4-hydroxybenzoic acid

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-01-20 to 2009-02-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: study according to OECD guideline 429 under GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 24 April 2002

Deviations:

no

Principles of method if other than guideline:

Deviations from the study plan: for few hours the rel. humidity rose to 88% but returned and remained at acceptable levels for the remaining whole test duration

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:

p-Hydroxybenzoesäure

IUPAC Name:

p-Hydroxybenzoesäure

Details on test material:

name of test item: p-Hydroxybenzoesäure
Sponsors Batch No.: 08/1000/10,1-20
production date: 20.10.2008
purity: 99,94%

storage: protected from light and moisture at room temperature

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories B.V., Postbus 6174, 5960 AD Horst, Netherlands
Strain: CBA/CaOlaHsd,
The female mice were housed in single cages under conventional conditions with 12h light/12 h dark at 19°C-25°C.
The female mice were 8-12 weeks old, nulliparous and non-pregnant and did not show any signs of illness. Acclimatisation was done minimum
5 days prior testing. Mice were weighed prior testing and prior administration of 3HTdR

- Weight at study initiation: 17.7 - 21.8 g (min - max)
- Diet (e.g. ad libitum): pelleted standard diet, ad libitum
- Water (e.g. ad libitum): tap water ad libitum

ENVIRONMENTAL CONDITIONS
- Humidity (%): 30 - 88 % (Deviations from the study plan: for few hours the rel. humidity rose to 88% but returned and remained at acceptable levels for the remaining whole test duration) In GL is stated: Humidity should preferably not exceed 70%.

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Concentration:

5%, 10%, 25% (w/v) test substance in DMSO
Test item preparation: The test item was placed into a volumetric flask on a tared balance and dimethylsulfoxide was quantitatively added.

No. of animals per dose:

4 animals

Details on study design:

Pretests:
1. solubility experiment: highest test item concentration: 25% solution in dimethylsulfoxide after warming to 37°C.
2. to determine the highest non irritant test concentration:
- 2 animals were treated with 10 and 25% each on three consecutive days.
- animals showed no signs of irritation or systemic toxicity (observation: within 1 hour, and 24 hours +/-4 hours after each application)

Main study:
Three test groups and one vehicle control group of female mice were used. Four females per group.
p-Hydroxybenzoic acid in DMSO was applied to both dorsal ear lobes (left and right) (8mm) epidermal, topically in an amount of 25 microlitres.
Five days after the first topical application, the mice were injected intravenously into a tail vein with radio labelled thymidine
(3H-methyl-thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised
and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were subsequently washed with phosphate buffered saline and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl-thymidine measured in a beta-scintillation counter.

Observations during main study:
- mortality and viability: once daily on week days.

Positive control substance(s):

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

Statistics:

Mean values and standard deviations were calculated for the body weight.
Results on LLNA are data on the pooled test groups.

Results and discussion

Positive control results:

5% ; 10%; 25% (w/v) Hexylcinnamaldehyde in acetone/olive oil (4/1) abbreviated 5%, 10%, 25% HCA in the following table;
SI: stimulation index is defined as the DPM per lymph node of test group devided by the DPM of a control

5% HCA: SI = 1.49
10% HCA: SI = 4.17
25% HCA: SI = 4.90

for an IS of 3 an estimated concentration of 7.8% was calculated (see OECD 429)

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Remarks on result:

other: 5% pHBA: SI = 0.94 10% pHBA: SI = 0.75 25% pHBA: SI = 0.99

Results 2

Key result

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: control: DPM: 761 5% pHBA: 712 10% pHBA: 571 25% pHBA: 754 background: (BG1 and BG2 both 1 ml 5% Trichloracetic acid BG1: 31 and BG2: 122 control: 761

Any other information on results incl. tables

The EC3 value could not be calculated, since all SIs were below 3.

The EC3 value is defined as the concentration that causes a threefold increase in DPM compared to control and is a threshold concentration for skin sensitisation.

Body weight of tested animals and control animals showed no significant differences nor did body weights prior and after the testing period. No clinical symptoms were observed during the study period.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

p-Hydroxybenzoic acid was not sensitising the skin in this assay.

Executive summary:

p-Hydroxybenzoic acid was tested in three concentrations (5%, 10%, 25%) in DMSO in the local lymph node assay with young female mice (strain CBA/CaOlaHsd) according to OECD guideline 429. Prior testing two animals were tested for irritation potential of p-HBA at doses of 10% and 25%, which was negative. 25% solution in DMSO is the technically highest possible concentration.

At all tested doses, no signs of skin sensitization based on DPM measurements per lymph node could be observed. There were no clinical symptoms at the ears nor mortality observed during the study period.

Tests, positive and negative controls were all valid.