2-Propenoic acid, 2-methyl-, 2-hydroxyethyl ester, reaction products with phosphorus oxide

Acute Toxicity: dermal

Administrative data

Endpoint:

acute toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From March 18, 2004 to April 29, 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1100 (Acute Oral Toxicity)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: JMAFF Japanese test guidelines

Deviations:

no

GLP compliance:

yes

Test type:

acute toxic class method

Limit test:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): PARAD substance 139.
- Molecular formula: C6H10O3.xH3O4P.
- Molecular weight: ~250.
- Appearance: Slightly yellowish viscous liquid.
- Analytical purity: n.a. (mixture).
- Density: 1.4488.
- Batch No.: 31104251.
- Expiration date: December 31, 2004.

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

Test animals
- Supplier: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: 9-12 weeks old.
- Fasting period: Food was withheld overnight (for a maximum of 20 h) prior to dosing and 3-4 h after dosing.
- Acclimatisation period: At least 5 d.
- Water: Free access to tap-water.
- Diet: Free access to standard pelleted laboratory animal diet.

Animal husbandry
- Animals per cage: 3 animals.
- Housing: Macrolon cages (type IV; height 18 cm) containing purified sawdust as bedding material.
- Diet supplier: Altromin (code VRF 1), Lage, Germany.

Environmental conditions
- Air changes: Approximately 15 air changes per h.
- Temperature: 21±3°C.
- Humidity: 30 - 70%.
- Photoperiod: 12 h artificial fluorescent light and 12 h darkness per d.

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on oral exposure:

Dosing method: Oral gavage, using a stainless steel stomach tube
Dose preparation: The formulations (w/w) were prepared in vehicle (propylene glycol) 4 h prior to dosing.

Doses:

300, 2,000 mg/kg

No. of animals per sex per dose:

3 females

Control animals:

not specified

Details on study design:

- Dosing: Initially, the test substance was administered by oral gavage to three female Wistar rats at 300 mg/kg bw. Further, in a stepwise procedure additional groups of females were dosed at 300, 2,000, and 2,000 mg/kg bw.
- Dose volume: 10 mL/kg

Frequency of observations:
- Mortality/viability- Twice daily
- Body weight: Days 1 (pre-administration), 8 and 15 and at death (if found dead after Day 1).
- Clinical signs: At periodic intervals on the day of dosing (Day 1) and once daily thereafter, until Day 15. The symptoms were graded according to fixed scales and the time of onset, degree and duration were recorded:
Maximum grade 4: grading slight (1) to very severe (4)
Maximum grade 3: grading slight (1) to severe (3)
Maximum grade 1: presence is scored
- Necropsy: Animals were sacrificed by asphyxiation using a oxygen/carbon dioxide procedure. All animals assigned to the study were subjected to necropsy and descriptions of all internal macroscopic abnormalities recorded.

Statistics:

No statistical analysis was performed (The method used is not intended to allow the calculation of a precise LD50 value).

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

1 animal died at dose level of 2,000 mg/kg bw administered at step 4.

Clinical signs:

other: At 300 mg/kg- Hunched posture and uncoordinated movements. At 2,000 mg/kg- Hunched posture, uncoordinated movements, lethargy, chromodacryorrhoea (snout), piloerection, flat gait and salivation. The surviving animals had recovered from the symptoms betwee

Gross pathology:

Macroscopic post mortem examination of the animal that was found dead during the study revealed abnormalities in the stomach (hemorrhage of glandular mucosa).

Macroscopic post mortem examination of the other animals at termination did not reveal any abnormalities.

Table 1: Incidence of mortality

Dose level	Mortality
300 mg/kg	0/3
300 mg/kg	0/3
2,000 mg/kg	0/3
2,000 mg/kg	1/3

Interpretation of results:

other: CLP criteria not met

Remarks:

does not have to be classified

Conclusions:

Under the study conditions, the oral LD50 of the test substance was determined to be ≥2,000 mg/kg bw

Executive summary:

A study was conducted to assess the acute oral toxicity of the test substance in female rats according to OECD Guideline 423, EU Method B.1 and EPA OPPTS 870.1100, in compliance with GLP. Initially, the test substance was administered by oral gavage to three female Wistar rats at 300 mg/kg bw. Further, in a stepwise procedure additional groups of females were dosed at 300, 2,000, and 2,000 mg/kg bw. Treated animals were then observed for mortality, clinical signs and body weight changes for 14 d and were then culled and subjected to gross pathological examination. In the rats treated at 300 mg/kg bw, clinical signs such as hunched posture and uncoordinated movements were observed. The rats treated at 2,000 mg/kg bw, revealed clinical signs that included hunched posture, uncoordinated movements, lethargy, chromodacryorrhoea (snout), piloerection, flat gait and salivation. Surviving rats had recovered from the symptoms between Day 1 and 4. The mean body weight gain in the treated rats was normal. One animal of the dose level 2,000 mg/kg at step 4 was found dead. Macroscopic examination of animal that was found dead revealed abnormalities in the stomach (hemorrhage of glandular mucosa). No abnormalities were found in other animals at termination. Based on the study results, LD50 of the test substance was ≥2,000 mg/kg bw (van Huygevoort AHBM, 2004).

Cross-reference 2

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From March 16, 2004 to March 26, 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2500 (Acute Dermal Irritation)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries (JMAFF), 12 Nousan, Notification No 8147, November 2000, including the most recent partial revisions

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

- Name of the test substance as cited in the report: PARAD Substance 139.
- Batch: 31104251.
- Molecular formula: C6H10O3.xH3O4P.
- Molecular weight: ~250.
- Description: Slightly yellowish viscous liquid.
- Purity: Not available (mixture).
- Test substance storage: At room temperature in the dark.
- Stability under storage conditions: Stable.
- Expiry date: December 31, 2004.

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

- Source: Charles River Deutschland, Kisslegg, Germany
- Age and body weight: Animals used within the study were at least 6 weeks old and body weights were at least 1 kg.
- Identification: Earmark
- Conditions: Animals were housed in a controlled environment, in which optimal conditions were considered to be approximately 15 air changes per h, a temperature of 21.0±3.0°C (actual range: 17.0 – 24.1°C), a relative humidity of 30-70% (actual range: 33 - 67%) and 12 h artificial fluorescent light and 12 h darkness per day.
- Accommodation: Individually in labelled cages with perforated floors (Scanbur, Denmark, dimensions 56x44x37.5 cm).
- Diet: Standard laboratory rabbit diet (Charles River Breeding and Maintenance Diet for Rabbits, Altromin, Lage, Germany) approximately 100 g per d. In addition, hay (BMI, Helmond, the Netherlands) was provided at least three times a week.
- Water: ad libitum
- Acclimatisation period: At least 5 d before start of treatment under laboratory conditions.

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

other: Adjacent areas of the untreated skin of each animal served as controls

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 0.5 mL
- Concentration (if solution): Undiluted

Duration of treatment / exposure:

4 h

Observation period:

72 h

Number of animals:

3 males

Details on study design:

A single rabbit was tested at the initial step. Two other rabbits were treated in a similar manner one week later, after considering the degree of skin irritation observed in the first rabbit.

Approximately 24 h before treatment, the dorsal fur was clipped with electric clippers, exposing an area of approximately 150 square centimeters (10x15 cm²). Whenever considered necessary the treated skin areas were re-clipped at least 3 h before the observations, to facilitate scoring.

A health inspection was performed prior to the commencement of treatment, to ensure that the animals were in a good state of health. Special attention was paid to the skin to be treated, which was intact and free from abnormalities. Each animal was treated by dermal application of 0.5 mL of the test substance. The test substance was applied to the skin of one flank, using a metalline patch of 2x3 cm. The patch was mounted on Micropore tape, which was wrapped around the abdomen and secured with Coban elastic bandage. Four hours after the application, the dressing was removed and the skin cleaned of residual test substance using water.

Observations:
Mortality/Viability: Twice daily.
Toxicity: At least once daily.
Body Weight: Day of treatment (prior to application) and at termination.
Irritation: The skin reactions were assessed at approximately 1, 24, 48 and 72 h and / or 7 d after the removal of the dressings and test substance. The irritation scores and a description of all other (local) effects were recorded. Adjacent areas of the untreated skin of each animal served as controls.

Numerical scoring system:
Erythema and eschar formation:

No erythema ................................................................................................... 0
Very slight erythema (barely perceptible) ...................................................1
Well-defined erythema ....................................................................................2
Moderate to severe erythema ........................................................................3
Severe erythema (beet redness) *.................................................................. 4
*. Where signs of necrosis or corrosion (injuries in depth) prevent erythema scoring, the maximum grade for erythema (= 4) is given.

Oedema formation:
No oedema ........................................................................................................ 0
Very slight oedema (barely perceptible) ....................................................... 1
Slight oedema (edges of area well-defined by definite raising) ................ 2
Moderate oedema (raised approximately 1 mm) ......................................... 3
Severe oedema (raised more than 1 mm and extending beyond the area of exposure) 4

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1.3

Max. score:

2

Reversibility:

fully reversible

Remarks on result:

other: The skin irritation had resolved within 7 d after exposure.

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1

Max. score:

2

Reversibility:

fully reversible

Remarks on result:

other: The skin irritation had resolved within 72hrs after exposure.

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.7

Max. score:

2

Reversibility:

fully reversible

Remarks on result:

other: The skin irritation had resolved within 72hrs after exposure.

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.3

Max. score:

1

Reversibility:

fully reversible

Remarks:

The skin irritation had resolved within 48hrs after exposure.

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1

Max. score:

2

Reversibility:

fully reversible

Remarks:

The skin irritation had resolved within 72hrs after exposure.

Irritation parameter:

edema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.3

Max. score:

1

Reversibility:

fully reversible

Remarks:

The skin irritation had resolved within 48hrs after exposure.

Irritant / corrosive response data:

Irritation:
Four hours exposure to 0.5 mL of test substance resulted in well-defined erythema and in slight or moderate oedema in the treated skin-areas of the three rabbits. The skin irritation had resolved within 72 h after exposure in two animals and within 7 d after exposure the remaining animal. Scaliness was noted in one animal at 72 h after exposure.

Corrosion:
There was no evidence of a corrosive effect on the skin.

Colouration / remnants:
No staining of the treated skin by the test substance was observed and no test substance remnants were seen.

Other effects:

Toxicity / mortality:
No symptoms of systemic toxicity were observed in the animals during the test period and no mortality occurred.

Table 1: Mean value irritation scores (24, 48 and 72 h)

 

Animal number	Mean 24 - 72 h
	Erythema	Oedema
1	1.3	0.3
2	1.0	1.0
3	0.7	0.3
Primary irritation index: 1.6

 

Table 2: Mean value irritation scores (24 and 72 h)

 

Animal number	Mean 24 - 72 h
	Erythema	Oedema
1	1.5	0.5
2	1.0	1.0
3	0.5	0.5
Primary irritation index: 1.7

 

Interpretation of results:

other: CLP criteria not met

Remarks:

does not have to be classified

Conclusions:

Under the study conditions, the test substance was considered to be not irritating to the skin.

Executive summary:

A study was conducted to assess the skin irritation potential of the test substance in rabbits according to OECD Guideline 404, EU Method B.4, US EPA 870.2500 and JMAFF Japanese test Guidelines, 2000, in compliance with GLP. A total of 0.5 mL of the test substance was applied onto the clipped skin of three animals for 4 h using a semi-occlusive dressing. The test substance was applied to the skin of one flank, using a metalline patch of 2x3 cm. After exposure for 4 h, the dressing was removed and residues of substance were carefully rinsed off with distilled water. Observations were made 1, 24, 48 and 72 h and / or 7 d after exposure. Exposure to test substance resulted in well-defined erythema and in slight or moderate oedema in the treated skin areas of the three rabbits. The skin irritation had resolved within 72 h after exposure in two animals and within 7 d after exposure in the remaining animal. Scaliness was noted in one animal at 72 h after exposure only. Based on the 24 and 72 h readings, the primary irritation index was calculated to be 1.7. Under the study conditions, the substance was considered to be not irritating to the skin (van Huygevoort AHBM, 2004).

Cross-reference 3

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From October 21, 2015 to October 27, 2015

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:

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

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:

- Name of test material (as cited in study report): PARAD Substance 139.
- Chemical name: Reaction product of 2-Hydroxyethyl methacrylate (HEMA) and phosphor pentoxide (P2O5).
- Batch No.: JBLB0008S.
- CAS No.: 1187441-10-6.
- Purity: 100%.
- Appearance: Clear colourless liquid.
- Manufacture date: February, 2014.
- Expiry date: February, 2016.
- Storage condition: Controlled room temperature (15 – 25ºC, below 70 RH%), protected from light.
- Safety precautions: Routine safety precautions (gloves, goggles, face mask, lab coat) for unknown materials were applied to assure personnel health and safety. Irritant, causes serious eye damage.

Species:

mouse

Strain:

other: CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories S.r.l., San Pietro al Natisone (UD), Zona Industriale Azzida, 57, 33049 Italy.
- Age at study initiation: 11 weeks old.
- Weight at study initiation: 19.1 - 20.4 g.
- Housing/ Enrichment: Group caging / mice were provided with glass tunnel-tubes.
- Cage type: Type II polypropylene / polycarbonate.
- Bedding: Lignocel 3/4-S Hygienic animal bedding.
- Source for bedding: J. Rettenmaier & Söhne GmbH & Co.KG (Holzmühle 1, 73494 Rosenberg, Germany) .
- Diet: ad libitum.
- Supplier of diet: ssniff spezialdiäten GmbH (Ferdinand-Gabriel-Weg 16, D-59494 Soest, Germany).
- Water: ad libitum.
- Acclimation period: 21 d.

ENVIRONMENTAL CONDITIONS
- Temperature: 17.9 – 25.9°C
- Humidity: 30 - 87%
- Air changes: 15 - 20 air exchanges per h
- Photoperiod: 12 h daily, from 6.00 a.m. to 6.00 p.m.
The temperature and relative humidity were recorded twice every day during the acclimatisation and experimental phases

Vehicle:

dimethylformamide

Concentration:

Preliminary test- 25 and 50% in DMF
Main test- 0, 10, 25 and 50% in DMF

No. of animals per dose:

Preliminary test- 2 animals/dose
Main test- 4 animals/dose

Details on study design:

PRELIMINARY STUDY
A preliminary study was performed with the test substance concentrations 25 and 50% (w/v) in DMF. It was conducted in a similar experimental manner to the main study, but terminated on Day 6 with a body weight measurement and the radioactive proliferation assay was not performed. All mice were observed daily for any clinical signs of systemic toxicity or local irritation at the application site. Both ears of each mouse were observed for erythema and scored. Ear thickness was also measured using a thickness gauge on Day 1 (pre-dose), Day 3 (approximately 48 h after the first dose) and Day 6. Additional quantification of the ear thickness was performed by ear punch weight determination after the euthanasia of the experimental animals.

MAIN STUDY
Based on results in preliminary test, 50% (w/v) dose was selected as top dose for the main test. The experimental groups and dose levels for the main experiment were:
1. Negative (vehicle) control (DMF)
2. 10% of test substance
3. 25% of test substance
4. 50% of test substance
5. Positive control (25% alpha-Hexylcinnamaldehyde solution in DMF)

Topical application: 25 µL of the appropriate formulation was applied using a pipette on the dorsal surface of each ear on Day 1, 2 and 3. No treatment was given on Day 4, 5 and 6.

Injection of tritiated Thymidine (3HTdR): On Day 6, animals were taken to the radioactive suite and each mouse was intravenously injected via the tail vein with 250 µL of sterile PBS (phosphate buffered saline) containing approximately 20 µCi of 3HTdR using a gauge 25G x 1" hypodermic needle with 1 mL sterile syringe. Once injected, the mice were left for 5h (±30 min).

Removal and Preparation of Draining Auricular Lymph Nodes: 5h (±30 min) after intravenous injection the mice were euthanized by asphyxiation with ascending doses of carbon dioxide (deep anaesthesia was confirmed before making incision, death was confirmed before discarding carcasses). The draining auricular lymph nodes were excised by making a small incision on the skin between the jaw and sternum, pulling the skin gently back towards the ears and exposing the lymph nodes. The nodes were then removed using forceps. The carcasses were discarded after cervical dislocation or after cutting through major cervical blood vessels. Once removed, the nodes of mice from each test group was pooled and collected in separate petri dishes containing a small amount (1 - 2 mL) of PBS to keep the nodes wet before processing.

Preparation of single cell suspension of lymph node cells: A single cell suspension (SCS) of pooled lymph node cells (LNCs) was prepared and collected in disposable tubes by gentle mechanical disaggregating of the lymph nodes through a cell strainer using the plunger of a disposable syringe. The cell strainer was washed with PBS (up to 10 mL). Pooled LNCs were pelleted with a relative centrifugal force (RCF) of 190 x g (approximately) for 10 min at 4ºC. After centrifugation supernatants were discarded. Pellets were gently resuspended and 10 mL of PBS was added to the tubes. The washing step was repeated twice. This procedure was repeated for each group of pooled lymph nodes

Determination of incorporated 3HTdR: After the final washing step, supernatants were removed. Pellets were gently agitated resuspended and 3 mL of 5% (w/v) TCA solution was added to the tubes for precipitation of macromolecules. After overnight (approximately 18 h) incubation at 2 - 8ºC, precipitates were centrifuged (approximately 190 x g for 10 min at 4ºC), and supernatants were removed. Pellets were resuspended in 1 mL of 5% (w/v) TCA solution and dispersed by using an ultrasonic bath. Samples were transferred into a suitable sized scintillation vial filled with 10 mL of scintillation liquid and thoroughly mixed. The vials were loaded into a β-scintillation counter and 3HTdR incorporation was measured (10 min measurement). The β-counter expresses the 3HTdR incorporation as the number of radioactive disintegrations per min (DPM). Background level was also measured in duplicates by adding 1 mL of 5% (w/v) TCA solution into a scintillation vial filled with 10 mL of scintillation liquid.

Positive control substance(s):

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

Positive control results:

A significant lymphoproliferative response (stimulation index value of 7.3) was noted for the positive control chemical, this result confirmed the validity of the assay.

Key result

Parameter:

SI

Value:

1.6

Test group / Remarks:

10%

Key result

Parameter:

SI

Value:

2.8

Test group / Remarks:

25%

Key result

Parameter:

SI

Value:

3.7

Test group / Remarks:

50%

Key result

Parameter:

EC3

Value:

30.6

PRELIMINARY IRRITATION/TOXICITY TEST:

No mortality or signs of systemic toxicity were observed. Slightly rigid ears were observed in the 50% (w/v) dose group on Day 4. No marked body weight loss (>5%) was detected on the mean body weight values of the groups; however one animal showed significant loss of body weight (5.1%) in the 50% (w/v) dose group.

MAIN TEST:

Clinical observation: No mortality or signs of systemic toxicity were observed during the study.

Body weight measurement: No treatment related effects were observed on the body weight changes of the experimental animals.

Table1: DPM, DPN and Stimulation Index values for all groups

Test Group Name	Measured DPM / group	DPM (Disintegration per minute)	Number of lymph nodes	DPN (DPM/No. of lymph nodes)	Stimulation Index
Background	36	-	-	-	-
(5% (w/v) TCA)	32
Negative (vehicle) control (DMF)	1864	1830.0	8	228.8	1.0
50% (w/v) in DMF	6733	6699.0	8	837.4	3.7
25% (w/v) in DMF	5128	5094.0	8	636.8	2.8
10% (w/v) in DMF	3049	3015.0	8	376.9	1.6
Positive control (25% (w/v) HCA in DMF)	13377	13343.0	8	1667.9	7.3

 

Interpretation of results:

other: category 1B

Remarks:

as per CLP criteria

Conclusions:

Under the study conditions, the test substance was shown to have sensitisation potential (sensitizer) in a local lymph node assay.

Executive summary:

The skin sensitization potential of the test substance was evaluated in a mouse local lymph node assay, conducted according to OECD Guideline 429 and EU Method B42, in compliance with GLP. Test substance concentrations selected for the main study were based on the results of a preliminary irritation test. In the main study, animals were treated topically with 10, 25 and 50% w/v of test substance on Days 1, 2 and 3. Negative control animals were similarly treated with vehicle alone (N,N-dimethylformamide (DMF)) and positive control animals received 25% alpha-hexylcinnamaldehyde. On Day 6, tritiated thymidine (3HTdR) was injected intravenously. 5h (±30 min) after intravenous injection, the mice were euthanized by asphyxiation and draining auricular lymph nodes were excised. Single cell suspension of lymph node cells was prepared. 3HTdR incorporation was measured by β-scintillation counter. The activity was expressed as the number of disintegrations per minute (DPM) and a stimulation index (SI) was subsequently calculated for each group. The EC3 value of the test substance (EC3 means the effective chemical concentration required for SI=3) was calculated by linear interpolation. No mortality or signs of systemic toxicity were observed during the study. No treatment related effects were observed on the body weight changes of the experimental animals. DPM for the experimental groups treated with test substance concentrations 10, 25 and 50% were 3,015, 5,094 and 6,699, respectively. The DPM values observed for the vehicle and positive control substance in this experiment were within the general historical control range. The stimulation index values determined were 1.6, 2.8 and 3.7 at concentrations of 10, 25 and 50% (w/v), respectively. These results show that the test substance at 50% concentration elicits a SI ≥3. The calculated EC3 value of test substance was 30.6% (w/v). Under the study conditions, the substance was shown to have sensitisation potential (sensitizer) in the local lymph node assay. (Váliczkó É, 2015).