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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 03 November 2008 and 28 November 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
GLP compliance:
yes (incl. QA statement)
Type of study:
other: maximisation method with Freund's complete adjuvant
Justification for non-LLNA method:
report available from 2009

Test material

Constituent 1
Chemical structure
Reference substance name:
Sodium glyoxylate
EC Number:
220-298-8
EC Name:
Sodium glyoxylate
Cas Number:
2706-75-4
Molecular formula:
C2H2O3.Na
IUPAC Name:
sodium oxoacetate
Specific details on test material used for the study:
Name: GOA-Na
Lot No.: C080901
Chemical name: Sodium glyoxylate
Appearance: White Powder
Purity: 100%

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male
Details on test animals and environmental conditions:
Source: LAB-ALL Bt. Budapest, 1174 Hunyadi u. 7.
Weight range at the beginning of the study: 331-379 g
Acclimatization time: 12 days
Animal health: Only animals in acceptable health condition were used for the test as certified by the veterinarian.
Cage type: Animals were housed in macrolon cages, size Ill., with 3 or 2 animals/cage (42 x 42 x 19 cm)
Bedding: Laboratory bedding, Lignocel 3-4 Fasern
Produced by: .T. RETTENMAIER & SOHNE GmbH+CO.KG, D-Rosenberg (Germany)
Light: 12 hours daily from 6 a.m. to 6 p.m. (artificial light)
Temperature: 17.9-24.7 °C
Relative humidity: 25-63 %
Food and Feeding:
Animals received PURINA Base Lap gr. diet for rabbits produced by AGRIBRANDS Europe Hungary PLC, H-5300 Karcag, Madarasi road, Hungary, ad libitum.
This diet is classified as being suitable for Guinea pigs, the vitamin D level is high enough to meet the needs of this species. This is the diet used by the breeder/supplier, so animals are fully adapted to this diet on arrival.
Water Supply: The animals received tap water, as for human consumption, ad libitum containing 50 mg/1 00 ml Ascorbic acid. The drinking water is routinely analysed and is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
Identification: The animals were marked individually with ear punching. The cages were marked with individual identity cards with infonnation about study code, sex, cage number, dose group and individual animal number.
Randomisation: All animals were sorted according to weight on the clay before the start of the treatment period. After this the animals were allocated to the test groups. The result of the randomisation was checked according to the actual body weights assunng an acceptable homogeneity and variability among the groups.
Body Weight: The body weights of individual animals were recorded at the beginning and at the end of the experiment. The mean values and the standard deviations were calculated in the test group as well as in the control.

Study design: in vivo (non-LLNA)

Induction
Route:
intradermal and epicutaneous
Vehicle:
other: methylcellulose (1%)
Concentration / amount:
1 % w/v for the intra-dermal treatment
75 % w/v for the dermal (occlusive) treatment
Day(s)/duration:
24 hrs (intra-dermal treatment) and 48 hrs (dermal treatment)
Adequacy of induction:
other: Intra-dermal: Highest technically applicable concentration used that caused local irritation. Dermal: substance not irritant dermally but test area pre-treated with 10% SDS
Challenge
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
other: Methylcellulose 1%
Concentration / amount:
75 % w/v
Day(s)/duration:
24 hrs
Adequacy of challenge:
not specified
No. of animals per dose:
Preliminary tests:
Intra-dermal - Two animals per dose (0.01, 0.1 and 1 % w/v)
Dermal - Two animals per dose (10, 25, 50 and 75 % w/v)

Main tests:
10 animals + 5 control animals
Details on study design:
Preliminary test and justification of test doses:
A series of test item concentrations were tested to identify any primary irritation by intra-dermal injection and dermal application. Three dose levels were tested in the preliminary dose range finding study lo identify any primary irritation by intra-dermal injection and four close levels by dermal application. For intra-dermal application 0.1 ml test item was injected at concentrations of 0.01, 0.1 and 1 % (w/v). Two animals were used to test each or the concentration. For dermal application 0.5 ml test item was applied to the skin of animals by closed patch exposure at concentrations of 10 and 25 % (w/v) for an exposure time of 24 hours and at concentrations of 50 and 75 % (w/v) for an exposure time or 48 hours. Two animals were used to test each of the concentrations.
The intra-dermal treatment of test item caused local initation at a concentration of 0.1 % (w/v). Very slight erythema (scores 1-1) was found in both animals 1 and 24 hours after the patch removal. At a concentration of 0.01 % very slight erythema (scores 1-1) was observed in both animals 1 hour after the patch removal. The test item was inappropriate at concentrations of 5 % (w/v) for the intra-dermal treatment due to the physical properties of the formulation. The intra-dermal treatment of the test item caused local irritation at a concentration of 1 % (w/v). Very slight erythema (scores 1) was found one animal (No.53) J and 24 hours after the patch removal and well defined erythema (scores 2) was found the other animal (No.55) 1 hour after the patch removal and very slight erythema (scores 1) was found at 24 hours after the patch removal. Dermal application of 0.5 ml test formulation at concentrations of 10 and 25, 50 and 75 % produced no reaction (scores 0-0) on the skin of guinea pigs. As a result of these preliminmy experiments, test item was administered at a
concentration of 1 % for intra-dermal treatment. The intra-dermal treatment was performed according to the Study Plan "Intra-dermal Induction Exposure". For dermal induction exposure, test item was applied at a concentration of 75 %. Control animals were treated with vehicle only. The dermal induction treatment was performed according to study plan "Dermal Induction Exposure". For the challenge exposure, all animals of the treatment and control group were treated with the test item at concentration of 75 %. The challenge exposure was performed according to study plan "Challenge Exposure".
Time of exposure: 24 hours (for the intra-dermal and challenge closes) and 48 hours (for the dermal induction close). Symptoms were examined and scored 1, 24, 48 and 72 hours after exposure.

Main study
See table in 'Any other information on materials and methods incl. tables' for study design.
Control animals were treated similarly to test animals, except that during the induction phase, the test item was omitted.
Induction involved two main procedures: intra-dermal treatments (Main Study I) and dermal exposure (Main Study II) with closed patch technique. The intra-dermal and the dermal induction treatments were observed and recorded.

Main Study I: Intra-dermal Induction Exposure
Approximately 24 hours before the treatment an area of approximately 5 x 5 cm on the scapular region of animals was clipped free of hair and shaven close with care.

Intra-dermal treatment
Test groups:
A series of three injections was administered on each side of treatment group animals, as follows, resulting in six injections per animal:
2 injections with 0.10 ml of Freund's Complete Adjuvanl mixed with physiological saline (I: 1 v/v),
2 injections with 0.10 ml of lhe test item in tvlethylcellulose I% at the selected concentration,
2 injections with 0.10 ml of !est item in selected concentration, formulated in a 1: 1 (v/v) mixture ofFreund's Adjuvant and physiological saline.
Control group:
The control animals were treated similarly as the test group however, the vehicle, without the test item was used for injections, as follows:
2 injections with 0.1 ml mix of Freund's Complete Adjuvant and physiological saline (NaCl 0.9 %) (1:1)(v/v),
2 injections with 0.1 ml of Me!hylcellulose 1% ,
2 injections with 0.1 ml of 50 w/v% fommlation of Me!hylcellulose 1 %, in a 1: 1 mixture (v/v) Preund's Adjuvant and physiological saline.

Main study II: Dermal Induction Exposure
Seven days after the intra-dermal injections, the tesl animals were exposed to test item on the other hand the control animals were treated wilh Methylcellulose 1% , as vehicle.
Closed patch was applied in lhe following manner: in case of lhe lest animals 0.5 ml or test item (at concentration of 75 %) was spread on lhe surface prepared previously and covered with a standard (5x5 cm) size of porous gauze patch. Control animals were treated dermally with 0.5 ml of Methylcellulose 1%, as vehicle and the dressing was prepared and applied as for the lest animals.
The test item was not a skin irritant in the range-finding study, so the test area was painted with 0.5 ml of 10 % sodium doclecyl sulphate in Vaseline 24 h prior to the topical induction application, in order to create a local irritation.
The exposed areas were covered for 48 hours with a porous gauze fastened with "Leucoplast" (Closed Patch Test). After the dermal induction exposition the rest of the test item was removed with water of body temperature. Following dermal induction treatments the animals were left untreated for 14 days prior to challenge applications.

Main study III: Challenge Exposure
Two weeks after the dermal treatment the animals were exposed to the challenge close, dermally. 24 hours before the challenge treatment the left and the right flank areas (5x5 cm) of each animal were prepared for application. The challenge was performed as a dermal exposure (Closed Patch Test). Left shaved flank areas of the animals (both the test and the control) were treated with 0.5 ml of the test item (at concentration of 75 %). The right shaved flank areas were treated with 0.5 ml of Methylcellulose 1% , in all cases. Time of exposure was 24 hours.
Challenge controls:
Control animals treated with vehicle in Main Study I and II were treated with 0.5 ml of the test item in the challenge control.
The right flank of all animals in the challenge test were treated with Methylcellulose 1% only.
Positive control substance(s):
yes
Remarks:
2-MERCAPTOBENZOTHIAZOLE, carried out between 20 October 2008 and 14 November 2008

Results and discussion

Positive control results:
In the test group, 10 animals were treated with the reference item. After the challenge with test item 2-MERCAPTOBENZOTHIAZOLE, positive response was seen in five out of ten animals in the test group (50 %). The mean of the scores were 0.50 and 0.30 according to the 24 and 48 hour results. The dermal scores represented discrete and discrete and confluent erythema developed on the skin of sensitised guinea pigs. On the opposite (right) side treated with vehicle no reaction was found.
A total of 5 control animals were exposed to vehicle for induction treatments and exposed to the reference item for challenge application. In the control group the mean of the scores was 0.00.

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
1%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no adverse clinical response to treatment
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no adverse clinical response to treatment
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
test chemical
Dose level:
75%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no adverse clinical response to treatment
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
0
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no adverse clinical response to treatment
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Group:
test chemical
Dose level:
75%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no adverse clinical response to treatment
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
24
Group:
positive control
Dose level:
75%
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no adverse clinical response to treatment
Remarks on result:
no indication of skin sensitisation

Any other information on results incl. tables

A group of 10 animals was treated with the test item during the induction phase of the study. Injections were given intra-dermally with and without FCA and one week after the test material was applied dermally on the same site. The animals were challenged by dermal exposure two weeks later.

Skin Effects Afier the Challenge Exposure

Test group

After the challenge with test item GOA-Na at a concentration of 75 %, positive response was not observed on the animals of the test group. The mean of the scores was 0.00 according to the 24 and 48 hour results.

On the opposite (right) side treated with vehicle no reaction was found.

Control group

Five control animals were exposed to vehicle during induction treatments and they were treated with the test item on the challenge day only.

No visible changes were found at the 24 and 48 hour examinations. During the challenge exposure, the test item GOA-Na at a concentration of 75 % did not evoke primary irritation.

Clinical Observations

There were no overt signs of an adverse clinical response to treatment with GOA-Na during the course of the study.

Mortality

There were no moribund or dead animals during the study.

Body Weight

The individual body weights of the guinea pigs were measured at the beginning and at the end of experiment. There were no notable differences between the test animal

group and the control group.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Challenge with test item GOA-Na evoked no positive responses in the test animals sensitised previously. At the same time, none of the animals proved to be positive in the control group. The net response value represented an incidence rate of 0 % and the net score value of 0.00.
The test item GOA-Na was shown to have no sensitisation potential and classified as a non-sensitizer, based on this test and according to current EU-regulations.
Executive summary:

A skin sensitisation study was performed according to the Magnusson-Kligman method, using a maximisation method with Freund's complete adjuvant to evaluate the sensitisation potential of test item GOA-Na in guinea pigs.

10 test animals were subjected to sensitisation procedures in a two-stage operation, i.e. an intra-dermal treatment and a topical application. The test item was used at a concentration of l% for intra-dermal injections and at a concentration of 75 % for dermal sensitisation treatment. Two weeks after the last induction exposure, a challenge close (at a concentration of 75 %) was administered. Challenge was perfomed by dermal application of the test item.

Five control guinea pigs were simultaneously exposed to vehicle during the sensitisation phase and they were treated with the test item (at a concentration of 75 %) only in the case of challenge.

Incidence Rate

No signs of contact sensitisation were detected in guinea pigs exposed previously to the test item during experiments.

Intensity of Sensitisation Response

In the control and treated animals the mean of the scores was 0.00 according to the 24h and 48h results.

Conclusion

The test item GOA-Na was shown to have no sensitisation potential and classified as a non-sensitiser, based on this test and according to current EU-regulations.