Barium(2+) 12-hydroxyoctadecanoate

Endpoint summary

Description of key information

In an in vivo skin sensitisation assay in mice (LLNA) according to OECD guideline 429, the test item induced an SI of 2.3. Thus, the test item is considered as not sensitising.

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:

2018-03-07 to 2018-04-19

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)

Version / remarks:

22 July 2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

20 July 2012

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
- Batch No.of test material: Lab NP_20171034-003
- Expiration date of the lot/batch: 2022-08-18

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: closed vessel at room temperature (20±5°C).

OTHER SPECIFICS:
white solid

Species:

mouse

Strain:

CBA/Ca

Remarks:

Ola Hsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source:TOXI-COOP ZRT., H-1103, Budapest, Cserkesz u. 90., Hungary
- Females nulliparous and non-pregnant: Yes
- Microbiological status of animals, when known: SPF
- Age at study initiation: 12 weeks old
- Weight at study initiation: 20.2 – 24.8 g
- Housing:
Housing during acclimatization period: Grouped caging in small groups
Housing during the test: Grouped caging (4 animals/cage)
Cage type: Type II. polypropylene/polycarbonate
- Diet: ssniff® Rat/Souris-Elevage E complete diet for rats and mice produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany, ad libitum.
- Water: tap water from watering bottles ad libitum.
- Acclimation period: 21 days
- Indication of any skin lesions: No

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 – 70
- Photoperiod (hrs dark / hrs light): 12/12, from 6.00 a.m. to 6.00 p.m.

Vehicle:

propylene glycol

Concentration:

maximum concentration of 10 % (w/v)

No. of animals per dose:

8 females/dose

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: Selection of the vehicle was performed following the recommendation of the relevant guidelines and based on a preliminary test item formulation evaluation which was performed before the study initiation date. Solubility of the test item was evaluated in the following vehicles (listed in order of preference) according to the relevant guidelines.
1. Acetone: Olive oil 4:1 (v/v) mixture (AOO)
2. N,N-Dimethylformamide (DMF)
3. Dimethyl sulfoxide (DMSO)
4. Methyl ethyl ketone (MEK)
5. Propylene glycol (PG)
6. Absolute ethanol: water 7:3 (v/v) mixture (EtOH)
7. Aqueous 1 % (w/v) Pluronic®PE 9200 (Plu)
No solubility was observed (the minimum evaluated concentration was 2.5 %, w/v). The formulations were suspensions with all vehicles. The most homogeneous suspension was achieved with PG at a maximum concentration of 10 % (w/v). Based on these observations the test item was examined in the LLNA as suspension formulation in PG.

- Irritation: Not observed
- Systemic toxicity: Not observed
- Ear thickness measurements: No increase observed
- Erythema scores: a max. of 2 was observed in 2 animals of the high dose group (10 %)

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: The animals were set in order of their body weights. The animals were randomly assigned to control and test groups using a randomization scheme. The randomization was checked by computer software [SPSS/PC+ (4.0.1)] according to the actual body weights verifying the homogeneity and deviations between the groups.
- Criteria used to consider a positive response:
The test item is considered as a skin sensitizer, if the following criterion is fulfilled:
- Exposure to at least one concentration (non-irritating, non-toxic) of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index (SI ≥ 3). However, the strength of the dose-response, the statistical significance and the consistency of the solvent/vehicle and PC responses may also be used when determining whether a borderline result is declared positive.

TREATMENT PREPARATION AND ADMINISTRATION:
Each mouse was topically treated with 25 μL of the appropriate formulations of the test item, the positive control substance or the vehicles (see Table 3) using a pipette, on the dorsal surface of each ear. After the treatments animals were returned to their cages. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There were no treatments on Days 4, 5 and 6.

OBSERVATIONS
During the main test (from Day 1 to Day 6) all animals were observed at least once a day for any clinical signs, including systemic toxicity and local irritation. Irritation was monitored by erythema scoring during the whole test. Individual records were maintained.Individual body weights were recorded on Day 1 (beginning of the test) and on Day 6 (prior to 3HTdR injection) with a precision of ± 0.1 g.

Positive control substance(s):

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

Positive control results:

The positive control group animals were treated with 25 % HCA solution (formulated in AOO) concurrent to the test item treated groups. No mortality, cutaneous reactions or signs of toxicity were observed in the positive control group. The positive control substance induced the appropriate stimulation compared to the relevant control (AOO). The calculated SI value was 10.3. The results of the positive control item demonstrated appropriate performance of the test in accordance with the relevant guidelines and confirmed sensitivity and validity of the assay.

Key result

Parameter:

SI

Value:

2.3

Test group / Remarks:

10 % test item in PG

Key result

Parameter:

SI

Value:

1.4

Test group / Remarks:

5.0 % test item in PG

Key result

Parameter:

SI

Value:

1.9

Test group / Remarks:

2.5 % test item in PG

Key result

Parameter:

SI

Value:

1.5

Test group / Remarks:

1.0 % test item in PG

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
No treatment group was excluded from the evaluation since no failed treatment and no sign of systemic toxicity or irritation were observed during the test. Visually larger lymph nodes compared to the relevant vehicle control (AOO) were observed in the positive control group only. Appearance of the lymph nodes was normal in the negative control groups (AOO or PG) and in the test item treated groups.
No significant lymphoproliferative response (SI ≥ 3) was observed for the test item at the tested concentrations. The observed stimulation index values were 2.3, 1.4, 1.9 and 1.5 at test item concentrations of 10 %, 5 %, 2.5 % and 1 % (w/v), respectively. Significance of the dose-response was evaluated by linear regression using the SI values. No significant dose-related response was observed (p = 0.3, r = 0.7).

EC3 CALCULATION
Not determinable since no SI > 3 was documented.

CLINICAL OBSERVATIONS:
No mortality or symptoms of systemic toxicity were observed in any treatment group. No sign of irritation (indicated by an erythema score ≥ 3) or any other local effect were observed in any treatment group.

BODY WEIGHTS
No significant, treatment related effect on the body weights was observed in any treatment group. Body weight decrease by > 5 % was observed in the following groups: positive control (2 of the 4 animals), vehicle control for the test item (PG, 1 of the 4 animals), 10 %, 2.5 % and 1 % (w/v) dose groups (1 of the 4 animals in each). The decrease was 6 % in all groups and was considered as insignificant.

Table 1: DPM and Stimulation Index Values for all Groups in the Main Test

Dose Group	Measured DPM/group	Group DPM *	DPM/mouse #	Stimulation Index Values
Vehicle control for the positive control: AOO	2863	2840.5	710.1	1.0
Positive control: 25 % HCA in AOO	29406	29383.5	7345.9	10.3
Vehicle control for the test item: PG	1290	1267.5	316.9	1.0
Test Item 10 % in PG	2995	2972.5	743.1	2.3
Test Item 5.0 % in PG	1751	1728.5	432.1	1.4
Test Item 2.5 % in PG	2372	2349.5	587.4	1.9
Test Item 1.0 % in PG	1886	1863.5	465.9	1.5
AOO = Acetone: Olive oil 4:1 (v/v) mixture HCA =α-Hexylcinnamaldehyde PG = Propylene glycol *Group DPM = measured DPMgroup- average DPMbackground Average DPMbackground = 22.5 # Number of animals/group = 4

Interpretation of results:

GHS criteria not met

Conclusions:

In an in vivo skin sensitisation assay in mice (LLNA) according to OECD guideline 429, the test item induced an SI of 2.3. Thus, the test item is considered as not sensitising.

Executive summary:

The skin sensitizing potential of the test item was assessed in an in vivo assay (LLNA) in mice. The pooled treatment group approach was used in this test.

The maximum dose selection was performed according to the relevant guidelines and based on results of a formulation evaluation and a Dose Range Finding test (DRF).

The test item was insoluble in all standard vehicles used in the LLNA. The most homogeneous formulation (suspension) was achieved with PG at a maximum concentration of 10 % (w/v). As no adverse effects (systemic toxicity or irritation) were observed up to this maximum concentration in the DRF the test item was examined in the main test at 10 %, 5 %, 2.5 % and 1 % (w/v) concentrations as suspension formulations in PG. The test item was applied topically to the mouse ear of 4/dose young adult (12 weeks old) female CBA/Ca mice (SPF). Appropriate positive control (α-Hexylcinnamaldehyde, HCA) and a negative control groups dosed with the vehicle of the positive control or the test item (AOO or PG, respectively) were employed. The positive control item (25 % (w/v) HCA in AOO) induced significant stimulation over the relevant control (SI = 10.3). Thus, confirming the sensitivity and validity of the assay. No mortality was observed during the main test. No significant, treatment related effect on body weights or any other sign of systemic toxicity were observed in any treatment group. No signs of irritation (monitored by erythema scoring) or any other local effect were observed at the treatment site (ears) in any treatment group. No significantly increased lymphoproliferation (indicated by an SI ≥ 3) compared to the control was noted for the test item at the applied test concentrations. The observed stimulation index values were 2.3, 1.4, 1.9 and 1.5 at test item concentrations of 10 %, 5 %, 2.5 % and 1 % (w/v), respectively. No significant dose-related response was observed (evaluated by linear regression using the SI values; p =0.3, r =0.7).

According to evaluation criteria of the relevant guidelines, the lack of a significantly increased lymphoproliferation up to the maximum feasible concentration of 10 % (w/v) and also the lack of a significant dose-related response are considered as evidence that the test item is not a skin sensitizer.

Under the conditions of the present assay, the test item tested at the maximum feasible concentration of 10 % (w/v) and also at concentrations of 5 %, 2.5 % or 1 % (w/v) as suspension formulations in a suitable vehicle (propylene glycol) was shown to have no skin sensitization potential in the Local Lymph Node Assay in mice.

Reference 2

Endpoint:

skin sensitisation: in vitro

Data waiving:

study technically not feasible

Justification for data waiving:

other:

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
A combination of non-animal methods within Integrated Approaches to Testing and Assessment (IATA) are needed to be able to fully substitute for the animal tests currently in use considering the restricted Adverse Outcome Pathway (AOP) mechanistic coverage of each of the currently available non-animal test methods (OECD TG 442C, 442D, 442E, addressing the first, second and third key event of the skin sensitisation AOP, respectively). The substance did not fulfill the requirements for solubility, indicated by the OECD guidelines 442 C, 442D or 442E. Thus the assays could not be performed. This justifies the use of the in vivo method for skin sensitisation in the absence of in chemico/in vitro data according to Annex VII, 8.3.1 of Regulation (EC) No 1907/2006. For further information please refer to the attached statement of the performing lab.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Additional information:

Skin sensitisation

The skin sensitizing potential of the test item was assessed in an in vivo assay (LLNA) in mice. The pooled treatment group approach was used in this test.

The maximum dose selection was performed according to the relevant guidelines and based on results of a formulation evaluation and a Dose Range Finding test (DRF).

The test item was insoluble in all standard vehicles used in the LLNA. The most homogeneous formulation (suspension) was achieved with PG at a maximum concentration of 10 % (w/v). As no adverse effects (systemic toxicity or irritation) were observed up to this maximum concentration in the DRF the test item was examined in the main test at 10 %, 5 %, 2.5 % and 1 % (w/v) concentrations as suspension formulations in PG. The test item was applied topically to the mouse ear of 4/dose young adult (12 weeks old) female CBA/Ca mice (SPF). Appropriate positive control (α-Hexylcinnamaldehyde, HCA) and a negative control groups dosed with the vehicle of the positive control or the test item (AOO or PG, respectively) were employed. The positive control item (25 % (w/v) HCA in AOO) induced significant stimulation over the relevant control (SI = 10.3). Thus, confirming the sensitivity and validity of the assay. No mortality was observed during the main test. No significant, treatment related effect on body weights or any other sign of systemic toxicity were observed in any treatment group. No signs of irritation (monitored by erythema scoring) or any other local effect were observed at the treatment site (ears) in any treatment group. No significantly increased lymphoproliferation (indicated by an SI ≥ 3) compared to the control was noted for the test item at the applied test concentrations. The observed stimulation index values were 2.3, 1.4, 1.9 and 1.5 at test item concentrations of 10 %, 5 %, 2.5 % and 1 % (w/v), respectively. No significant dose-related response was observed (evaluated by linear regression using the SI values; p =0.3, r =0.7).

According to evaluation criteria of the relevant guidelines, the lack of a significantly increased lymphoproliferation up to the maximum feasible concentration of 10 % (w/v) and also the lack of a significant dose-related response are considered as evidence that the test item is not a skin sensitizer.

Under the conditions of the present assay, the test item tested at the maximum feasible concentration of 10 % (w/v) and also at concentrations of 5 %, 2.5 % or 1 % (w/v) as suspension formulations in a suitable vehicle (propylene glycol) was shown to have no skin sensitization potential in the Local Lymph Node Assay in mice.

Justification for classification or non-classification

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

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. In vitro results with the test item were negative. As a result the test substance is considered not to be classified for skin sensitisation under Regulation (EC) No 1272/2008, as amended for the twelfth time in Regulation (EU) No 2019/521.