Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 September 2011 to 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:
2012
Report date:
2012

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
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

Constituent 1
Chemical structure
Reference substance name:
N-tert-butylglycyl chloride hydrochloride (1:1)
EC Number:
618-771-2
Cas Number:
915725-52-9
Molecular formula:
C6H13Cl2NO
IUPAC Name:
N-tert-butylglycyl chloride hydrochloride (1:1)
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
Purity Not indicated by the sponsor; treated as 100% pure Test substance storage In refrigerator (2-8°C) in the dark Stability under storage conditions Stable Expiry date 21 July 2012

In vivo test system

Test animals

Species:
mouse
Strain:
CBA:J
Sex:
female
Details on test animals and environmental conditions:
Species Mouse, CBA/J strain, inbred, SPF-Quality. Recognized by the international guidelines as the recommended test system (e.g. OECD, EC, EPA). Source: Janvier, Le Genest-Saint-Isle, France Number of animals 20 females (nulliparous and non-pregnant), five females per group. Age and body weight 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. Health inspection A health inspection was performed prior to treatment, to ensure that the animals were in a good state of health. Special attention was paid to the ears, which were intact and free from any abnormality. Reliability check The results of a reliability test with three concentrations of Hexylcinnamaldehyde in Acetone/Olive oil (4:1 v/v), performed not more than 6 months previously and using the same materials, animal supplier, animal strain and essential procedures is summarized in the appendix of this report. For both scientific and animal welfare reasons, no concurrent positive control group was added to the study. An extensive data base is available with reliability checks performed each half year during at least the recent 9 years showing reproducible and consistent positive results.

Animal husbandry
Conditions 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.

Accommodation Animals were group housed in labeled Makrolon cages (MIII type; height 18 cm) containing sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France). Paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) and shelters (disposable paper corner home, MCORN 404, Datesand Ltd, USA) were supplied as cage-enrichment. 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. Certificates of analysis were examined and then retained in the NOTOX archives.

Diet Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).

Water Free access to tap water.

Results of analysis for diet (nutrients and contaminants), sawdust, paper, shelters and water were assessed and did not reveal any findings that were considered to have affected the study integrity. All certificates and results of analysis are retained in the NOTOX archives.

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Acetone/Olive oil (4:1 (v/v)
No. of animals per dose:
5f/group
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 (see section 6.7) 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 (MII type, height 14 cm). 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.

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) (Merck, Darmstadt, Germany) containing 20 μCi of 3H-methyl thymidine (PerkinElmer Life and Analytical Sciences, Boston, MA, US).

After approximately five hours, all animals were killed by intraperitoneal injection (0.2 mL/animal) with Euthasol® 20% (AST Farma BV, Oudewater, The Netherlands). 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) (Merck, Darmstadt, Germany) 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 (PerkinElmer Life and Analytical Sciences, Boston, MA, US) 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).

Interpretation
DPM values are presented for each animal and for each dose group. A Stimulation Index (SI) is calculated for each group. The SI is the ratio of the DPM/group compared to DPM/vehicle control group.

If the results indicate a SI ≥ 3, the test substance may be regarded as a skin sensitizer.

The results were evaluated 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 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of substances and mixtures.

Consideration was given to the EC3 value (the estimated test substance concentration that will give a SI =3)

Test substance preparation
Vehicle Acetone/Olive oil (4:1 v/v) (Acetone p.a.: Merck, Darmstadt, Germany; Olive oil: Sigma-Aldrich, Steinheim, Germany). Rationale The vehicle was selected based on trial formulations performed at NOTOX and on test substance data supplied by the sponsor. Preparation The test substance formulations (w/w) were prepared within 4 hours prior to each treatment. No adjustment was made for specific gravity of the vehicle. Homogeneity was obtained to visually acceptable levels.
Positive control substance(s):
other: For both scientific and animal welfare reasons, no concurrent positive control group was added to the study.

Results and discussion

Positive control results:
For both scientific and animal welfare reasons, no concurrent positive control group was added to the study.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
ca. 0.9
Test group / Remarks:
10 % test substance group
Key result
Parameter:
SI
Value:
ca. 0.8
Test group / Remarks:
25 % substance test group
Key result
Parameter:
SI
Value:
ca. 1.3
Test group / Remarks:
50 % test substance group
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA

DETAILS ON STIMULATION INDEX CALCULATION

EC3 CALCULATION
No irritation and no signs of systemic toxicity were observed in any of the animals examined. Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values. Based on these results, the highest test substance concentration selected for the main study was a 50% concentration.

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. The body weight loss noted for one individual animal was considered not toxicologically significant since the change was slight in nature and no concentrationrelated incidence was apparent.

Macroscopy of the auricular lymph nodes and surrounding area
Auricular lymph nodes of the animals of the experimental and control groups were considered normal in size, except for one node of one animal treated at 10% which was considered enlarged No macroscopic abnormalities of the surrounding area were noted in any of the animals.

Radioactivity measurements
Mean DPM/animal values for the experimental groups treated with test substance concentrations 10, 25 and 50% were 325, 279 and 463 DPM respectively. The mean DPM/animal value for the vehicle control group was 351 DPM.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The SI values calculated for the substance concentrations 10, 25 and 50% were 0.9, 0.8 and 1.3 respectively.

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

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 hypersensitivity.

Based on these results, N-t-butylglycine acid chloride 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.
Executive summary:

Assessment of Contact Hypersensitivity to N-t-butylglycine acid chloride HCl in the Mouse (Local Lymph Node Assay).  

The study was carried out based on the guidelines described in:  OECD, Section 4, Health Effects, No.429 (2010),  EC, No 440/2008; B42: "Skin Sensitization: Local Lymph Node Assay" EPA, OPPTS 870.2600 (2003) “Skin Sensitization”.

Test substance concentrations selected for the main study were based on the results of a pre-screen test.

In the main study, three experimental groups of five female CBA/J mice were treated with test substance concentrations of 10, 25 or 50% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with vehicle alone (Acetone/Olive oil (4:1 v/v)).

Three days after the last exposure, all animals were injected with 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of Disintegrations Per Minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.

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. The body weight loss noted for one individual animal was considered not toxicologically significant since the change was slight in nature and no concentrationrelated incidence was apparent.

Auricular lymph nodes of the animals of the experimental and control groups were considered normal in size, except for one node of one animal treated at 10% which was considered enlarged  No macroscopic abnormalities of the surrounding area were noted in any of the animals.

Mean DPM/animal values for the experimental groups treated with test substance concentrations 10, 25 and 50% were 325, 279 and 463 DPM respectively. The mean DPM/animal value for the vehicle control group was 351 DPM.  

The SI values calculated for the substance concentrations 10, 25 and 50% were 0.9, 0.8 and 1.3 respectively.  

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

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 hypersensitivity.

Based on these results, N-t-butylglycine acid chloride 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.