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Skin sensitisation

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skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
2 (reliable with restrictions)

Data source

Reference Type:
Irritancy and sensitization potential of Glyoxylic acid
Anderson SE
Bibliographic source:
Journal of Immunotoxicity, 5, 93-98

Materials and methods

Test guideline
equivalent or similar to
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Principles of method if other than guideline:
The aim of the study was to define the potential adverse effects associated with exposure to Glyoxylic Acid 50% by examining the irritancy and sensitization potential after dermal exposure to Glyoxylic Acid 50% using a combined murine LLNA.
The LLNA was performed following the method described in the ICCVAM Peer Review Panel report (NIEHS, 1999) with minor modifications
GLP compliance:
not specified
Type of study:
mouse local lymph node assay (LLNA)

Test material

Details on test material:
Glyoxylic Acid 50% (CAS 298-12-4) purchased from Sigma Aldrich Chemical Company (St. Louis, MO),
alpha-Hexylcinnamaldhyde (HCA, CAS 101-86-0) and Toluene 2,4-diisocyanate (TDI, CAS 584-84-9) purchased from Sigma Aldrich Chemical Company, Inc. (Milwaukee, WI)

In vivo test system

Test animals

Details on test animals and environmental conditions:
- Source: Taconic (Hudson, NY)
- Age at study initiation: 8-12-week
- Housing: Cages, with hardwood chip bedding (actively ventilated, cleaned and sanitized weekly)
- Diet: modified NIH-31 6 % irradiated rodent diet (Harlan Teklad #7913) ad libitum
- Water: tap water ad libitum
- Acclimation period: Mice were quarantined for 1 week upon arrival and maintained under conditions specified by NRC guidelines
- Mice were weighed, tail-marked for identification, and assigned to homogenous weight groups (n=5) before each experiment.

- Temperature (°C): 18-26°C
- Humidity (%): 30-70%
- Photoperiod : 12-hr intervals (light: 6:00-18:00)

Study design: in vivo (LLNA)

other: acetone
For range finding studies, irritancy measurement and phenotyping analysis, Glyoxylic Acid 50% was tested at the following concentrations: 10, 20, and 40%
For derivation of the EC3 value Glyoxylic Acid 50% was tested at the following concentrations: 1.25, 10, 20, and 40%
No. of animals per dose:
Details on study design:
The Local Lymph Node assay (LLNA) was performed following the method described in the ICCVAM Peer Review Panel report (NIEHS, 1999) with minor modifications. Briefly, mice were exposed topically to acetone, increasing concentration of Glyoxylic Acid 50%, or positive control (30 % HCA) on the dorsal surface of each ear (25 µL per ear) for three consecutive days. Animals were allowed to rest for 2 d following the last exposure. On day 6, mice were injected intravenously via the lateral tail vein with 20 µCi [3H]-thymidine.
Five hours after [3H]-thymidine injection, animals were euthanized via CO2 inhalation, and the left and right cervical draining lymph nodes (DLNs) located at the bifurcation of the jugular vein were excised and pooled for each animal. Single cell suspensions were made and following overnight incubation in 5 % trichloroacetic acid (TCA), samples were counted using a Packard Tri-Carb 2500TR liquid scintillation analyzer. Stimulation indices (SI) were calculated by dividing the mean disintegrations per minute (DPM) per test group by the mean DPM for the vehicle control group. EC3 values (concentration of chemical required to induce a 3-fold increase over the vehicle control) were calculated based on the equation for Basketter et al (1999). The EC3 value was derived by interpolation between two points on the SI axis, one immediately above and one immediately below the SI value of 3. For these studies Glyoxylic acid 50% was tested at concentrations between 1.25 – 40 %.

Mice were weighted, tail marked for identification, and assigned to homogenous weight groups (n=5) before each experiment.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistical analysis was performed using Graph Pad Prism version 3.0 (San Diego, CA). All data were analyzed by one-way analysis of variance (ANOVA) and when significant differences were detected (p=0.05), Dunnett’s test was used to compare treatment groups with the appropriate control group. If the assumptions were not able to be met by parametric analysis, the nonparametric Kruskal-Wallis k-sample test was utilized followed by the Mann-Whitney U-test for pairwise comparisons with the controls. Statistical significance is designated by *p ≤ 0.05 and ** p ≤ 0.01.

Results and discussion

Positive control results:
HCA (30 %) was used as a positive control for the LLNA and resulted in an average stimulation index (SI) value of 23.4.

In vivo (LLNA)

Resultsopen allclose all
Remarks on result:
other: 2.5 at 5 %; 10.7 at 10 %; 20.3 at 20 %; 23.9 at 40 % EC3: 5.05 %
other: disintegrations per minute (DPM)
Remarks on result:
other: DPM/2 nodes: control (vehicle): 672 ± 83 , 10 % : 5420 ± 1337** ; 20 % : 7516 ± 831** ; 40 % : 6379 ± 987** (means ± SE , n=5 , ** value significantly differnet from acetone controls at p < 0.01)

Any other information on results incl. tables

Irritancy as indicated by Ear Swelling

Although not statistically significant, a trend in ear swelling 24 hr post-final chemical exposure was observed after exposure to 20 % and 40 % Glyoxylic acid. Observations of the 20% and 40 % exposure group did identify two of the five animals in each group to display common signs of irritation including redness and swelling. In several of mice exposed to 40 % Glyoxylic acid, one or both of the ears were red and blistered after the dermal exposure. These observations were consistent with the observation after animal exposure for the LLNA and phenotype studies. TDI (2.5 %) was used as positive control for irritancy studies and resulted in an averaged significant increase of 68 % ear swelling post exposure.


Sensitization Potential Determined by the LLNA

Initial concentration tested (10 - 40%) generated a greater than 3-fold stimulation index, therefore the experiment was repeated using concentrations between 1.25-10 %. No changes in body weight were observed following exposure to the highest concentration of 40 %.

Exposure to Glyoxylic acid resulted in a calculated EC3 value of 5.05 %.

HCA (30 %) was used as a positive control for the LLNA and resulted in an average stimulation index (SI) value of 23.4.


Lymph Node Phenotyping and Analysis of Total Serum IgE

Phenotype analysis of the lymphocytes from draining lymph nodes following exposure to Glyoxylic acid resulted in significant increases in the B220+ cell population at all concentrations tested. However, there was no significant increase in the IgE+B220+ cells or total serum IgE. This results suggests that the glyoxylic acid response is a TH1 response rather tah a TH2 response. TDI (2.5%) was used as a positive control for phenotyping experiments and resulted in significant elevations of IgE+B220+ (17.8 %) and B220+ (33.3 %) cell populations. TDI (2.5 %) was also used as a positive control for the total IgE ELISA and resulted in a significant elevation of total IgE (175 ng/mL) when compared to vehicle).

Applicant's summary and conclusion

Interpretation of results:
Migrated information
Executive summary:
In a dermal sensitisation study with Glyoxylic acid in acetone 8 - 12 weeks old female Balb/c mice were tested using a combined Local Lymph Node Assay. The test concentrations were 1.25 , 2.5, 5.0, 10 , 20 or 40 %. The stimulation index was > 3, with a maximal value of 23.9 achieved at the maximum applied concentration of 40 %. Therefore Glyoxylic acid tested positive in the LLNA with an EC3 value of 5.05 %.

Significant increases were observed in the B220+ cell population in the draining lymph nodes. No changes were identified in the IgE+B220+ cell population in the draining lymph nodes or total serum IgE levels; this suggests that Glyoxylic acid functions as a T-cell-mediated contact sensitizer.