Aspartic acid

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Test material

Test material information

Constituent 1

Reference substance name:

Aspartic acid

EC Number:

200-291-6

EC Name:

Aspartic acid

Cas Number:

56-84-8

Molecular formula:

C4H7NO4

IUPAC Name:

aspartic acid

Details on test material:

Identity:L-aspartic acid
Chemical name: L-aminosuccinic acid L-asparagic acid L-asparaginic acid 2-aminobutanedioic acid
Batch number: ASP 26111
Purity: 99.0 to 101.0%
Appearance: White crystals
Storage conditions: 4°C in the dark
Date received:16 November 1993

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

Fifteen healthy female nulliparous and non-pregnant albino guinea-pigs of the Dunkin/Hartley strain
were obtained from D. Hall, Newchurch, Staffordshire, England.
The animals were in the weight range of 308 to 351 g on arrival and approximately six to seven
weeks of age. All the guinea-pigs were acclimatised to the experimental environment for 5 days prior
to allocation to the main study.
An additional ten animals, from the same supplier, were used for the preliminary investigations.
The animals on the main study were allocated without conscious bias to two groups as follows:
Group
Control animals
Test animals
Number of
animals
5
10
Animal
numbers
4435 to 4439
4450 to 4459
The guinea-pigs were housed in groups of ten in suspended metal cages with wire mesh floors in
Building R 17 Room 14.
A vitamin C enriched guinea-pig diet FDI and drinking water were provided ad libitum. Hay was
given weekly.
The batch of diet used for the study was not analysed for nutrients, possible contaminants or microorganisms.
Results of routine physical and chemical examination of drinking water at source, as conducted
usually weekly by the supplier, are made available to Huntingdon Research Centre Ltd. as quarterly
summaries.
Animal room temperature was maintained at approximately 21 °C and relative humidity at 30 - 70 %.
These environmental parameters were recorded daily. Air exchange was maintained at approximately
15 air changes per hour and lighting was controlled by means of a time switch to give 12 hours of
artificial light (0700 - 1900 hours) in each 24 hours period.
Each animal was identified by ear tattoo number. This number was unique within the HRC Industrial
Toxicology Department throughout the duration of the study. Each cage was identified by a coloured
label displaying the study schedule number, animal numbers and the initials of the Study Director and
Home Office licensee

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal

Vehicle:

water

Induction 2

Concentration / amount:

Induction intradermal injection - 0.01 % w/v in water for irrigation
Induction topical application - 70% w/v in distilled water
This was the maximum practical concentration that could be prepared and dosed topically and
did not give rise to irritating effects.
Topical challenge - 70 and 35% w/v in distilled water
From preliminary investigations 70% w/v in distilled water was the maximum practical
concentration and did not give rise to irritating effects.

Challenge

Challenge 1

Route:

epicutaneous, semiocclusive

Vehicle:

water

Challenge 2

Concentration / amount:

Induction intradermal injection - 0.01 % w/v in water for irrigation
Induction topical application - 70% w/v in distilled water
This was the maximum practical concentration that could be prepared and dosed topically and
did not give rise to irritating effects.
Topical challenge - 70 and 35% w/v in distilled water
From preliminary investigations 70% w/v in distilled water was the maximum practical
concentration and did not give rise to irritating effects.

No. of animals per dose:

10

Details on study design:

The procedure may be considered in two parts, Induction and Challenge.
Induction
*
Induction intradermal injections - test animals
A 40 x 60 mm area of dorsal skin on the scapular region of the guinea-pig was clipped free
of hair with electric clippers. Three pairs of intradermal injections were made into a 2 x 4
cm area within the clipped area as shown in Figure 1.
Injectables for the test animals were prepared as follows:
1. Freund's complete adjuvant* was diluted with an equal volume of water for irrigation
(Ph.Eur.).
2. L-aspartic acid, 0.01 % w/v in water for irrigation.
3. L-aspartic acid, 0.01 % w/v in a 50 : 50 mixture of Freund's complete adjuvant and
water for irrigation.
Induction topical application - test animals
The preliminary investigations indicated that the maximum practical concentration of the test
suhstance for topical application (70%) did not produce skin irritation. Therefore, six days
after the injections, the same 40 x 60 mm interscapular area was clipped and shaved free of
hair and the site was pre-treated by gentle rubbing with 0.2 ml per site of 10% w/w sodium
lauryl sulphate in petrolatum. Twenty-four hours later a 20 x 40 mm patch of Whatman No.
3 paper was saturated with approximately 0.4 ml of L-aspartic acid, 70% w/v in distilled water.
The patch was placed on the skin of the test animals and covered by a length of impermeable
plastic adhesive tape (50 mm width "Blenderm"). This in turn was firmly secured by elastic
adhesive bandage (50 mm width "Elastoplast") wound round the torso of the animal and fixed
with "Sleek" impervious plastic adhesive tape. The dressing was left in place for 48 hours.
Induction - control animals
During the induction phase, the control animals were treated similarly to the test animals with
the exception that the test substance was omitted from the intradermal injections and topical
application.
The dermal reactions observed after each induction phase in both control and test animals by
group are shown in Table I.

Challenge controls:

Challenge - control and test animals
The control and test animals were challenged topically two weeks after the topical induction
application using L-aspartic acid, 70 and 35% w/v in distilled water.
Hair was removed by clipping and then shaving from an area on the left flank of each
guinea-pig. A 20 x 20 mm patch of Whatman No.3 paper was saturated with approximately
0.2 ml of L-aspartic acid, 70% w/v in distilled water and applied to an anterior site on the
flank. L-aspartic acid, 35% w/v in distilled water was applied in a similar manner to a
posterior site. The patches were sealed to the flank for 24 hours under strips of "Blenderm"
covered by "Elastoplast" wound round the trunk and secured with "Sleek".

Positive control substance(s):

yes

Remarks:

Formalin

Results and discussion

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

70% in distilled water

No. with + reactions:

0

Total no. in group:

15

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 70% in distilled water. No with. + reactions: 0.0. Total no. in groups: 15.0.

Results 2

Reading:

2nd reading

Hours after challenge:

72

Group:

test chemical

Dose level:

70% in distilled water

No. with + reactions:

0

Total no. in group:

15

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: test group. Dose level: 70% in distilled water. No with. + reactions: 0.0. Total no. in groups: 15.0.

Any other information on results incl. tables

According to HRC report (see reference), L-Aspartic acid does not require labelling with the risk phrase R 43 "May
cause sensitization by skin contact" in accordance with Council Directive 79/831/EEC, Annex VI, Part II (D) as described in the Commission Directive 93/21/EEC.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

Classification: not sensitizing
In this study, L-aspartic acid did not produce evidence of skin sensitisation (delayed contact
hypersensitivity) in any of the test animals.

Executive summary:

A study was performed to assess the skin sensitisation potential of L-aspartic acid using the guineapig. The method followed was that described in: EEC Methods for the determination of toxicity, Annex of Directive 92169/EEC (OJ No: L383A, 29.12.92), Part B, Method B.6. Acute toxicity (skin sensitisation). Based on the results of a preliminary study and in compliance with the guideline, the following dose levels were selected: Intradermal injection: 0.01 % w/v in water for irrigation Topical application: 70 % w Iv in distilled water Challenge application: 70 and 35% w/v in distilled water Ten test and five control guinea-pigs were used in this study. In this study L-aspartic acid did not produce evidence of skin sensitisation (delayed contact hypersensitivity) in any of the test animals. L-aspartic acid does not require labelling with the risk phrase R43 "May cause sensitization by skin contact" in accordance with Council Directive 79/8311EEC Annex VI, Part II(D) as described in The Commission Directive 93/211EEC.