Reaction mass of lauric acid, compound with morpholine (1:1) and 2-ethylhexyl dihydrogen phosphate, compound with morpholine (1:2) and bis(2-ethylhexyl) hydrogen phosphate, compound with morpholine (1:1)

Endpoint summary

Description of key information

Under the conditions of the present Local Lymph Node Assay, the test item tested at 5 % (as the maximum feasible non-toxic, non-irritant concentration) and at 2.5%, 1% and 0.5 % (w/v) concentrations as formulations (apparently solutions) in a suitable vehicle (aqueous 1% (w/v) Pluronic®PE 9200) showed to have skin sensitization potential. Based on the EC3 value of 2.6 % the test item was considered a moderate skin sensitizer in this LLNA. Based on this EC3 value the test item can be allocated to Category 1 and sub-category 1B according to the effective regulations.

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:

2016-04-18 to 2016-06-02

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:

2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2012

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

2003

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA/Ca

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: at arrival: SPF; during test: good convential
- Age at study initiation: Young adult mice 8-9 or 12 weeks old (at start of the first DRF); 10-11 weeks old (at start of the second DRF)
- Weight at study initiation: 19.0 – 22.7 g; the weight variation in animals involved in the study did not exceed ± 20 % of the mean weight.
- Housing: Grouped caging (5 animals/cage); cage: Type II. polypropylene/polycarbonate. Mice are group-housed to allow social interaction, and with deep wood sawdust bedding, to allow digging and other normal rodent activities. Bedding: Lignocel Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co.KG (Holzmühle 1, D-73494 Rosenberg, Germany) was available to animals during the study.
- Diet: Animals received ssniff® Rat/Souris-Elevage E complete diet for rats and mice produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany, ad libitum. The food is periodically analysed and is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. Copies of the relevant Certificates of Analysis are maintained in TOXI-COOP ZRT.’s archive.
- Water: Animals received tap water from watering bottles ad libitum. The drinking water is periodically analysed and is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. Copies of the relevant Certificates of Analysis are maintained in TOXI-COOP ZRT.’s archive.
- Acclimation period: 7 days
- Indication of any skin lesions: none

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30 – 70 %
- Photoperiod: 12 hours daily, from 6.00 a.m. to 6.00 p.m.

Vehicle:

other: PluronicPE9200 (Plu)

Concentration:

1 % (w/v)

No. of animals per dose:

5 animals (1 or 2 animals/group) in the first Dose Range Finding Test (DRF)
8 animals (2 animals/group) in the second DRF

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: The test item was appropriately miscible with Plu at a maximum concentration of 75 % (w/v) and below (75 %, 50 %, 25 % and 10 % (w/v) concentrations were examined)
- Irritation: As a sign of a significant irritation significantly increased ear thickness values (compared to the initial values) were observed in all dose groups. All animals were affected: the maximum increase was 165.0 % while the minimum increase was 95.2 %. Additionally exfoliation (2/2 animals) and scab (1/2 animals) were observed at the treatment site (ears) in the 75 % (w/v) dose group on the non-treatment days (Days 4, 5 and 6). Similar effect was observed in the 50 % (w/v) dose group: exfoliation was observed for 2/2 animals and scab was observed for 1/2 animals on Days 4, 5 and 6. Exfoliation on the ears was observed for the animal treated with the 25 % (w/v) formulation on Day 6.
- Systemic toxicity: No mortality was observed during the test. Significant (≥ 5 %) loss of body weights were observed in the 50 % (w/v) dose group (1/2 animals, 11 % decrease) and in the 25 % (w/v) dose group (1/1 animal, 8 % decrease). No other sign of systemic toxicity was observed.
- Erythema scores: No erythema was observed during the test.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response: The test item is considered as a skin sensitizer, if the following criterion is fulfilled:
That 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, of the positive control substance or of the negative controls (Plu or AOO, see Table 3). The formulations were applied, with a pipette, on the dorsal surface of each ear. 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.

Positive control substance(s):

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

Statistics:

The measured DPM values corrected with the mean background value were used for statistical analysis of the proliferation data. Statistical analysis was performed by SPSS/PC+ (4.0.1) software package.
The heterogeneity of variance between the groups treated with the test item or the vehicle control (Plu) was checked by Bartlett's test. Since significant heterogeneity was detected, the normal distribution of data was examined by Kolmogorow-Smirnow test followed by the non-parametric method of Kruskal-Wallis One-Way analysis of variance. As a result of this analysis the inter-group comparison was performed using Mann-Whitney U-test to assess the significance of inter-group differences. Significance of the positive control response was evaluated by t-test versus the relevant vehicle control (AOO).
Significance of the dose-response was evaluated by linear regression made with Microsoft Excel Software.

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. Statistically significant increase of the proliferation values was observed in the positive control group by t-test versus AOO control (p < 0.01). The calculated SI value was 9.1. 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:

1

Test group / Remarks:

Vehicle control for the positive control: AOO

Key result

Parameter:

SI

Value:

9.1

Test group / Remarks:

Positive control: 25 % HAC in AOO

Key result

Parameter:

SI

Value:

1

Test group / Remarks:

Vehicle control for the test item: Plu

Key result

Parameter:

SI

Value:

8

Test group / Remarks:

Test item 5 % in Plu

Key result

Parameter:

SI

Value:

2.8

Test group / Remarks:

Test item 2.5 % in Plu

Key result

Parameter:

SI

Value:

2.5

Test group / Remarks:

Test item 1 % in Plu

Key result

Parameter:

SI

Value:

1.8

Test group / Remarks:

Test item 0.5 % in Plu

Key result

Parameter:

EC3

Value:

2.6

Test group / Remarks:

Test item

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
Visually larger lymph nodes compared to the relevant vehicle controls (AOO or Plu) were observed in the positive control group and in the 5 % (w/v) test item treated group. Visual appearance of the lymph nodes was normal in the negative (vehicle) control groups and in the 2.5 %, 1 % or 0.5 % (w/v) test item treated groups. Significant lymphoproliferation (SI ≥ 3) was observed for the test item at test concentration of 5 % (w/v). No significant lymphoproliferation was observed at the other test concentrations (2.5 %, 1 % or 0.5 %, w/v).
The corresponding stimulation index values were 8.0, 2.8, 2.5 and 1.8 at treatment concentrations of 5 %, 2.5 %, 1 % and 0.5 % (w/v), respectively.

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 other local effects were observed in any treatment groups.

BODY WEIGHTS
Body weights decreased by ≥ 5 % were observed in the following treatment groups: positive control (2/5 animals, 5 % or 7 % decrease), vehicle control for the test item (Plu; 2/5 animals, 7 % decrease for both), 2.5 % (w/v) dose group (1/5 animals, 6 % decrease), 1 % (w/v) dose group (1/5 animals, 7 % decrease).

Table 1 Mean DPM and DPN ± SD

Test group	Mean DPM ± SD	Mean DPN ± SD
Positive control: 25% HCA in AOO	13147.9 ± 6485.9	6574 ± 3242.9
Vehicle control for positive control: AOO	1449.5 ± 1031.8	725 ± 515.9
Vehicle control for test item: Plu	364.1 ± 181.4	182 ± 90.7
Test item 5 % Plu	2917.9 ± 1391.7	1459 ± 695.8
Test item 2.5 % in Plu	1020.7 ± 568.5	510 ± 284.3
Test item 1 % in Plu	913.9 ± 561.7	457 ± 280.8
Test item 0.5 % in Plu	651.5 ± 261.0	326 ± 130.5

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

Under the conditions of the present Local Lymph Node Assay, the test item tested at 5 % (as the maximum feasible non-toxic, non-irritant concentration) and at 2.5%, 1% and 0.5 % (w/v) concentrations as formulations (apparently solutions) in a suitable vehicle (aqueous 1% (w/v) Pluronic®PE 9200) showed to have skin sensitization potential. Based on the EC3 value of 2.6 % the test item was considered a moderate skin sensitizer in this LLNA. Based on this EC3 value the test item can be allocated to Category 1 and sub-category 1B according to the effective regulations.

Executive summary:

The aim of this study was to determine the skin sensitization potential of the test item following dermal exposure in the Local Lymph Node Assay. An individual approach was used in this test. The test item was a highly viscous liquid.Based on chemical information, supplied by the Sponsor, aqueous 1 % (w/v) Pluronic®PE 9200 (Plu) was considered to be the most relevant vehicle for formulation of the test item.The test item was appropriately miscible with the vehicle. The maximum dose selection was based on results of two consecutive Dose Range Finding tests performed according to the relevant guidelines. Adverse effects (systemic toxicity and/or irritation) was observed at test concentrations of 75 %, 50 %, 25 % or 10 % (w/v), hence the test item was examined in the main test at 5 % (as the maximum feasible non-toxic, non-irritant concentration) and at 2.5 %, 1 % and 0.5 % (w/v) concentrations as formulations in Plu. Appropriate positive control, furthermore two negative control groups (dosed with the vehicles of the test and positive control groups, respectively) were employed. The positive control item (α-Hexylcinnamaldehyde [HCA]; 25 % (w/v) in Acetone: Olive oil 4:1 (v/v) mixture [AOO]) induced the appropriate stimulation over the control (SI = 9.1), thus confirming the validity of the assay.No mortality was observed during the main test. No significant, treatment related effect on body weights was considered during the test. No other signs of systemic toxicity were observed in any treatment group. No visible signs of irritation or other local effects were observed in any treatment group.Significant lymphoproliferation (SI ≥ 3) was observed for the test item at test concentration of 5 % (w/v). No significant lymphoproliferation was observed at the other test concentrations (2.5 %, 1 % or 0.5 %, w/v).The corresponding stimulation index values were 8.0, 2.8, 2.5 and 1.8 at treatment concentrations of 5 %, 2.5 %, 1 % and 0.5 % (w/v), respectively.The measured individual DPM values corrected with the mean background value were statistically evaluated.Statistically significant increase of the proliferation values was observed in the positive control group by t-test versus AOO control (p < 0.01). Statistically significant difference compared to the relevant vehicle control (Plu) was observed in the 5 % (w/v) test group (p < 0.01, evaluated by Mann-Whitney U-test).The dose-response relationship, evaluated by linear regression using the SI values, was statistically significant (p = 0.049, r = 0.95). According to evaluation criteria of the relevant guidelines, the significantly increased lymphoproliferation observed at 5 % (w/v) concentration and the significant dose-response correlation are considered evidence that the test item is a skin sensitizer. Chemicals can be classified according to their relative skin-sensitization potency using EC3 value (dose calculated to induce a stimulation index of 3) calculated by linear interpolation using data points lying immediately above and below the SI value of 3 on the LLNA dose-response curve according to published method.The calculated EC3 value for the test item was 2.6 % (w/v) in this LLNA.Using this value the test item can be ranked among moderate skin sensitizers according to the published data for classification of contact allergens.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

LLNA

The aim of this study was to determine the skin sensitization potential of the test item following dermal exposure in the Local Lymph Node Assay. An individual approach was used in this test. The test item was a highly viscous liquid.Based on chemical information, supplied by the Sponsor, aqueous 1 % (w/v) Pluronic®PE 9200 (Plu) was considered to be the most relevant vehicle for formulation of the test item.The test item was appropriately miscible with the vehicle. The maximum dose selection was based on results of two consecutive Dose Range Finding tests performed according to the relevant guidelines. Adverse effects (systemic toxicity and/or irritation) was observed at test concentrations of 75 %, 50 %, 25 % or 10 % (w/v), hence the test item was examined in the main test at 5 % (as the maximum feasible non-toxic, non-irritant concentration) and at 2.5 %, 1 % and 0.5 % (w/v) concentrations as formulations in Plu. Appropriate positive control, furthermore two negative control groups (dosed with the vehicles of the test and positive control groups, respectively) were employed. The positive control item (α-Hexylcinnamaldehyde [HCA]; 25 % (w/v) in Acetone: Olive oil 4:1 (v/v) mixture [AOO]) induced the appropriate stimulation over the control (SI = 9.1), thus confirming the validity of the assay.No mortality was observed during the main test. No significant, treatment related effect on body weights was considered during the test. No other signs of systemic toxicity were observed in any treatment group. No visible signs of irritation or other local effects were observed in any treatment group.Significant lymphoproliferation (SI ≥ 3) was observed for the test item at test concentration of 5 % (w/v). No significant lymphoproliferation was observed at the other test concentrations (2.5 %, 1 % or 0.5 %, w/v).The corresponding stimulation index values were 8.0, 2.8, 2.5 and 1.8 at treatment concentrations of 5 %, 2.5 %, 1 % and 0.5 % (w/v), respectively.The measured individual DPM values corrected with the mean background value were statistically evaluated.Statistically significant increase of the proliferation values was observed in the positive control group by t-test versus AOO control (p < 0.01). Statistically significant difference compared to the relevant vehicle control (Plu) was observed in the 5 % (w/v) test group (p < 0.01, evaluated by Mann-Whitney U-test).The dose-response relationship, evaluated by linear regression using the SI values, was statistically significant (p = 0.049, r = 0.95). According to evaluation criteria of the relevant guidelines, the significantly increased lymphoproliferation observed at 5 % (w/v) concentration and the significant dose-response correlation are considered evidence that the test item is a skin sensitizer. Chemicals can be classified according to their relative skin-sensitization potency using EC3 value (dose calculated to induce a stimulation index of 3) calculated by linear interpolation using data points lying immediately above and below the SI value of 3 on the LLNA dose-response curve according to published method.The calculated EC3 value for the test item was 2.6 % (w/v) in this LLNA.Using this value the test item can be ranked among moderate skin sensitizers according to the published data for classification of contact allergens.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

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. Based on available data on sensitizing properties, the test item is classified and labelled as skin sensitizer Category 1B (H317: "May cause an allergic skin reaction") according to Regulation (EC) No 1272/2008 (CLP), as amended for the eighth time in Regulation (EU) No 2016/918.