Methyl 2,4-dihydroxy-3,6-dimethylbenzoate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Remarks:

LLNA was performed in 2004

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 April, 2003 - 15 April, 2003

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

information is sufficiently adequate

Justification for type of information:

LLNA was performed before 2016

Data source

Reference

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2002

Deviations:

yes

Remarks:

No details on test material, no details on clinical signs or local irritation at application site, positive control study performed 9 month before main study instead of 6 months, no dose relationship in response was observed in the positive control test.

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

1

Reference substance name:

Methyl 2,4-dihydroxy-3,6-dimethylbenzoate

EC Number:

225-193-0

EC Name:

Methyl 2,4-dihydroxy-3,6-dimethylbenzoate

Cas Number:

4707-47-5

Molecular formula:

C10H12O4

IUPAC Name:

methyl 2,4-dihydroxy-3,6-dimethylbenzoate

Test material form:

other: solid

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/Ca/Ola/Hsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Interfauna UK Limited, Blackthorne, Bicester, Oxon, UK
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: Young adults
- Weight at study initiation: 14.4 - 19.6 g
- Housing: A maximum of 4 mice was housed per cage, in cages suitable for animals of this strain and weight range.
- Diet: Free access to RM1 diet (supplied by Special Diets Services Limited, Witham, Essex, UK
- Water: Free access to mains water
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS set to maintain
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): A minimum of 15
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

other: 25% ethanol/ 75% diethyl phthalate

Concentration:

Undiluted test item or the test item at concentrations of 1%, 2.5%, 5%, 10% or 25% w/v in vehicle.

No. of animals per dose:

Groups of four mice were treated.

Details on study design:

TREATMENT PROCEDURES:
TOPICAL APPLICATION:
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with different test item concentrations of 1 %, 2.5%, 5%, 10 %, 25% and 100 % (undiluted) in 25% ethanol/ 75% diethyl phthalate (w/v). The application volume, 25 μL, was spread over the entire dorsal surface of each ear lobe once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals).
ADMINISTRATION OF 3H-METHYL THYMIDINE:
Three days after the third application, all mice were administered with approximately 250 μL of phosphate buffered saline (PBS) containing approximately 20 μCi of a 2.0 Ci/mmol specific activity 3H-methyl thymidine (3HTdR) by intravenous injection via the tail vein.
DETERMINATION OF INCORPORATED 3HTdR:
Approximately five hours after treatment with 3HTdR all mice were euthanized by inhalation of halothane vapour followed by cervical dislocation. The draining auricular lymph nodes were removed from each animal and, together with the nodes from the other animals in the group, were placed in a container of PBS.

A single cell suspension was prepared by mechanical disaggregation of lymph nodes through stainless steel gauze (200 μm mesh size). After washing three times with phosphate buffered saline (approx. 10 mL) the lymph node cells were resuspended in 5% trichloroacetic acid (approx. 3 mL) and incubated at approximately +4 °C overnight, the samples were pelleted by centrifugation and the supernatant was discarded. for precipitation of macromolecules. The precipitates were then resuspended in 5% trichloroacetic acid (1 mL) and transferred to glass scintillation vials with 10 mL of scintillation liquid (Optiphase) and thoroughly mixed.
The level of 3HTdR incorporation was then measured on a β-scintillation counter (Packard Tri-Carb 2500TR Liquid Scintillation Counter).

OBSERVATIONS:
Body weights: The bodyweight of each animal was recorded prior to dosing on day 1 and prior to injection of 3HTdR on day 6.
Clinical signs (local / systemic): Animals were checked at least once daily for signs of systemic toxicity.

Positive control substance(s):

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

Statistics:

Not performed.

Results and discussion

Positive control results:

The application of hexylcinnamaldehyde at concentrations of 2.5%, 5% or 10% w/v in acetone resulted in a greater than 3-fold increase in isotope incorporation at all three concentrations (no dose relationship in response was observed). Therefore, hexylcinnamaldehyde was shown to be a skin sensitiser, confirming the validity of this study. See section "Any other information on results incl. tables".

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

EC3

Remarks:

% w/v

Value:

19

Results 2

Key result

Parameter:

other: NOEC %

Value:

10

Results 3

Parameter:

SI

Value:

1.59

Remarks on result:

other: 1%

Results 4

Parameter:

SI

Value:

2.25

Remarks on result:

other: 2.5%

Results 5

Parameter:

SI

Value:

1.99

Remarks on result:

other: 5%

Results 6

Parameter:

SI

Value:

1.41

Remarks on result:

other: 10%

Results 7

Parameter:

SI

Value:

3.94

Remarks on result:

other: 25%

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
See section "Any other information on results incl. tables"

DETAILS ON STIMULATION INDEX CALCULATION
See section "Any other information on results incl. tables"

EC3 CALCULATION
See section "Any other information on results incl. tables"

CLINICAL OBSERVATIONS: No data on results.

BODY WEIGHTS:
The body weight of the animals, recorded at the start of application and on day 6, was within the range commonly recorded for animals of this strain and age.

Any other information on results incl. tables

Skin sensitisation potential of Methyl atrarate:

Concentration of test substance (% w/v)	Number of lymph nodes assayed	Disintegration per minute (dpm)	dpm per lymph node	Test control ratio
0 (vehicle only)	8	3050	381	N/A
1	8	4846	606	1.59
2.5	8	6866	858	2.25
5	8	6052	757	1.99
10	8	4314	539	1.41
25	8	12008	1501	3.94
EC3	19% w/v

N/A: not applicable

Skin sensitisation potential of the positive control substance hexylcinnamaldehyde:

Concentration of hexylcinnamaldehyde (% w/v)	Number of lymph nodes assayed	Disintegration per minute (dpm)	dpm per lymph node	Test control ratio
0 (vehicle only)	8	2570	321	N/A
2.5	8	16088	2011	6.26
5	8	15659	1957	6.10
10	8	15611	1951	6.08

N/A: not applicable

Applicant's summary and conclusion

Interpretation of results:

other: Sensitiser 1B

Remarks:

According to EU CLP (EC No. 1272/2008 and its amendments).

Conclusions:

The SI values calculated for the substance concentrations 1, 2.5, 5, 10 and 25 % were 1.59, 2.25, 1.99, 1.41 and 3.94, respectively. These results show that the test substance could elicit a SI ≥ 3. An EC3 value of 19% w/v was calculated. A NOEC of 10% is derived. The test isubstance was considered to be a sensitiser under the conditions of the test.

Executive summary:

The skin sensitisation potential of the substance has been tested according to OECD TG 429 test guideline and GLP principles. At 1, 2.5, 5, 10 and 25% the substance showed SI values of 1.59, 2.25, 1.99, 1.41 and 3.94, respectively. Reliable negative and positive controls were included. These results show that the test substance could elicit a SI ≥ 3. An EC3 value of 19% w/v was calculated. A NOEC of 10% is derived. Based on the results, the substance was considered to be a sensitiser and should be classified as skin sensitizer (Category 1B) and labeled as H317: May cause an allergic skin reaction according to Regulation (EC) No. 1272/2008 and GHS.