Carbamic acid, N-[(1R)-2-hydroxy-1-phenylethyl]-, 1,1-dimethylethyl ester

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 October 2015 to 19 February 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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:

Carbamic acid, N-[(1R)-2-hydroxy-1-phenylethyl]-, 1,1-dimethylethyl ester

EC Number:

600-273-1

Cas Number:

102089-74-7

Molecular formula:

C13H19NO3

IUPAC Name:

Carbamic acid, N-[(1R)-2-hydroxy-1-phenylethyl]-, 1,1-dimethylethyl ester

Test material form:

solid: particulate/powder

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

EXPERIMENTAL ANIMALS
Species and strain: CBA/CaOlaHsd mice
Source: Harlan Laboratories S.r.l.
San Pietro al Natisone (UD)
Zona Industriale Azzida, 57,
33049 Italy
Hygienic level: SPF at arrival; standard housing conditions during the study
Justification of strain: On the basis of OECD Guideline, mice of CBA/Ca or CBA/J strain can be used. Females are used because the existing database is predominantly based on females.
Number of animals: 4 animals / group
Sex: Female, nulliparous, non pregnant
Age of animals at starting: 11 weeks old (age-matched, within one week)
Body weight range at starting: 21.1 – 23.2 grams (The weight variation in animals in the study did not exceed ± 20 % of the mean weight.)
Acclimatization time: 21 days

Husbandry
Animal health: Only healthy animals were used for the study. Health status was certified by the veterinarian.
Housing / Enrichment: Group caging / mice were provided with glass tunnel-tubes
Cage type: Type II. polypropylene / polycarbonate
Bedding: Bedding was available to animals during the study
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 19.2 – 25.9°C
Relative humidity: 30 - 71 %
Ventilation: 15-20 air exchanges/hour
The temperature and relative humidity were recorded twice every day during the acclimatisation and experimental phases.
Room/Cabinet (preliminary experiment): 244/9
Room/Cabinet (main test, non-radioactive phase): 244/4
Room/Cabinet (main test, radioactive phase): 139 – 140

Food and feeding
Animals received ssniff SM Rat/Mouse – “Breeding & Maintenance, 15 mm, autoclavable Complete diet for rats/mice” (Batch number: 930 3907, Expiry date: 31 December 2015 and Batch number: 575 4308, Expiry date: 31 March 2016) produced by ssniff Spezialdiäten GmbH (Ferdinand-Gabriel-Weg 16, D-59494 Soest, Germany), ad libitum. The food was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. The contents of the standard diet are detailed in Appendix 3.

Water supply
Animals received tap water from the municipal supply from 500 mL bottle, ad libitum. Water quality control analysis was performed once every three months and microbiological assessment was performed monthly by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József Attila u. 36., Hungary). Copies of the relevant Certificates of Analysis are retained in the Archive at CiToxLAB Hungary Ltd.

Bedding
Bedding of certified wood chips especially designed to keep animals in the best natural environment was provided for animals during the study. Lignocel 3/4-S Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH & Co.KG (D-73494 Rosenberg, Germany) was available to animals during the study. Nest building material was also provided for animals (Enviro-Dri produced by LBS).

Identification and randomisation
A unique number written on the tail with a permanent marker identified each animal. The animal number was assigned on the basis of CiToxLAB Hungary Ltd.’s master file. The cages were marked with identity cards with information including study code, cage number, and dose group, sex and individual animal number. The animals were randomised and allocated to the experimental groups. The randomisation was checked by computer software using the body weight to verify the homogeneity and variability between the groups.

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Concentration:

50 % (w/v), 25% (w/v), 10% (w/v)

No. of animals per dose:

4 animals / group

Details on study design:

Dose Selection and Justification of Dose Selection
The Preliminary Irritation/Toxicity Test was started according to the Study Plan on CBA/CaOlaHsd mice using two doses (2 animals/dose) at test item concentrations of 50 and 25 % (w/v) in DMSO. The preliminary experiment was conducted in a similar experimental manner to the main study, but it was terminated on Day 6 and the radioactive proliferation assay was not performed.
The maximum concentration of test item in an acceptable solvent was established according to OECD guideline 429. Based on the observation of the solubility test, the maximum available concentration was 50 % (w/v).
In the Preliminary Irritation / Toxicity Test, all mice were observed daily for any clinical signs of systemic toxicity or local irritation at the application site. Both ears of each mouse were observed for erythema and scored using Table 2 [3]. Ear thickness was also measured using a thickness gauge on Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose) and Day 6. Additional quantification of the ear thickness was performed by ear punch weight determination after the euthanasia of the experimental animals.
During the Preliminary Irritation / Toxicity Test, no mortality or signs of systemic toxicity were observed. Test item precipitate or minimal amount of test item precipitate was observed on the ears of the animals in the 50 % (w/v) dose group on Days 1-3 and in the 25 % (w/v) dose group on Days 2-3. Clinical observations are summarized in Table 8 of Appendix 4. No marked body weight loss (>5%) was detected in the experimental animals (Table 6 of Appendix 4).
The ear thickness values and the weights of the ear punches (2 per animal) are summarized in Table 7 of Appendix 4. The ear thickness values and ear punch weights were within the acceptable range. There were no indications of any irritancy at the site of application on the experimental animals.
The draining auricular lymph nodes of the animals were visually examined: they were normal in both dose groups (subjective judgement by analogy with observations of former experiments).
Based on these observations, the 50 % (w/v) was selected as top dose for the main test. The experimental groups and dose levels for the main experiment are summarized in Table 3.

Topical application
During the study, animals were topically dosed with 25 μL of the appropriate formulation using a pipette on the dorsal surface of each ear. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6.

Positive control substance(s):

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

Results and discussion

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1.2

Test group / Remarks:

50% (w/v)

Remarks on result:

no indication of skin sensitisation based on QSAR/QSPR prediction

Results 2

Key result

Parameter:

SI

Value:

1.5

Test group / Remarks:

25% (w/v)

Remarks on result:

no indication of skin sensitisation based on QSAR/QSPR prediction

Results 3

Key result

Parameter:

SI

Value:

1.7

Test group / Remarks:

10% (w/v)

Remarks on result:

no indication of skin sensitisation based on QSAR/QSPR prediction

Cellular proliferation data / Observations:

8.1. CLINICAL OBSERVATION
No mortality or signs of systemic toxicity were observed during the study. Test item precipitate or minimal amount of test item precipitate was observed on the ears of the experimental animals in the 50 and 25 % (w/v) dose groups on Days 1-3 and in the 10 % (w/v) dose group on Days 2-3. There were no indications of any irritancy at the site of application. The results of the observations are summarized in Appendix 5.

BODY WEIGHT MEASUREMENT
No treatment related effects were observed on the mean body weight changes, however marked body weight loss (≥5%) was observed for one animal in the 50 % (w/v) group, for two animals in the 10 % (w/v) dose group and for one animal in the positive control group. Individual and mean body weights are given in Table 4.

PROLIFERATION ASSAY
The results of the proliferation assay are summarized in Table 5 and Figure 1. The appearance of the lymph nodes was normal in the negative and in all the test item treated dose groups. Larger than normal lymph nodes were observed in the positive control group.

INTERPRETATION OF OBSERVATIONS
The test item was a powder, which was used formulated in DMSO. Since there were no confounding effects of irritation or systemic toxicity at the applied concentrations, the proliferation values obtained are considered to reflect the real potential of the test item to cause lymphoproliferation in the Local Lymph Node Assay. The resulted stimulation indices observed under these exaggerated test conditions was considered to be good evidence that N-Boc-D-phenylglycinol is a non-sensitizer.

Any other information on results incl. tables

RELIABILITY OF THE TEST

The result of the positive control substance α-Hexylcinnamaldehyde (HCA) dissolved in the same vehicle was used to demonstrate the appropriate performance of the assay [1]. The positive control substance was examined at a concentration of 25 % in the relevant vehicle (DMSO) using CBA/CaOlaHsd mice.

No mortality, cutaneous reactions or signs of toxicity were observed for the positive control substance in the study. A lymphoproliferative response in line with historic positive control data (stimulation index value of 7.1) was noted for HCA in the main experiment. This value was considered to confirm the appropriate performance of the assay.

Furthermore, the DPN values observed for the vehicle and positive control substance in this experiment were in harmony with the historical control data. Each treated and control group included 4 animals.

Historical control data for the positive and negative control substances are shown in Appendix 6.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, under the conditions of the present assay, N-Boc-D-phenylglycinol, tested in a suitable vehicle, was shown to have no sensitisation potential (non-sensitizer) in the Local Lymph Node Assay.