Glycerol, propoxylated, esters with acrylic acid

Endpoint summary

Description of key information

Skin sensitization was observed in an LLNA assay (BASF SE, 2005).

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 16, 2004 - June 24, 2004

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2002

Deviations:

yes

Remarks:

only cell count, lymph node weight and ear weight assessed

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

2003

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA

Remarks:

CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH / Borchen / Germany
- Age at study initiation: About 6 - 8 weeks
- Weight at study initiation: 16.5 g - 21.1 g
- Housing: Makrolon type 1 cage, 1 animal per cage
- Diet: Kliba-Labordiät Kliba SA / Kaiseraugst / Basel / Switzerland; ad libitum
- Water: Tap water ad libitum
- Acclimation period: 14 days (2nd step) resp. 15 days (1st step) before the first test substance application.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24"C
- Humidity (%): 30 - 70%.
- Photoperiod (hrs dark / hrs light): 12 h light (6.00 a.m. - 6.00 p.m.); 12 h darkness (6.00 p.m. - 6.00 a.m.)

Vehicle:

other: acetone

Concentration:

1 st step: 1%, 3% and 10% in acetone
2 nd step: 0.1%, 0.3% and 1 % in acetone

No. of animals per dose:

6

Details on study design:

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- animal assignment: random
- Criteria used to consider a positive response: statistically significance / evaluation by using historical control data

TREATMENT PREPARATION AND ADMINISTRATION:
- TI Prepartion: TI is soluted in acetone
- Application volume: 25 µL per ear
- Site of application: dorsal part of both ears
- Frequency of application: 3 consecutive applications (day 0 - day 2) to the same application site

Terminal procedures:
The animais were sacrificed on study day 5 by cervical dislocation.
Ear weight:
lmmediately after the death of each animal a circular piece of tissue (diameter 0.8 cm) was punched out of the apical part of each ear. The weight of the pooled punches was determined for each animal.
Lymnph node weight: Immediately after removal of the ear punches the left and right auricular Iymph nodes were dissected. The weight of the pooled lymph nodes from both sides was determined for each animal.
Preparation of celI suspension and determination of ceII count:
After weight determination, the pooled lymph nodes of each animal were stored in phosphate buffered saline in an ice-water bath until further preparation. A single ceII suspension was prepared as soon as possible after disseotion by carefuhly passing the
Iymph nodes through an iron mesh (mesh size 200,pm) into 6 mL of phosphate buffered saline. For determination of ceIl count the standard suspension was further diluted with Casyton in a ratio 1:500 or 1: 1000. The celI count was determined using a Casy®-Counter. Each suspension was measured in triplicate (3 dilutions of the standard suspension). The mean of the triplicate measurements was used tor further evaluation.

EVALUATION OF RESULTS
In order to reveal a possible induction of sensitization, the response in the draining lymph node after epicutaneous administration of several concentrations of the test substance to the skin of the ear backs is determined. The parameters used to characterize the response are lymph node celI count and to a certain extent weight. Because not only sensitization induction but also Irritation of the ear skin by the test substance may induce lymph node responses, the weight of ear punches taken from the area of test substance application 18 determined as a parameter tor inflammatory ear swelling serving as an indicator tor the irritant action of the test substance. If a test substance does not show a statistically significant increase in ceII count and/or lymph node weight as compared to the vehicle control in the presence of statistically significantly increased ear weights as indication of skin irritation, it is considered not to be a sensitizer. If at least one concentration tested causes a concentration dependent statistically significant inorease in cell count and/or lymph node weight without being accompanied by a statistically significant increase in ear weight, the test substance is considered to be a sensitizer. If statistically signiticant increases in ear weights are running in parallel to the increase in cell count and/or lymph node weight, it cannot be ruled out, that the lymph node response was caused by irritation and not by skin sensitization. Then, tor identitication of the relevance of the statistical evaluation, a comparison of the results of the present test to appropriate historical control is pertormed. lt one or a combination of the measured parameters change statistical signiticance, evaluation on basis of the
criteria described above may be possible. lt the statistical comparison with the historical control does not yield results usetul for evaluation, further investigations may be necessary to ditterentiate between irritation and sensitization response. lt a test substance does not elicit a statistical signiticant increase in lymph node weight and/or cell count but shows a clear concentration related inorease in response, turther investigating ot the sensitization potential at higher concentrations should be considered.

Positive control substance(s):

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

Statistics:

Lymph node weight, celI count and ear weight: WILCOXON - Test

Positive control results:

A concurrent positive control (reliability check) with a known sensitizer was not included. Studies using the positive control substance Alpha-Hexylcinnamaldehyde, techn. 85% are performed twice a year in the Iaboratory in order to show that the test system is able to detect sensitizing compounds under the test conditions chosen.
Alpha-Hexylcinnamaldehyde, techn. 85% induced a statistically significant and biologically relevant response in the auricular lymph nodes, when applied as 3% or 10% preparations in acetone.

Key result

Parameter:

other: cell count index

Value:

1.86

Test group / Remarks:

1% in acetone

Key result

Parameter:

other: cell count index

Value:

2.47

Test group / Remarks:

3% in acetone

Key result

Parameter:

other: cell count index

Value:

3.14

Test group / Remarks:

10% in acetone

Parameter:

SI

Test group / Remarks:

1% in acetone

Remarks on result:

not measured/tested

Parameter:

SI

Test group / Remarks:

3% in acetone

Remarks on result:

not measured/tested

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
Treatment of the mice with 1% 3% and 10% test substance preparations induced statistically significant increase in Iymph node ceIl counts and Iymph node weights as compared to the vehicle control group. Both parameters display concentration related changes in concentrations of 1% - 10%.
Treatment of the mice with a 0.3% test substance preparation caused a slight significant increase in lymph node weights as compared to the vehicle control group, while no effect on cell counts could be noticed.
Treatment of the mice with 1 % up to 10% test substance preparations induced statistically significant increase in ear weight as compared to the vehicle control group (1st step, only). On the day of Iymph node removal incrustation in five animais and scaling in one animal were noticed on the ears that were treated with a 10% test substance preparation.

DETAILS ON STIMULATION INDEX CALCULATION
not determined

EC3 CALCULATION
not calculated

CLINICAL OBSERVATIONS:
No abnormalities were observed during general observation. No signs of systemnic toxicity were noticed.

BODY WEIGHTS
The expected body weight gain was generally observed in the course of the study.

The mean indices (fold of change as compared to the vehicle control) for lymph node weight, cell count and ear weight are summarized for each test group in the table below.

An increase in ear weight by more than 25% is considered excessive.

Test group	Treatment	Lymph node Weight  Index	Cell Count  Index	Ear Weight Index
1	vehicle acetone	1.00	1.00	1.00
2	1 % in acetone	1.35 #	1.86 #	1.05 #
3	3 % in acetone	1.85 ##	2.47 ##	1.17 ##
4	10 % in acetone	2.25 ##	3.14 ##	1.43 ##
5	vehicle acetone	1.00	1.00	1.00
6	0.1 % in acetone	1.05	1.10	1.00
7	0.3 % in acetone	1.14 #	1.14	1.01
8	1 % in acetone	1.52 ##	1.84 ##	1.04

                            

Results of the positive control

Test group	Treatment	Lymph node Weight  Index	Cell Count  Index	Ear Weight Index
1	vehicle acetone	1.00	1.00	1.00
2	1 % in acetone	1.08	1.25	1.07 #
3	3 % in acetone	1.21##	1.56 #	1.14 ##
4	10 % in acetone	1.73 ##	2.58 ##	1.14 ##

The statistical evaluations were performed using the WILCOXON-test (# for p ≤0.05, ## for p ≤0.01)

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Reference 2

Endpoint:

skin sensitisation: in vitro

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro skin sensitisation study does not need to be conducted because adequate data from an in vivo skin sensitisation study are available

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

The skin sensitizing potential of the test item was assessed using the Murine Local Lymph Node Assay similar to OECD guideline 429 (BASF, 2005). The response of the auricular lymph nodes on repeated application of the test substance to the dorsal skin of the ears was determined. The test was performed in two steps. In the first step groups of 6 female CBA/Ca mice each were treated with 1 %, 3% and 10% w/w preparations of the test substance in acetone or with the vehicle alone. As the treatment of the mice induced statistically significant increase in lymph node cell counts, lymph node weights and ear weights at all concentrations, in the second step concentrations of 0.1%, 0.3% and 1 % w/w were examined in order to determine the no effect concentration. The study used 6 test groups and 2 control groups. Each test animal was applied with 25 µL per ear of the respective test substance preparation to the dorsum of both ears for three consecutive days. The control groups were treated with 25 µL per ear of the vehicle alone. Three days after the last application the mice were sacrificed and the auricular lymph nodes were removed. Lymph node response was evaluated by measuring the cellular content (indicator of cell proliferation) and weight of each animal's pooled lymph nodes. Moreover, a defined area with a diameter of 0.8 cm was punched out of the apical part of each ear and for each animal the weight of the pooled punches was determined in order to obtain an indication of possible skin irritation. No signs of systemic toxicity were noticed. The test substance induced a statistically significant increase in lymph node weights when applied as 0.3%, 1%, 3% or 10% preparation, though the increase at 0.3% was not considered relevant, since it was slight and within the historical control range. Cell counts were statistically significantly and biologically relevantly increased when compared to the vehicle control group after application of 1 % 3% or 10% test substance preparations. The statistically significant increases in ear weights indicate irritation of the ear skin at the concentrations of 1%, 3% or 10%, which was excessive (i.e. more than 25% increase) at 10%. The ears of the animals of this group also showed incrustations or scaling on the day of lymph node removal. According to the OECD 429, a substance is considered a skin sensitizer, if cell proliferation is increased by a factor greater than 3. No such factor can be calculated from the available data, since no measurement of cell proliveration was performed other than determination of the cell count and lymph node weight. Consequently, though skin sensitization was definitely achieved in this study, no threshold concentration can be determined, for which cell proliferation exceeds a factor of 3, and no subcategory can be assigned.

Another study (Cytec, 1981) was performed to determine the delayed contact hypersensitivity potential of GPOTA in 10 male albino guinea-pigs (Hartley/Dunkin strain) with a different protocol (Guinea pig maximization test similar to OECD 406). No definite conclusion can be drawn from this study as most of the control animals also reacted during the challenge procedure. But there seems to be evidence of sensitization in two of the treated animals, so this study was included as support for the sensitzing potential already observed in the LLNA assay described above.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008

The available test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on this information, the substance is considered to be classified for skin sensitisation Cat.1 (H317) under Regulation (EC) No 1272/2008, as amended for the twelfth time in Regulation (EU) 2019/521. No subcategory can be assigned solely on the available data on cell count and lymph node weight, since no EC3 value for proliferation can be calculated.