(2S)-5-amino-2-[(aminoacetyl)amino]-5-oxopentanoic acid

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-07-15 to 2009-08-13

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 24 April, 2002

Deviations:

yes

Remarks:

Proliferation was assessed on study Day 4 and not on Day 6 as recommended by guideline; different quantification of cell proliferation, resulting in a positive response in case of SI >= 1.4 (historical control data not shown)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

adopted 2004

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

adopted August, 1998

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Note for Guidance on Non-Clinical Tolerance Testing of Medicinal Products CPMP/SWP/2145/00

Version / remarks:

adopted 1 March, 2001

GLP compliance:

yes (incl. QA statement)

Remarks:

Behörde für Soziales, Familie, Gesundheit und Verbraucherschutz, Hamburg, Germany

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

(2S)-2-(2-aminoacetamido)-4-carbamoylbutanoic acid

EC Number:

700-144-0

Cas Number:

13115-71-4

Molecular formula:

C7H13N3O4

IUPAC Name:

(2S)-2-(2-aminoacetamido)-4-carbamoylbutanoic acid

Test material form:

solid: crystalline

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland GmbH, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approximately 10 weeks
- Weight at study initiation: 20 - 24 g
- Housing: individually in Macrolon type III cages measuring 39 x 23 x 15 cm with granulated textured wood as bedding
- Diet: ssniff R/M-H V1530 (ssniff Spezialdiäten GmbH, D-59494 Soest), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 15
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

other: PEG 300

Concentration:

10, 25, 50% (w/v) test item glycyl-L-glutamine monohydrate, equivalent to 9.2, 23, 46% (w/v) anhydrous glycyl-L-glutamine

No. of animals per dose:

6

Details on study design:

PRE-SCREEN TESTS:
In order to determine the concentrations to be employed in the main study, a preliminary dose-range-finding study was conducted in 1 animal per dose level. Four dose levels of 5, 10, 25 and 50% test substance in PEG 300 were examined. No irritating potential was noted.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: mouse local lymph node assay
Ear - draining lymph node cell counts and lymph node weight were used to assess lymph node cell proliferation. In addition, the acute inflammatory skin reaction was measured by ear weight determination of circular biopsies of the ears and ear thickness measurements on test day 1 and test day 4 to identify skin irritation properties of the test item. Threshold values of the stimulation indices for the lymph node cell count and ear weight were calculated by dividing the average values per group of the test item treated animals by the vehicle treated ones. Values above 1.4 (cell count) are considered positive for a skin sensitizing potential. In addition, the lymph node weights were determined for concentration-related properties. Stimulation indices above 1.1 for the ear weight indicate a potential for irritation.

TREATMENT PREPARATION AND ADMINISTRATION:
The test item was dissolved in PEG 300 and administered to the dorsum of both ears of the animals at an application volume of 25 µL/ear. The animals were observed daily for their activities and for any clinical signs of local irritation at the application site or of systemic toxicity. Cageside observations included skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behaviour patterns. The onset, intensity and duration of any signs observed were recorded for each animal. Individual body weights were recorded prior to application (Day 1) and at the time of necropsy (Day 4).

The experimental schedule of the assay was as follows:
Day 1:
- The weight of each animal was individually identified and recorded. In addition, ear swelling measurements were carried out at the helical edge of both ears using an Oditest micrometer.
- Open application of 25 µL of the appropriate dilution of the test item, the vehicle alone or the positive control were administered to the dorsum of each ear.
Days 2 and 3: The application procedure carried out on day 1 was repeated.
Day 4 (24 hours after the last application the animals were sacrificed under ether anaesthesia by cutting the aorta abdominalis): Ear swelling measurements were carried out at the helical edge of both ears using an Oditest micrometer.
Punch biopsies of 8 mm in diameter of the apical area of both ears were prepared and immediately weighed on an analytical balance. Lateral pairs of auricular lymph nodes draining the ear tissue were excised, carefully separated from remaining fatty tissue and weighed on an analytical balance immediately following preparation. The lymph nodes were then stored on ice in PBS / 0.5% BSA and subjected to the preparation of single cell suspensions by mechanical tissue disaggregation. The cells were counted automatically in a cell counter.
The so-called stimulation (or LLN-) indices to determine the sensitising potential (this value was fixed empirically during the inter-laboratory validation of this method) were calculated by dividing the average absolute lymph node weight or lymph node cell counts per group of the test item treated lymph nodes by the vehicle treated ones. Thus, in case of no stimulating effect the index for the lymph node cell count is always below 1.4 (cut-off value). An index above 1.4 is considered positive. For lymph node weight a significance at p ≤ 0.01 is considered positive (U-test according to MANN and WHITNEY). A possible concentration-response-relationship for the lymph node weight in order to determine a possible sensitising potential was examined by linear regression analysis employing PEARSON's correlation coefficient. Outliers were determined according to the Nalimov test.
In addition, the acute inflammatory skin reaction (irritating potential) was measured by ear weight determination of circular biopsies of the ears and ear thickness measurements on test day 1 and test day 4 to identify skin irritation properties of the test item employing the U-test according to MANN and WHITNEY by comparing the test groups to the vehicle control.

Positive control substance(s):

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

Statistics:

For lymph node weight: U-test according to MANN and WHITNEY. For possible concentration-response-relationship for the lymph node weight: linear regression analysis employing PEARSON's correlation coefficient. Outliers were determined according to the Nalimov test.
Ear weight determination: U-test according to MANN and WHITNEY by comparing the test groups to the vehicle control.

Results and discussion

Positive control results:

The positive control substance alpha-hexyl cinnamic aldehyde (30% in acetone:olive oil (3:1 v/v), 25 µL/ear) was considered to be a sensitiser under the conditions of the test. A measurement of 6916667 cells/ml resulted in a stimulation index of 1.853 (vehicle control: 3733333 cells/mL = stimulation index of 1.0).

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1.196

Test group / Remarks:

10%

Results 2

Key result

Parameter:

SI

Value:

1.339

Test group / Remarks:

25%

Results 3

Key result

Parameter:

SI

Value:

1.272

Test group / Remarks:

50%

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
Treatment with test item formulations of 10%, 25% or 50% (w/w) did not reveal any significant increased values (p ≤ 0.01) for the lymph node cell count. Lymph node weights were significantly increased (p ≤ 0.01) at all 3 concentrations, however, ear weight and ear thickness were not affected, hence, no clear overall irritating potential was noted. The stimulation indices for the lymph node cell count and ear weight did not exceed the threshold levels of 1.4 or 1.1, respectively.

DETAILS ON STIMULATION INDEX CALCULATION :
Threshold values of the stimulation indices for the lymph node cell count and ear weight were calculated by dividing the average values per group of the test item treated animals by the vehicle treated ones.

CLINICAL OBSERVATIONS AND BODY WEIGHTS: There were no changes in behaviour and no changes in body weights observed.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance at concentrations of 10, 25, 50% (w/w) test item glycyl-L-glutamine monohydrate, equivalent to 9.2, 23, 46% (w/w) anhydrous glycyl-L-glutamine in PEG 300 did not reveal any sensitising properties in the local lymph node assay in mice.
CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No. 1272/2008