Reaction products of amines, dicoco alkyl and glycollic acid

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2006-03-29 to 2006-08-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP, Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

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:

EPA OPPTS 870.2600 (Skin Sensitisation)

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Details on test material:

- Substance type: amber liquid
- Analytical purity: Stable in accordance with MSDS
- Storage condition of test material: room temperature and humidity

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: The Jackson Laboratory, Bar Harbor, Maine, USA
- Age at study initiation: 10 weeks
- Weight at study initiation: 16 to 22 g
- Housing: 1 per cage, suspended wire cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): temperature controlled
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle

IN-LIFE DATES: From: 2006-03-29 To: 2006-04-07

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

1%, 2.5%, 5%, 10%, or 25% v/v in vehicle

No. of animals per dose:

5 per dose.

Details on study design:

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay in the Mouse. The assay has undergone extensive inter-laboratory validation and has been shown to reliably detect test materials that are moderate to strong sensitisers.
- Criteria used to consider a positive response: For each animal, the LNC suspension was analyzed for BrdU incorporation and total number of LNC by flow
cytometry. The amount of proliferating (#BrdU+) LNC was determined as a measure of the proliferative response of the local lymph node. The stimulation index (SI) was calculated by dividing the proliferative response (BrdU incorporation) of each test article group by the proliferative response of the vehicle
control group. Test articles that yielded a SI ≥ 3 were characterized as sensitizing substances.


TREATMENT PREPARATION AND ADMINISTRATION: Groups of 5 mice were treated with increasing concentrations of 1%, 2.5%, 5%, 10% and 25% v/v in acetone/olive oil 4:1. 25 µL of test material to dorsal surface of each ear for three consecutive days. Admisistered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette. Further groups of five mice received either the vehicle alone or the positive control (25% HCA in the vehicle) in the same manner. All animals were observed once daily throughout the study and any siigns of toxicity or ill health recorded. Bodyweights were recorded on Day 1 (prior to dosing) and on Day 6. Ear measurements were taken from all animals and recorded on Days 1, 3 and 6.

Five days following the initial dose, and five hours prior to sacrifice, the mice were given an intraperitoneal injection of the thymidine analog 5-bromo-2’-deoxy-uridine (BrdU), and at sacrifice the auricular lymph nodes were isolated and single-cell suspensions of lymph node cells (LNC) were generated.
The concentration at which SI = 3 (EC3) was calculated for each test article.

Positive control substance(s):

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

Statistics:

Eliminating outliers: One value, from the 5% OS 21 5085 group, was identified as an outlier by the statistical Q-test’ for rejection of data points from a sample group. This animal exhibited an uncharacteristic (out of historical range) lymph node cell number and %BrdU+ counts. This may be due to a pre-existing viral or bacterial infection, or other physiological or immune system aberration. Inclusion or rejection of the outlier value did not alter the conclusion or the prediction of sensitizing potential.

Results and discussion

Positive control results:

25% HCA: SI = 12.0 (S.D. = ±3.7)

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

8.5

Variability:

3.6

Test group / Remarks:

25%

Results 2

Key result

Parameter:

SI

Value:

3.3

Variability:

1.8

Test group / Remarks:

10%

Results 3

Key result

Parameter:

SI

Value:

1

Variability:

0.4

Test group / Remarks:

5%

Results 4

Key result

Parameter:

SI

Value:

2.3

Variability:

1.6

Test group / Remarks:

2.5%

Results 5

Key result

Parameter:

SI

Value:

1.3

Variability:

0.5

Test group / Remarks:

1%

Any other information on results incl. tables

The EC3 value for test article could be determined because all test article concentrations below 5% had a SI ‘3, and all test article concentrations above 10% had SI values 23. The dose response was calculated to cross the threshold (SI=3) at 9.4%

Table 1: Stimulation Index

Treatment	SI	S.D
Acetone/olive oil 4:1	1.0	(± 0.4)
25% HCA (+ve control)	^12.0a	(± 3.7)
1%	^1.3a	(± 0.5)
2.5%	^2.3	(± 1.6)
5%	1.0	(±0.4)
10%	^3.3a	(± 1.8)
25%	^8.5a	(± 3.6)

a: A SI≥3 indicates a sensitising response. The SI was calculated by dividing the mean number of proliferating lymph node cells (#BrdU+) from each treatment group by the mean number of proliferating cells from the vehicle group.

All animals survived the in-life phase of the study and appeared normal.

Body weight changes were normal. Ear swelling measurements and individual animal observations indicated that dermal irritation occurred at the 25% treatment.

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Topical application of the test article resulted in dermal (ear) irritation at the highest dose tested, 25%. In addition, at concentrations between 10% and 25% of the test article, Stimulation Index values were greater than 3 (SI > 3.0), and therefore this test article is a dermal sensitizer in the Local Lymph Node Assay. The EC3 value for the test article was calculated to be 9.4%.

Executive summary:

Test Guidance

OECD 429 and US EPA OPPTS 870.2600

Method and materials

For the definitive study, ten separate groups of five healthy female CBNJ mice were treated with increasing concentrations of the test article by topical application to the dorsum of each ear, once daily for three consecutive days. A vehicle control group of five mice was treated with Acetone/Olive Oil (4:l) (AOO) and another group of five mice were treated with the positive control, 25%

HCA (in AOO), in the exact same manner.

Five days following the initial dose, and five hours prior to sacrifice, the mice were given an intraperitoneal injection of the thymidine analog 5-bromo-2’-deoxy-uridine (BrdU), and at sacrifice the auricular lymph nodes were isolated and single-cell suspensions of lymph node cells (LNC) were generated. For each animal, the LNC suspension was analyzed for BrdU incorporation and total number of LNC by flow cytometry. The amount of proliferating (#BrdU+) LNC was determined as a measure of the proliferative

response of the local lymph node. The stimulation index (SI) was calculated by dividing the proliferative response (BrdU incorporation) of each test artrcle group by the proliferative response of the vehicle control group. Test articles that yielded a SI 2 3 were characterized as sensitizing substances.

The concentration at which SI = 3 (EC3) was calculated for each test article.

Results

All animals survived the in-life phase of the study and appeared normal. There were no difficulties in administration of the test article or with its adherence to the dosed ears. For all animals, body weight changes were normal. Ear swelling measurements

and individual animal observations indicated that the 25% treatment for the test article resulted in dermal irritation.

The SI of the positive control, 25% HCA, was 12.0, consistent with historical data.

The SI values for the test article at 1%, 2.5,% , 5%, 10% and 25% were 1.3, 2.3, 1.0, 3.3 and 8.5, respectively.

Conclusions

Topical application of the test article resulted in dermal (ear) irritation at the highest dose tested, 25%. In addition, at concentrations between 10% and 25% of the test article, Stimulation Index values were greater than 3 (SI > 3.0), and therefore this test article is a dermal sensitizer in the Local Lymph Node Assay. The EC3 value for thes test article was calculated to be 9.4%.