2-(tert-butylamino)acetic acid hydrochloride

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 September - 03 October 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Principles of method if other than guideline:

Protocol deviations:
1. Temporary deviations from the maximum level of relative humidity occurred.
Evaluation: Laboratory historical data do not indicate an effect of the deviations. The study integrity was not adversely affected by the deviation.

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:

2-(tert-butylamino)acetic acid hydrochloride

EC Number:

614-959-3

Cas Number:

6939-23-7

Molecular formula:

C6H14ClNO2

IUPAC Name:

2-(tert-butylamino)acetic acid hydrochloride

Test material form:

solid: particulate/powder

Details on test material:

White powder
Test substance storage: In refrigerator (2-8°C) in the dark

Specific details on test material used for the study:

White powder
Test substance storage: In refrigerator (2-8°C) in the dark
Batch #: 11681004

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

20 females (nulliparous and non-pregnant) mice, CBA/J strain, inbred, SPF-Quality, five females per group from Janvier, Le Genest-Saint-Isle, France. Young adult animals (approx. 9 weeks old) were selected. Body weight variation was within +/- 20% of the sex mean. Identification tail mark with marker pen.
Recognized by the international guidelines as the recommended test system.
Animals were housed in a controlled environment, in which optimal conditions were considered to be approximately 15 air changes per hour, a temperature of 21.0 ± 3.0ºC (actual range: 20.2 - 23.6ºC), a relative humidity of 40-70% (actual range: 41 - 77%) and 12 hours artificial fluorescent light and 12 hours darkness per day. Due to a technical failure of the sensors in the study room A0.18 from 05 July 2011 until 09 September
2011, temperature and relative humidity data for part of the pre-screen test were used from room A0.19 which is adjacent to the study room A0.18 and connected to the same temperature and relative humidity regulation system as the study room A0.18. Temperature and relative humidity inside room A0.19 is therefore considered representative for the conditions in the study room A0.18.
Animals were group housed in labeled Makrolon cages containing sterilised sawdust as bedding material. The acclimatization period was at least 5 days before the start of treatment under laboratory conditions. On Day 6, the animals were group housed in Makrolon MII type cages with a sheet of paper instead of sawdust and cage enrichment. Free access to pelleted rodent diet and free access to tap water.

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

Test substance concentrations of 10, 25 or 50% w/w.

No. of animals per dose:

Three groups of five animals were treated with one test substance concentration per group. One group of five animals was treated with vehicle.
Group 1 = vehicle (dimethly formamide)
Group 2 = 10 % test substance concentration
Group 3 = 25 % test substance concentration
Group 4 = 50 % test substance concentration

Details on study design:

Pre-screen test:
A pre-screen test was conducted in order to select the highest test substance concentration to be used in the main study. In principle, this highest concentration should cause no systemic toxicity, may give moderate irritation at the most (maximum grade 2 and/or an increase in ear thickness < 25%) and is the highest possible concentration that can technically be applied. Two test substance concentrations were tested; a 25% and 50% concentration. The highest concentration was the maximum concentration as required in the test guidelines (50% for solids). The test system, procedures and techniques were identical to those used in the main study except that assessment of lymph node proliferation and necropsy were not performed. Two young adult animals per concentration were selected (in the range of 8 to14 weeks old). Each animal was treated with one concentration on three consecutive days. Animals were group housed in labeled Makrolon cages. Ear thickness measurements were conducted using a digital thickness gauge prior to dosing on Days 1 and 3, and on Day 6. Animals were sacrificed after the final observation.
Main study:
Three groups of five animals were treated with one test substance concentration per group. The highest test substance concentration was selected from the pre-screen test. One group of five animals was treated with vehicle.

Allocation
Group animal numbers induction (test substance; % w/w)
1 01 - 05 0 (Dimethyl formamide)
2 06 - 10 10
3 11 - 15 25
4 16 - 20 50

Induction - Days 1, 2 and 3:
The dorsal surface of both ears was topically treated (25 μL/ear) with the test substance concentration, at approximately the same time on each day. The concentrations were stirred with a magnetic stirrer immediately prior to dosing. The control animals were treated in the same way as the experimental animals, except that the vehicle was administered instead of the test substance.
Excision of the nodes - Day 6:
Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) containing 20 μCi of 3H-methyl thymidine.
After approximately five hours, all animals were killed by intraperitoneal injection (0.2 mL/animal) with Euthasol® 20%. The draining (auricular) lymph node of each ear was excised. The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in approximately 3 mL PBS.
Tissue processing for radioactivity - Day 6:
A single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (diameter 125 μm). LNC were washed twice with an excess of PBS by centrifugation at 200g for 10 minutes at 4ºC. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) and stored in the refrigerator until the next day.
Radioactivity measurements - Day 7:
Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail as the scintillation fluid. Radioactive measurements were performed using a Packard scintillation counter (2800TR). Counting time was to a statistical precision of ± 0.2% or a maximum of 5 minutes whichever came first. The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).
Observations:
Mortality/Viability: Twice daily
Body weights: On Day 1 (pre-dose) and Day 6 (prior to necropsy).
Clinical signs: Once daily on Days 1-6 (on Days 1-3 between 3 and 4 hours after dosing).
Irritation: Once daily on Days 1-6 (on Days 1 - 3 immediately after dosing) according to the following numerical scoring system. Furthermore, a
description of all other (local) effects was recorded.

Results and discussion

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

2.2

Test group / Remarks:

10%

Remarks on result:

no indication of skin sensitisation based on QSAR/QSPR prediction

Results 2

Key result

Parameter:

SI

Value:

1.9

Test group / Remarks:

25 %

Remarks on result:

no indication of skin sensitisation based on QSAR/QSPR prediction

Results 3

Key result

Parameter:

SI

Value:

1.4

Test group / Remarks:

50%

Remarks on result:

no indication of skin sensitisation based on QSAR/QSPR prediction

Any other information on results incl. tables

No irritation of the ears was observed in any of the animals examined.

No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the

main study. Body weights and body weight gain of experimental animals remained in the same range

as controls over the study period.

Mean DPM/animal values for the experimental groups treated with test substance concentrations 10,

25 and 50% were 449, 402 and 293 DPM respectively. The mean DPM/animal value for the vehicle

control group was 207 DPM.

The SI values calculated for the substance concentrations 10, 25 and 50% were 2.2, 1.9 and 1.4

respectively.

The six-month reliability check with Alpha-hexylcinnamicaldehyde indicates that the Local Lymph

Node Assay as performed at NOTOX is an appropriate model for testing for contact hypersensi

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Since there was no indication that the test substance elicited an SI ≥ 3 when tested up to 50%, Nbutylglycine HCl was considered to be a non skin sensitizer.

Executive summary:

Based on these results, N-butylglycine HCl would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. The test substance does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2011) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures.