouse Local Lymphnode Assay was conducted to determine the allergenic potential of the test chemical
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA:J
Sex:
female
Details on test animals and environmental conditions:
Nulliparous and non-pregnant female mice were used in the study.
Age Range: 8-9 weeks at start of dosing
Body Weight Range: 19-26 grams at the outset (Day 1) of the study. The body weight variations between the mice on Day 1 did not exceed ±20% of the mean weight
Animal Source: Jackson Laboratories, Bar Harbor, ME 04609

Housing: Animals were group housed 5 per cage upon receipt in compliance with National Research Council "Guide for the Care and Use of Laboratory
Animals". The room in which the animals were kept was documented in the study records. No other species were kept in the same room.
Lighting: 12 hours light/12 hours dark
Room temperature: 26 to 29°C
Relative humidity: 16 to 76% in aeroneg enclosure but animals were housed in micro-isolator cages.
Food: Animals had access to Harlan Teklad Certified Rodent Chow 2016C ad libitum. No contaminants were known to be present in the certified diet at levels that
would be expected to interfere with the results of this study. Analysis of the diet was limited to that performed by the manufacturer.
Water: Tap water was available ad libitum, via water bottles. The water is routinely analyzed for contaminants. No contaminants were known to be present in the water at levels that would be expected to interfere with the results of this study.
Acclimation: Study animals were acclimated to their housing for 14 days prior to their first day of dosing
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Group 1 - 0%
Group 2 - 25%
Group 3 - 50%
Group 4 - 100%
No. of animals per dose:
5
Details on study design:
DOSING
Route: Topically on the dorsal surface of both ear
Frequency: Once daily for 3 consecutive days (Days 1-3). The timing of dose administration remained consistent (± 3 hours) during the dosing phase.
Procedure: A volume of 25 uL/ear was applied using a micropipette.

IN-LIFE OBSERVATIONS
Mortality/Morbidity: Daily on Days 1 to 6
Clinical observations: Observations were performed prior to dose administration and immediately following dose administration on Days 1-3. Clinical observations
were also performed once daily on Days 4-6. Particular attention was given to the application sites. Any significant alterations to the application sites, and the general appearance of the pinnae, including build up of test article, was recorded.
Dermal irritation: Animals were examined daily for signs of erythema and edema. Irritation was scored and recorded using the Draize scoring system. Scoring was performed prior to dosing on Days 1-3.
Body weight: Animals were weighed on Days 1 and 6.

METHOD OF PERFORMANCE
Mice were treated on the dorsal surface of both ears, once per day on Days 1, 2, and 3. Approximately 24±3 hours separated each application of test article. On Day 6, the mice were injected i.v. with 20 uCi of 3H-thymidine in 250 uL of sterile saline. Five hours later the mice were euthanized by C02 asphyxiation and the draining auricular lymph nodes were removed. At removal, the number of nodes collected per animal was recorded, and the nodes were examined for size/appearance and the data recorded. Any unexpected observations were noted in study records. A single cell suspension was prepared from the lymph nodes of each mouse. Cells were washed twice with phosphate buffered saline (PBS) and precipitated with 5% trichloroacetic acid (TCA) overnight at 4-5°C. The pellets were recovered by centrifugation and resuspended in 1 ml of TCA and transferred to a vial containing scintillation fluid. An additional 1 mL of TCA
was used to rinse the tube, and it was also transferred to the scintillation fluid. Incorporation of 3H-thymidine was measured in a beta-scintillation counter and
reported as DPM per mouse.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
All data were collected manually except for the data generated by the beta-scintillation counter (Beckman LS 6000 SC). SYSTAT version 9.01, developed by SPSS, Inc was used for statistical analysis. The mean DPM and standard error (sem) for each group was determined. Increases in 3H-thymidine incorporation relative to the vehicle-treated control was derived for each group and recorded as stimulation indices (SI). The criterion for a positive response was that one or more concentrations of a test article elicits a 3-fold or greater increase in isotope incorporation relative to the vehicle control.
Body weights and body weight changes on Days 1 and 6 were also evaluated using SYSTAT version 9.01, developed by SPSS, Inc. The evaluation of the equality of means for body weight data was made by a one-way analysis of variance using the F distribution to assess statistical significance. If statistically significant differences between the means were found, a Dunnett's test was used to determine the degree of significance from the control means.
Individual DPM values were analyzed by log transformation (base 1 0) of the data. The evaluation of the equality of means for the DPM was made by a one-way analysis of variance using the F distribution to assess statistical significance. If statistically significant differences between the means were found, a Dunnett's test was used to determine the degree of significance from the control means.
Positive control results:
SI (35%) = 7.1
Key result
Parameter:
SI
Value:
ca. 1.3
Test group / Remarks:
25%
Remarks on result:
other: not sensitizing
Key result
Parameter:
SI
Value:
ca. 0.9
Test group / Remarks:
50%
Remarks on result:
other: not sensitizing
Key result
Parameter:
SI
Value:
ca. 1.3
Test group / Remarks:
100%
Remarks on result:
other: not sensitizing
Cellular proliferation data / Observations:
MORTALITY AND CLINICAL OBSERVATIONS
There was no mortality and all animals appeared normal throughout the study.
DERMAL IRRITATION SCORES AND DOSE APPLICATION SITE OBSERVATIONS
No erythema or edema was noted in any of the mice in the vehicle group, those dosed with the test article at 25%, 50% or 100% (v/v) or the HCA group at 35%. The ears of all mice treated with the test article at 50% or 100% and those dosed with HCA appeared wet on Days 2-4. There were no other findings.
BODY WEIGHTS
Body weights at Day 1 and Day 6 and body weight changes from Day 1 to Day 6 were evaluated. Statistically significant differences in mean body
weights on Day 1 were observed for the 50%, 100% and HCA groups when compared to the mean body weight for the vehicle control for Day 1. As these
differences were only for the initial body weight they were not biologically relevant. There were no statistically significant differences observed between
any of the groups at Day 6. When the change in body weights were evaluated, the only statistically significant difference observed was in the positive control group. Therefore, the test article did not appear to cause any overt toxicity.
LOCAL LYMPH NODE ASSAY
At termination the lymph nodes in the mice treated with the vehicle and test article group at 25% (v/v) were normal in size and appearance. The lymph nodes from one of five treated with the test article at 50% and three of five treated with the test article at 100% were enlarged but otherwise normal in appearance. The lymph nodes from all five treated with HCA at 35% were enlarged but otherwise normal in appearance. The positive control, 35% (v/v) HCA, resulted in a stimulation index (SI) of 7.1. A 3-fold or greater increase in proliferative activity relative to the concurrent vehicle control is considered a positive response. In addition, the response with HCA was also statistically significant (p<0.001) when the log DPM for this group was compared to the vehicle group. Thus, the sensitivity of the test system was demonstrated.

Group

Treatment

Dose

DPM

(MEAN ±SEM)

SI

(TEST/CONTROL RATIO)

Results*

1

Vehiclea

-

908± 176

-

-

2

Test

25%

1198± 231

1.3

-

3

Test

50%

780 ±191

0.9

-

4

Test

100%

1208± 361

1.3

-

5

HCA

35%

6472± 684***

7.1

+

*- Test/control ratio of 3.0 or greater represents a positive result

a- AOO(4:1)W/V

***- Statistically significant difference when log DPM compared to the vehicle control groups (Group 1)(p<0.001)

Interpretation of results:
other: not sensitizing
Conclusions:
A test material is considered to have skin sensitizing activity if, at one or more concentrations, it induces a 3-fold or greater increase in proliferative activity relative to the concurrent vehicle treated control. Thus, a stimulation index >= 3.0 is regarded as a positive response.
Therefore, based on the criteria of this study, treatment with test chemical at concentrations of 25%, 50% and 100% did not result in a stimulation index of 3.0 or greater and hence the substance at concentrations up to 100% did not have skin sensitizing activity.
Executive summary:

Mouse Local Lymphnode Assay was conducted to determine the allergenic potential of the test chemical. The assay was performed according to OECD 429 Guidelines. 25 Nulliparous and non-pregnant female CBA:J mice were used in the study.

A volume of 25 uL/ear of the test chemical at concentrations of 25%, 50% and 100% in acetone: olive oil(4:1) was applied topically on the dorsal surface of both ears once daily for 3 consecutive days (Days 1-3). The timing of dose administration remained consistent (± 3 hours) during the dosing phase.

On Day 6, the mice were injected i.v. with 20 uCi of 3H-thymidine in 250 uL of sterile saline. Five hours later the mice were euthanized by C02 asphyxiation and the draining auricular lymph nodes were removed. At removal, the number of nodes collected per animal was recorded, and the nodes were examined for size/appearance and the data recorded. Any unexpected observations were noted in study records. A single cell suspension was prepared from the lymph nodes of each mouse. Cells were washed twice with phosphate buffered saline (PBS) and precipitated with 5% trichloroacetic acid (TCA) overnight at 4-5°C. The pellets were recovered by centrifugation and resuspended in 1 ml of TCA and transferred to a vial containing scintillation fluid. An additional 1 mL of TCA was used to rinse the tube, and it was also transferred to the scintillation fluid. Incorporation of 3H-thymidine was measured in a beta-scintillation counter and reported as DPM per mouse.

At termination the lymph nodes in the mice treated with the vehicle and test article group at 25% (v/v) were normal in size and appearance. The lymph nodes from one of five treated with the test article at 50% and three of five treated with the test article at 100% were enlarged but otherwise normal in appearance. The lymph nodes from all five treated with HCA at 35% were enlarged but otherwise normal in appearance. The positive control, 35% (v/v) HCA, resulted in a stimulation index (SI) of 7.1. A 3-fold or greater increase in proliferative activity relative to the concurrent vehicle control is considered a positive response. In addition, the response with HCA was also statistically significant (p<0.001) when the log DPM for this group was compared to the vehicle group. Thus, the sensitivity of the test system was demonstrated.

A test material is considered to have skin sensitizing activity if, at one or more concentrations, it induces a 3-fold or greater increase in proliferative activity relative to the concurrent vehicle treated control. Thus, a stimulation index >= 3.0 is regarded as a positive response.

Therefore, based on the criteria of this study, treatment with test chemical at concentrations of 25%, 50% and 100% did not result in a stimulation index of 3.0 or greater and hence the substance at concentrations up to 100% did not have skin sensitizing activity.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

 

Various studies have been reviewed to determine the allergenic potential of the test chemical in living organisms. These include in vivo experimental results conducted on mice, guinea pigs and humans. The studies are summarized as follows:

Mouse Local Lymphnode Assay was conducted to determine the allergenic potential of the test chemical. The assay was performed according to OECD 429 Guidelines. 25 Nulliparous and non-pregnant female CBA:J mice were used in the study.

A volume of 25 uL/ear of the test chemical at concentrations of 25%, 50% and 100% in acetone: olive oil(4:1) was applied topically on the dorsal surface of both ears once daily for 3 consecutive days (Days 1-3). The timing of dose administration remained consistent (± 3 hours) during the dosing phase.

On Day 6, the mice were injected i.v. with 20 uCi of 3H-thymidine in 250 uL of sterile saline. Five hours later the mice were euthanized by C02 asphyxiation and the draining auricular lymph nodes were removed. At removal, the number of nodes collected per animal was recorded, and the nodes were examined for size/appearance and the data recorded. Any unexpected observations were noted in study records. A single cell suspension was prepared from the lymph nodes of each mouse. Cells were washed twice with phosphate buffered saline (PBS) and precipitated with 5% trichloroacetic acid (TCA) overnight at 4-5°C. The pellets were recovered by centrifugation and resuspended in 1 ml of TCA and transferred to a vial containing scintillation fluid. An additional 1 mL of TCA was used to rinse the tube, and it was also transferred to the scintillation fluid. Incorporation of 3H-thymidine was measured in a beta-scintillation counter and reported as DPM per mouse.

At termination the lymph nodes in the mice treated with the vehicle and test article group at 25% (v/v) were normal in size and appearance. The lymph nodes from one of five treated with the test article at 50% and three of five treated with the test article at 100% were enlarged but otherwise normal in appearance. The lymph nodes from all five treated with HCA at 35% were enlarged but otherwise normal in appearance. The positive control, 35% (v/v) HCA, resulted in a stimulation index (SI) of 7.1. A 3-fold or greater increase in proliferative activity relative to the concurrent vehicle control is considered a positive response. In addition, the response with HCA was also statistically significant (p<0.001) when the log DPM for this group was compared to the vehicle group. Thus, the sensitivity of the test system was demonstrated.

A test material is considered to have skin sensitizing activity if, at one or more concentrations, it induces a 3-fold or greater increase in proliferative activity relative to the concurrent vehicle treated control. Thus, a stimulation index >= 3.0 is regarded as a positive response.

Therefore, based on the criteria of this study, treatment with test chemical at concentrations of 25%, 50% and 100% did not result in a stimulation index of 3.0 or greater and hence the substance at concentrations up to 100% did not have skin sensitizing activity.

This is supported by another Mouse local lymphnode assay (LLNA) conducted to determine the allergenic potential of the test chemical.

30 CBA/CaHsdRcc(SPF) female mice were used in the experiment (nulliparous and non-pregnant).

Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with different test item concentrations of 5%, 10%, 15%, 25% and 50 % (w/v) in acetone/olive oil (4/1, v/v). The application volume, 25 uL, was spread over the entire dorsal surface (0 - 8 mm) of each ear lobe once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals.A hair dryer was passed briefly over the ear's surface to prevent the loss of any of the test item applied. Five days after the first topical application, all mice were administered with 250 uLof 77.68 uCi/mL 3HTdR (equal to 19.4 uCi 3HTdR) by intravenous injection via a tail vein.

Approximately five hours after treatment with 3 HTdR all mice were euthanized by inhalation of C02 (dry ice). The draining lymph nodes were rapidly excised and pooled for each animal (2 nodes per animal). Single cell suspensions (phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 um mesh size). After washing twice with phosphate buffered saline ( approx. 10 mL) the lymph node cells were resuspended in 5% trichloroacetic acid (approx. 3 mL) and incubated at approximately +4 oc for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5% trichloroacetic acid (1 mL) and transferred to glass scintillation vials with 10 mL of 'lrga-Safe Plus' scintillation liquid and thoroughly mixed. The level of 3HTdR incorporation was then measured on a beta-scintillation counter. Similarly, background 3 HTdR levels were also measured in two 1ml-aliquots of 5% trichloroacetic acid. The beta-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (dpm).

A test item is regarded as a sensitizer in the LLNA if the exposure to one or more test concentrations resulted in 3-fold or greater increase in incorporation of 3

HTdR compared with concurrent controls, as indicated by the STIMULATION INDEX (S.I.).

In this study STIMULATION INDICES of 0.8, 0.9, 0.8, 1.0 and 1.2 were determined with the test item at concentrations of 5%, 10%, 15%, 25% and 50% (w/v), respectively.

No dose-response relationship was observed. Calculation of the EC3 value was not performed because no test concentrations produced a STIMULATION INDEX (S.I.) of 3 or higher.

The test chemical was therefore considered to be a non-sensitizer when tested at up to the concentration of 50% (w/v) in acetone/olive oil (4/1, v/v).

These are further supported by another Mouse Local Lymphnode Assay conducted to determine the sensitization potential of the test chemical. 20 Nulliparous, non-pregnant female mice of the Crl:CBAJCa Cru.BR strain were obtained from Charles River (UK) Ltd, Margate.

 

A preliminary screening test was performed with one mouse. The mouse was treated by daily application of 25 uL of the test article at a concentration of 50% v/v in dimethyl formamide to the dorsal surface of each ear for three consecutive days (Days 1, 2 and 3). The mouse was observed daily for five days from the initiation of treatment. Any signs of toxicity or irritation during this period were recorded. The body weight was recorded on Day 1 and not Day -1 as detailed in the protocol. This was considered not to have affected the purpose or integrity of the study. The animal was killed by exposure to a rising concentration of carbon dioxide at the end of the observation period.

Doses were selected from the concentration series 100%, 50%, 25%, 10%, 5%, 2.5%, 1.0%, 0.5% etc. Three consecutive concentrations were selected on the basis of the preliminary screening test and from existing acute toxicity and dermal irritation data which indicated that the undiluted test article would not cause systemic toxicity and excessive local irritation. It is considered that, had the preliminary screening test used the undiluted test article, the group size of one mouse may not have reliably predicted the presence of overt systemic toxicity.

Formulations were freshly prepared as required, using dimethylformamide on Days 1, 2 and 3. The formulations were stored at room temperature, in sealed, airtight containers prior to dosing and were used within two hours of preparation. The formulations were mixed by multiple inversion of the containers prior to administration to ensure homogeneity. Concentrations of test article were expressed volumetrically and in terms of test article received (without regard to purity or active content).

The five groups of five female mice were subjected to application of the vehicle control, positive control or one of the test formulations to the outer aspect of the auditory pinnae, once daily on Days 1, 2 and 3. On Day 6 the mice were placed under an infra-red heat lamp. This was intended to cause dilatation of the peripheral blood vasculature and thus facilitate intravenous dosing. Each mouse was transferred to a cylindrical restrainer. A plastic syringe and fine gauge hypodermic needle were used to administer 0.25 mL phosphate buffered saline incorporating 20 uCi of 3HTdR into a tail vein of each mouse by slow bolus injection. After this treatment, the mice were returned to their cages.

Approximately five hours after intravenous injection of the 3HTdR, all mice were killed by exposure to a rising concentration of carbon dioxide. Killing was organised to minimise the interval between death and the recovery of the auricular lymph nodes to no more than fifteen minutes.

The Local Lymph Node Assay demonstrated that the test chemical has the potential to cause skin sensitisation.

Buehler test was also performed to determine the allergic reactions caused by the test chemical in guinea pigs. The study was performed according to EPA OPPTS 870.2600 (Skin Sensitisation) Guidelines.

10 healthy male and 10 healthy female Hartley Albino guinea pigs were assigned to test and control groups.

The day prior to the first induction application, Site 1 of all animals was clipped free of hair with an electric clipper. The clipped area was approximately 5 x 10 em. Any animal with skin irregularities or irritation was eliminated from the study. The treated sites were reclipped the day prior to each induction application.

Thirteen days after the last induction application, a naive site (site 3) on each animal was clipped free of hair. Sites 2 and 4 (right shoulder and hip areas, respectively) were reserved for alternate sites in the event that eschar was noted during the induction phase and/or a re-challenge was required. The day prior to screen dosing, the dorsal area of each animal was clipped free of hair. The clipped area was approximately 10 x 10 cm.

Group 1, 100%: Ten males and ten females in Group 1 were dosed with 0.4 ml of the test article. The dose was applied to the left shoulder area (Site 1) using a 25 mm Hilltop Chamber which is designed to keep the test article on a 25 mm area of the site. The chamber contained a cotton pad used to facilitate contact of the liquid test article with the site. The chamber was covered with a strip of rubber dental dam sufficient to cover the treated sites. The torso was wrapped with non-irritating tape to provide occlusion. After 6 hours, the dams were removed. Any residual test article was cleansed from the sites with distilled water and the sites were dried with soft toweling. This procedure was performed once/week on the same day each week for a three week period, a total of 3 six hour insults.

Group 2: Five males and five females were untreated for the three week induction period and served as the naive control. Four animals received four concentrations of the test article, one/site as follows: (25, 50, 75, & 90%) and another four animals received three concentrations of the test article, one/site as follows: ( 1 0, 75, 100 & 100% Vehicle Concentration) was placed in a 25 mm Hilltop Chamber, designed to keep the test article on a 25 mm area of the site. The chamber contained a cotton pad which aided in the retention of the liquid sample on the site. The test sites were covered with a strip of rubber dental dam sufficient to cover the treated sites and wrapped with non-irritating tape to provide occlusion. After six hours, the dams and test article were removed, the sites cleansed with distilled water and dried with soft toweling. Fourteen days after the last induction exposure, animals in Groups 1 and 2 were challenged using the same dosing procedure as in the induction phase. Based on the results of the screen, 25% was chosen as the highest non-irritating concentration for the challenge. The doses were applied to a naive site on the left hip area (Site 3).

To aid in the analysis of the data, two indices were calculated from the erythema scores: one to evaluate incidence and the other to evaluate severity. The indices were calculated for both the Test Article Group and the Naive Control Group using the 24 and 48 hour scores following challenge patch removal.

The Incidence Index is the number of animals with a score of 1 or greater divided by the number of animals examined following challenge. The Severitv Index is the total of all erythema scores following challenge (per time period) divided by the number of scores added.

One female died on Day 23; diarrhea in one male of Day 7; soiled anogenital area in 1 male on Days 18-22, 26-27 and in 1 female on Day 30. The incidence and severity Index for the test group at 24 and 48 hours post challenge exposure were 0.053 and 0.105 respectively. Hence, the test chemical can be considered to be not sensitizing to skin.

The above results are further supported by a human maximization test performed to evaluate the dermal sensitization potential of the test chemical. 8% test chemical in petrolatum was applied to the skin of 22 human volunteers and observed for dermal reactions (duration of exposure, observation period not mentioned).

Since none of the volunteers had developed any signs of contact sensitization, the test chemical was considered to be not sensitizing to the skin.

Even though one of LLNA study reports a possibility of the test chemical to be sensitizing to skin, but results of the other studies suggest otherwise. Hence, the test chemical can be considered to be not sensitizing to skin. Comparing the above annotations with the criteria of CLP regulation, the test chemical can be classified under the category “Not Classified”.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Even though one of LLNA study reports a possibility of the test chemical to be sensitizing to skin, but results of the other studies suggest otherwise. Hence, the test chemical can be considered to be not sensitizing to skin. Comparing the above annotations with the criteria of CLP regulation, the test chemical can be classified under the category “Not Classified”.