[No public or meaningful name is available]

Endpoint summary

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

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

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

CAS No.: 078567-28-9; Batch No.: WDJ 1853 D-3; Appearance: clear liquid

Species:

mouse

Strain:

NMRI

Sex:

female

Details on test animals and environmental conditions:

Adaptation: after their arrival, the animals intended for the study were allowed to adapt to the conditions of the animal room for at least 7 days and their state of health was monitored,
Health status: only healthy animals showing no signs of disease were used in the study. The animals were not vaccinated or treated with antiinfectives either before their arrival or during the adaptation or study period. The females were nulliparous and nonpregnant.
Age and Body weight: the mice exhibited a weight of 26 — 32 grams at the beginning of the study. The age of the animals was 8 — 9 weeks.
Housing of the animals:
During the adaptation period the animals were housed in conventional Makrolon type HI cages up to 8 mice and during the study period in type II cages, one animal being kept in each cage. The cages were changed at least twice a week.
Low-dust wood granulate from J. Rettenmaier & Sane Ftillstoff-Fabriken, Germany, was used as bedding. At the instigation of the Laboratory Animal Services (LAS) the wood granulates was analyzed at random for contaminants.
At times animals taking part in other toxicological studies were kept in the same room, but adequate spatial separation and appropriate organization of the work procedures ensured that animals could not be confused.
Room temperature: 22 ± °C
Relative humidity: 40 – 70 %
Light/dark cycle: 12h/12h with artificial illumination
Air throughput: about 10 changes per hour
Diet: PROVIMI KLIBA SA 3883 maintenance diet for rats and mice and tap water (drinking water) provided ad libitum.

Vehicle:

methyl ethyl ketone

Concentration:

The concentrations of 0 %; 3 %; 10 % and 30 % of the test substance.

No. of animals per dose:

6

Details on study design:

Modified Local Lymph Node Assay (IMDS):
A modification of the assay by measuring the cell counts instead of radioactive labeling provides comparable sensitivity, and has the advantage that the cell suspension can be further analysed by different methods (flow cytometry, chemiluminescence responses, immunofluorescence) to gain an insight into mechanistic events.
A further modification was done by including the measurement of the ear swelling after treatment leading to a much more simplified and reliable assay (Integrated Model for the Differentiation of Skin reactions (IMDS). By comparing the specific immune reaction induced by the test substance in the draining lymph nodes (LN; cell counts / LN weights) with the immediate unspecific acute skin reaction (ear swelling / ear weight) it is possible to discriminate the irritating potential from the sensitizing potential of the compound tested.

The test substance was applied epicutaneously onto the dorsal part of both ears of the animals. This treatment was repeated on three consecutive days. The volume administrated was 25 µL/ear. The animals were anaesthetized by inhalation of the carbon dioxide and sacrificed one day after last application (day 4). The appropriate organs were then removed. Lymphatic organs (the auricular lymp nodes) were transferred into PBS.

LLN weigh and cell count determination:
The weights of the lymph nodes were determined on a Mettler semiautomatic balance and stored in a IBM compatible PC. After crushing the lymph nodes through a plastic sieve in a 12-well plate, the cell counts per ml were determined using a Culter Counter. These data were also processed by the computer. Special BASIC programs (GWBASIC complier) were used to calculate indices, means and standard deviations.
The so called stimulation or LLN- index is calculating by dividing the absolute number of weight or cell counts of the substance treated lymph nodes by the vehicle treated ones.
The samples (cell suspensions) of this study have been analysed by flow cytometry (FACScan) in addition.
Ear swelling: Before the first treatment and before sacrifice the thickness of both auricles of the animals was measured using a spring-loaded micrometer.
Ear weight: on day 4 te ear weight o the sacrificed animals was measured using a punch to take of the piece of every ear wit a diameter of 8 mm.
Body weight: the body weights of the animals were recorded initiating the and at the end of the study.

Key result

Parameter:

other: cell counts

Value:

1.3

Test group / Remarks:

all test groups, statistical significance in mid and high test group

Key result

Parameter:

other: ear swelling

Remarks:

[mm]

Value:

0.02

Test group / Remarks:

high dose group

Key result

Parameter:

SI

Remarks on result:

not measured/tested

Cellular proliferation data / Observations:

The results show that the test substance has a sensitizing potential in mice after dermal application.
- Compared to vehicle treated animals there was a clear increase regarding the weights of the draining lymph nodes, that is of statistical significance in the highest dose group and a clear increase in the cell counts in the mid and highest dose group.
- The "positive level" of index 1.3 was exceeded for the cell counts in all dose groups.
- A significant increase compared to vehicle treated animals regarding ear swelling (2-E2 mm) and ear weight was detected in the mid and the highest dose group. An increase in this parameter to point of an acute irritating (inflammatory) response. However, such an irritating property is also combined with a strong skin sensitizing potential of a test substance.

Interpretation of results:

other: CLP

Remarks:

classified as 1B sensitizer

Conclusions:

Under the study conditions, the test substance was considered to be sensitising to skin.

Executive summary:

A study was conducted to determine the skin sensitisation potential of the test substance according to OECD Guideline 429 (modified Local Lymph Node Assay), EU Method B.42 and EPA OPPTS 870.2600. Four groups of 6 female NMRI mice of the strain Hsd Win:NMRI were treated at concentrations of 0 %, 3%, 10% and 30% of the test substance formulated in methyl ethyl ketone (MEK). The test substance was applied epicutaneously on the dorsal part of both ears of the animals. This treatment was repeated on three consecutive days. Following exposure, the animals were anaesthetized by inhalation of carbon dioxide and sacrificed one day after last application (Day 4). The ear lymph nodes were then removed and transferred into phosphate buffered saline. Compared to vehicle treated animals there was a clear increase in the weights of the draining lymph nodes (statistically significant in the highest dose group). Also, there was a clear increase in the cell counts in the mid and highest dose groups. The cell count index exceeded 1.3 in all dose groups. The ear swelling and ear weight was also increased in the mid and the highest dose groups. Under the study conditions, the test substance was considered to be sensitising to skin (Vohr, 2003).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

A study was conducted to determine the skin sensitisation potential of the test substance according to OECD Guideline 429 (modified Local Lymph Node Assay), EU Method B.42 and EPA OPPTS 870.2600. Four groups of 6 female NMRI mice of the strain Hsd Win:NMRI were treated at concentrations of 0 %, 3%, 10% and 30% of the test substance formulated in methyl ethyl ketone (MEK). The test substance was applied epicutaneously on the dorsal part of both ears of the animals. This treatment was repeated on three consecutive days. Following exposure, the animals were anaesthetized by inhalation of carbon dioxide and sacrificed one day after last application (Day 4). The ear lymph nodes were then removed and transferred into phosphate buffered saline. Compared to vehicle treated animals there was a clear increase in the weights of the draining lymph nodes (statistically significant in the highest dose group). Also, there was a clear increase in the cell counts in the mid and highest dose groups. The cell count index exceeded 1.3 in all dose groups. The ear swelling and ear weight was also increased in the mid and the highest dose groups. Under the study conditions, the test substance was considered to be sensitising to skin (Vohr, 2003).

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The available in vivo LLNA study with the test substance indicated increase in ear swelling and ear weight accompanied by increase in the cell count index above 1.3 in the treated mice. Based on a conservatice approach, th test substance warrants a classification of Skin sens. 1B (H317: May cause an allergic reaction).