Oxazolidine, diheptyl~

Endpoint summary

Description of key information

Skin Sensitization

REACH_sensitising | mouse | OECD 429 | #key study#

REACH_not sensitising | human | HRIPT |

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Feb. - March 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2002

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2004

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

2003

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

ANIMALS
- Species: Mouse, CBA strain, inbred, SPF-Quality. Recognized by the international guidelines as the recommended test system (e.g. OECD, EC, EPA); source: Charles River France, L'Arbresle Cedex, France.
- Number of animals: 20 females (nulliparous and non-pregnant), five females per group.
- Age and bodyweight: Young adult animals (approx. 11 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 are in a good state of health. Special attention was paid to the ears, which were intact and free from any abnormality.
- Reliability check: The reliability test with Hexylcinnamic aldehyde was performed not more than 6 months previously or 2 months afterwards. Similar procedures were used in the reliability test and in this study.

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 ± 3.0°C (actual range: 20.6 - 23.2°C), a relative humidity of 30-70% (actual range: 35 - 63%) and 12 hours artificial fluorescent light and 12 hours darkness per day.
- Accommodation: Individual housing in labeled Macrolon cages (MI type; height 12.5 cm) containing sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France).
- Acclimatization period: The acclimatization period was at least 5 days before the start of treatment under laboratory conditions. Accommodation was as described above except that the animals were group housed in Macrolon cages (MIII type; height 18 cm). Paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ud), Surrey, United Kingdom) was supplied as cage-enrichment.
- 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 each batch of diet (nutrients) and results of quarterly analysis of diet (contaminants), sawdust, paper 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.

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

25 % open epicutaneous
50 % open epicutaneous
100 % open epicutaneous

No. of animals per dose:

5

Details on study design:

Three groups of five animals were treated with one test substance concentration (25%, 50%, 100%) per group. The highest test substance concentration was selected from the preliminary irritation study. One group of five animals was treated with vehicle.

INDUCTION - Days 1, 2 and 3
The dorsal surface of both ears was epidermally treated (25 µL/ear) with the test substance concentration, at approximately the same time per day. The concentrations were mixed thoroughly using a vortex mixer immediately prior to dosing. The control animals were treated the same as the experimental animals, except that, instead of the test substance, the vehicle alone was administered.

TREATMENT- Day 6
All animals: 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 (GE Health care,
Buckinghamshire, UK). After approximately five hours, all animals were killed by intraperitoneal injection with pentobarbital (0,2 mL/animal Euthesate®; Sanofi Sante BV, Maassluis, The Netherlands).
The draining (auricular) Iymph 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 - Day 6
A single cell suspension of Iymph 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. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) (Merck, Darmstadt, Germany) at 4° C during the night.

RADIOACTIVITY MEASUREMENTS - Day 7
Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail (perkinEImer 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 comes first The Packard 2800TR was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).

Positive control substance(s):

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

Positive control results:

The six monthly reliability check with Hexylcinnamic aldehyde (NOTOX Project 477214) indicates that the Local Lymph Node Assay as performed at NOTOX is an appropriate model for testing for contact hypersensitivity

Key result

Parameter:

EC3

Value:

42.3

Key result

Parameter:

SI

Value:

12

Test group / Remarks:

100%

Key result

Parameter:

SI

Value:

3.4

Test group / Remarks:

50%

Parameter:

SI

Value:

2.1

Test group / Remarks:

25%

Preliminary irritation study

Results of the epidermal exposures for the selection of highest test substance concentration to be tested in the main study:

- Animal 1 (50%): erythema grade 2, edema grade 0 (day 3)

- Animal 2 (100%): erythema grade 2, edema grade 0 (day 3)

Based on the results, the highest test substance concentration selected for the main study was a 100% concentration.

Skin reactions / Irritation

A dose related increase in irritation of the ears was seen, from slight erythema in all animals at low dose up to well-defined erythema in all animals at high dose. No oedema was observed in any of the animals examined.

Macroscopy of the auricular Iymph nodes and surrounding area

The majority of nodes were considered normal in size, except for the nodes of all animals treated at 100% that were enlarged. No macroscopic abnormalities of the surrounding area

were noted.

Body weights

Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. The slight body weight loss, noted in some animals, was

considered not toxicologically significant.

Radioactivity measurements

Mean DPM/animal values for the experimental groups treated with test substance concentrations 25, 50 and 100% were 676, 1102 and 3878 respectively. The mean DPM/animal

value for the vehicle control group was 323.

Toxicity and Mortality

No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study.

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

Based on the study results:
- according to the recommendations made in the test guidelines, the test substance would be regarded as skin sensitizer.
- according to the Globaily Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations (New York and Geneva, 2017), the test substance should be classified as skin sensitizer (Category 1B).
- according to the EC criteria for classification and labeling requirements for dangerous substances and preparations (Council Directive 67/548/EEC), the test substance should be labeled as: may cause sensitization by skin contact (R 43).
- according to the ECETOC classification scheme for potency, the test substance would be regarded as weak skin sensitizer.

Executive summary:

Assessment for Contact Hypersensitivity to Sa 34 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 (2002),

EC, Council Directive 67/548/EEC, Annex V, B.42 (2004);

EPA, OPPTS 870.2600 (2003) "Skin Sensitisation".

Test substance concentrations selected for the main study were based on the results of a preliminary study.

In the main study, three groups of five experimental animals were treated with test substance concentrations of 25%, 50% or 100% 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, ail animals were injected with H-methyl

thymidine and after five hours the draining (auricular) Iymph nodes were excised.

After precipitating the DNA of the Iymph node ceils, 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.

A dose related increase in irritation of the ears was seen, from slight erythema in all animals at low dose up to well-defined erythema in all animals at high dose. No oedema was observed in any of the animals examined.

The majority of nodes were considered normal in size, except for the nodes of all animals treated at 100% that were enlarged.

Mean DPM/animal values for the experimental groups treated with substance concentrations 25, 50 and 100% were 676, 1102 and 3878 respectively. The mean DPM/animal value for the

vehicle control group was 323.

The SI values calculated for the substance concentrations 25, 50 and 100% were 2.1, 3.4 and 12.0 respectively.

These results indicate that the test substance could elicit an SI >= 3. The data showed a dose-response and an EC3 value of 42.3% was calculated.

This conclusion should be taken with care, since a dose related increase in irritation of the ears was seen. It is known that irritation might stimulate the nodes, causing a false positive outcome of the local Iymph node assay. Based on the laboratory experience, irritation might induce an SI up to approximately 4. The SI of 12.0 in this study was considered indicative for sensitisation.

Based on these results:

- according to the recommendations made in the test guidelines, the test substance would be regarded as skin sensitizer.

- according to the Globaily Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations (New York and Geneva, 2017), the test substance should be classified as skin sensitizer (Category 1B).

- according to the EC criteria for classification and labeling requirements for dangerous substances and preparations (Council Directive 67/548/EEC),

the test substance should be labeled as: may cause sensitization by skin contact (R 43).

- according to the ECETOC classification scheme for potency,

the test substance would be regarded as weak skin sensitizer.

Reference 2

Endpoint:

skin sensitisation, other

Remarks:

HRIPT

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

August - October 2007

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

according to guideline

Guideline:

other: HRIPT (Human repeated insult patch test)

GLP compliance:

yes

Type of study:

other: Patch Test

Positive control results:

not applicable

RESULTS

Erythema grade

Grade	Visible effect	Induction Phase (number of subjects: 108)	Challenge Phase (number of subjects: 106)
0	None	108	106
1	Faint redness, entire site might not be involved	0	0
2	Moderate redness, entire site involved	0	0
3	Intense redness	0	0
-	Number of subjects manifesting effects	0	0

Incidence of Sensitisation

Grade	Effect	Induction Phase (Presumptive effects)	Challenge Phase (Confirmed effects)
0 -3	No visible effect or effects not deemed indicative of a sensitised status	108	106
4	Weak positive	0	0
5	Strong positive	0	0
6	Extreme positive	0	0
-	Number of subjects sensitised	0	0

Incidence of miscellaneous effects

In regard to the test item, no other effects (edema, papules, vesicles, spreading, soap effect, fissuring, desquamation, dryness, pigmentation, folliculitis, tape reaction, crusting) were observed during the complete study.

In regard to Paraffin viscous (vehicle control), faint, spotty redness was detected on one subject on Thursday of Week#1.

In regard to Saline 0.9% (negative control), faint, spotty redness was detected on one subject on Thursday and Friday of Week #1.

SUMMARY

Induction Phase:

No adverse effects were detected on any of the one-hundred-and-eight (108) subjects concerning whom data were acquired.

1292 post-application assessments were conducted during this phase.

Challenge Phase:

No adverse effects were detected on any of the one-hundred-and-six (106) subjects concerning whom data were acquired.

424 post-application assessments were conducted during this phase.

Significance of the effects:

The data indicate that the test article may be considered innocuous in so far as it's capabilities to elicit the signs or symptoms of inflammation under the conditions of this patch test regimen.

FINDlNGS

The investigator met Sponsor's requirement to provide data on one hundred compliant subjects .

The data strongly support a finding that the test item, as tested, is not a skin irritant.

The data strongly support a finding that the test item is not a skin sensitizer.

Interpretation of results:

GHS criteria not met

Conclusions:

On the basis of the study's observations and the lack of any data obtained during the follow-up period to the contrary, the test item, as tested, was found to possess neither skin sensitizing nor skin irritating propensities.

Executive summary:

ABSTRACT

The test item, a pale yellow liquid, was received by Product Investigations on 24 August 2007. The sample was submitted by Henkel KGaA for a patch test to determine whether it possesses any skin-irritating and/or sensitizing propensities. Also submitted was a sample of Paraffin viscous to serve as a vehicle control. At the request of Sponsor, the investigator provided sterile physiological saline solution to serve as a negative control.

To accomplish this, Product Investigations initiated a repeated insult patch test of the study and control articles. The regimen for each article was identical and conducted concurrently on the same study population consisting of one-hundred-and-nine (109) adult volunteers at the outset. The study was conducted in three phases, an Induction Phase of three weeks, an Intermediate/Rest Phase of two weeks, and a Challenge Phase of one week.

The regimen comprised four (4) consecutive 24-hour application/assessment cycles conducted during Weeks #1, #2, and #3. Such cycles were initiated on Monday, Tuesday, Wednesday, and Thursday of Weeks #1 and #3; and on Tuesday, Wednesday, Thursday, and Friday of Week #2, the clinic being closed on Monday of that week because of the Labor Day Holiday.

The procedure for preparing and applying a patching device was as follows: a technician delivered 40 µL of the study article evenly over the surface of the 2cm x 2cm non-woven fabric pad of an occlusive, stand-alone patching device; the technician applied the prepared device on the upper portion of the left side of a presenting subject's back. For all applications, save #8, the technician rernoved the device in the clinic after it had been in situ for approximately twenty-four hours. Five to ten minutes after she had removed the device, the technician assigned a grade in accordance with her assessment of the magnitude of the effect being manifested by the skin. The device constituting Application #8 was applied on Friday of Week #2 and removed on Saturday by the subject or helper at home. When the subject returned to the clinic on Monday of Week #3, the effect being manifested by the skin at that time was assessed and graded by a technician. The assessment and grading of effects on Friday of Week #3 marked the end of the Induction Phase for the one-hundred-and-two (102) subjects who had undergone all twelve application/assessment cycles and for whom further surveillance was not indicated.

Starting on Monday ofWeek #4, applications were continued on six subjects to make up for applications missed because of absences during the scheduled regimen.

After the effects of Application #12 were assessed and graded, a subject was given a recess until Monday, 1 October 2007.

The Challenge Phase regimen comprised four (4) consecutive 24-hour application/assessment cycles conducted on the naive skin of the study article's designated challenge site on the upper portion of the left side of each presenting subject's back.

Data were acquired on one-hundred-and-eight (108) subjects during the Initial/Induction Phase.

No data were acquired concerning the one subject who failed to return after receiving the initial application.

In regard to the test item, no adverse effects were detected on any of the one-hundred-and-eight (108) subjects during the Initial/lnduction Phase.

In regard to Paraffin viscous (vehicle control), faint, spotty redness was detected on one subject on Thursday of Week#1.

In regard to Saline 0.9% (negative control), faint, spotty redness was detected on one subject on Thursday and Friday of Week #1.

Data were acquired on one-hundred-and-six (106) subjects during the Challenge Phase. No data were acquired concerning three subjects who failed to return after the hiatus.

No adverse effects attributable to any of the articles under study were detected on any of the one-hundred-and-six (106) subjects conceming whorn data were acquired during the Challenge Phase.

On the basis of the above-cited observations and the lack of any data obtained during the follow-up period to the contrary, the test item, as tested, was found to possess neither skin sensitizing nor skin irritating propensities.

The investigator concludes that the data do not contraindicate uses entailing repeated applications of the test item under conditions comrmensurate with those that prevailed during the course of this study.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The test item was found to be a skin sensitiser in the LLNA assay and an EC3 value of 42.3 % was derived. The HRIPT test in humans was performed with a concentration of 0.1 % of the test item, which is to low to do the assumption that the substance could not be skin sensitising.  

According to the Regulation (EC) No 1272/2008 on classification, labeling and packaging (CLP) of substances and mixtures, the test item should be classified as a weak skin sensitiser (Category 1B).