Soybean meal, protein extn. residue

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 June 2018 to 04 July 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2010

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Soybean meal, protein extn. residue

EC Number:

293-693-6

EC Name:

Soybean meal, protein extn. residue

Cas Number:

91081-84-4

Molecular formula:

UVCB substance

IUPAC Name:

Not determined

Test material form:

solid: particulate/powder

Details on test material:

- Physical state/Appearance: Beige-yellow/tan powder
- Storage Conditions: Room temperature in the dark

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test material was formulated within 2 hours of being applied to the test system. It was assumed that the formulation was stable for this duration.
No analysis was conducted to determine the homogeneity, concentration or stability of the test material formulation.

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Remarks:

CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: 8 to 12 weeks old
- Weight at study initiation: 15 to 23 g
- Housing: The animals were group housed in suspended solid floor polypropylene cages furnished with softwood woodflakes.
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: At least 5 days.
- The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature: 19 - 25 °C
- Humidity: 30 - 70 %
- Air changes: At least fifteen changes per hour.
- Photoperiod: 12 hours continuous light and 12 hours darkness.

Study design: in vivo (LLNA)

Vehicle:

other: 1 % pluronic L92 in distilled water

Concentration:

25, 10 and 5 % w/w

No. of animals per dose:

Four

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: For the purpose of the study, the test material was freshly prepared as a suspension in 1 % pluronic L92 in distilled water. This vehicle was chosen as it produced the highest concentration that was suitable for dosing compared with the other vehicles examined in the Vehicle Determination. Other vehicles tested were: acetone/olive oil (4:1), dimethyl formamide, butanone, dimethyl sulphoxide, acetone, ethanol/distilled water (7:3) and propylene glycol.
- Preliminary Screening Test: As no toxicological information was available regarding the systemic toxicity of the test material, a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 µL of the test material at a concentration of 25 % w/w in 1 % pluronic L92 in distilled water, to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Local skin irritation was scored daily. Any clinical signs of toxicity, if present, were also recorded. The body weight of the mouse was recorded on Day 1 (prior to dosing) and on Day 6. The thickness of each ear was measured using a Mitutoyo 547 300S gauge (Mitutoyo Corporation), pre dose on Day 1, post dose on Day 3 and on Day 6. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between time periods Days 1 and 3 and Days 1 and 6. A mean ear thickness increase of equal to or greater than 25 % was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitisation.


MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay in the Mouse – Pooled Method
- Criteria used to consider a positive response: The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (disintegrations per minute/node) and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index). The test material will be regarded as a sensitiser if at least one concentration of the test material results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test material failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non sensitiser".

TREATMENT PREPARATION AND ADMINISTRATION:
- Groups of four mice were treated with the test material at concentrations of 25, 10 or 5 % w/w in 1 % pluronic L92 in distilled water. The preliminary screening test suggested that the test material would not produce systemic toxicity or excessive local skin irritation at the highest suitable concentration. The mice were treated by daily application of 25 µL of the appropriate concentration of the test material to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test material formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette. A further group of four mice received the vehicle alone in the same manner.
- 3H-Methyl Thymidine Administration: Five days following the first topical application of the test material or vehicle (Day 6) all mice were injected via the tail vein with 0.25 mL (250 µL) of phosphate buffered saline (PBS) containing 3H methyl thymidine (3HTdR: 80 µCi/mL, specific activity 2.0 Ci/mmoL, ARC UK Ltd) giving a total of 20 µCi to each mouse.

OBSERVATIONS
- Clinical Observations: All animals were observed twice daily on Days 1, 2 and 3 and on a daily basis on Days 4, 5 and 6. Any signs of toxicity or signs of ill health during the test were recorded.
- Body Weights: The body weight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).

TERMINAL PROCEDURES
- Termination: Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation followed by cervical separation. The draining auricular lymph nodes from the four mice were excised and pooled for each experimental group. For each group 1 mL of PBS was added to the pooled lymph nodes.
- Preparation of Single Cell Suspension: A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through a 200 mesh stainless steel gauze. The lymph node cells were rinsed through the gauze with 4 mL of PBS into a petri dish labelled with the study number and dose concentration. The lymph node cell suspension was transferred to a centrifuge tube. The petri dish was washed with an additional 5 mL of PBS to remove all remaining lymph node cells and these were added to the centrifuge tube. The pooled lymph node cells were pelleted at 1400 rpm (approximately 190 g) for 10 minutes. The pellet was re suspended in 10 mL of PBS and re-pelleted. To precipitate out the radioactive material, the pellet was re suspended in 3 mL of 5 % Trichloroacetic acid (TCA).
- Determination of 3HTdR Incorporation: After approximately 18 hours incubation at approximately 4 °C, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for 10 minutes, re suspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid. 3HTdR incorporation was measured by β-scintillation counting. The "Poly Q™" vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left to stand in darkness for approximately 20 minutes. The purpose of this period of time in darkness was to reduce the risk of luminescence, which has been shown to affect the reliability of the results. After approximately 20 minutes, the vials were shaken vigorously. The number of radioactive disintegrations per minute was then measured using the Beckman LS6500 scintillation system (Beckman Instruments Inc, Fullerton, CA, USA).

Positive control substance(s):

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

Results and discussion

Positive control results:

A group of five animals was treated with 50 µL (25 µL per ear) of α-Hexylcinnamaldehyde, tech., 85 % as an emulsion in 1 % pluronic L92 in distilled water at a concentration of 25 % v/v. A further control group of five animals was treated with 1 % pluronic L92 in distilled water alone.
The Stimulation Index expressed as the mean radioactive incorporation for the treatment group divided by the mean radioactive incorporation of the vehicle control group is as follows:
25 % v/v positive control in 1 % pluronic L92 in distilled water had a Stimulation Index of 6.48 and was therefore considered to be a sensitiser under the conditions of the test.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1.13

Test group / Remarks:

5 % w/w in 1 % pluronic L92 in distilled water

Results 2

Key result

Parameter:

SI

Value:

0.93

Test group / Remarks:

10 % w/w in 1 % pluronic L92 in distilled water

Results 3

Key result

Parameter:

SI

Value:

1.9

Test group / Remarks:

25 % w/w in 1 % pluronic L92 in distilled water

Cellular proliferation data / Observations:

PRELIMINARY SCREENING TEST
- No signs of systemic toxicity, visual local skin irritation or irritation indicated by an equal to or greater than 25 % increase in mean ear thickness were noted.
- Based on this information the dose levels selected for the main test were 25, 10 and 5 % w/w in 1 % pluronic L92 in distilled water.

CELLULAR PROLIFERATION DATA
- The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows: 5 % test material concentration SI = 1.13, 10 % test material concentration SI = 0.93 and 25 % test material concentration SI = 1.90. The SI values gave negative results at all concentrations tested.

CLINICAL OBSERVATIONS:
- There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.

BODY WEIGHTS
- Body weight change of the test animals between Day 1 and Day 6 was comparable to that observed in the corresponding control group animals over the same period.

Any other information on results incl. tables

Table 1: Results of the Main Test

Concentration (% w/w) in 1 % pluronic L92 in distilled water	Stimulation Index	Result
5	1.13	Negative
10	0.93	Negative
25	1.90	Negative

Applicant's summary and conclusion

Interpretation of results:

other: Not classified in accordance with EU criteria

Conclusions:

Under the conditions of this study, the test material was not-sensitising.

Executive summary:

The potential of the test material to be sensitising to the skin was investigated in accordance with the standardised guidelines OECD 429 and EU Method B42, under GLP conditions.

A Local Lymph Node Assay was performed in the CBA/Ca strain of mouse with topical application of the test material to the dorsal surface of the ear.

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 25 % w/w of the test material in 1 % pluronic L92 in distilled water, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of four animals, were treated with 50 µL (25 µL per ear) of the test material as a suspension in 1 % pluronic L92 in distilled water at concentrations of 25, 10 or 5 % w/w. A further group of four animals was treated with 1 % pluronic L92 in distilled water alone.

A positive control study was performed to assess the sensitivity of the strain of mouse used at these laboratories using α- Hexylcinnamaldehyde. The SI was 6.48 and therefore α-Hexylcinnamaldehyde, tech., 85 % was considered to be a sensitiser under the conditions of the test.

The Stimulation Index of the test material expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows: 5 % test material concentration SI = 1.13, 10 % test material concentration SI = 0.93 and 25 % test material concentration SI = 1.90. The SI values gave negative results at all concentrations tested.

Under the conditions of this study, the test material was not-sensitising.