Tall oil, reaction products with diethylenetriamine

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 Feb 2007 - 28 Feb 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

This study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Cross-reference

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

It is considered appropriate to address the data requirements for DTO_DETA by read-across to the available studies on the main components of DTO_DETA: AAI_DETA and Rosin.
DTO_DETA contains comparatively lower levels of imidazolines and higher levels of resin acids than AAI_DETA and therefore consideration of data for resin acids is also considered necessary. The main resin acid in DTO_DETA is abietic acid, but abietic acid derivatives and other acids, such as pimaric acid, are also found in notable quantities, and the resin acids collectively are known as ‘rosin’. DTO_DETA contains up to 25% unreacted rosin, and taking into account the compositional information available for the rosin in DTO_DETA and Rosin (CAS# 8050-09-07, EC# 232-475-7), the latter was considered appropriate for read-across to DTO_DETA.
An LLNA (OECD 429) is available for AAI_DETA (CIT, 2007), the main component in DTO_DETA. Based on the results of this study, AAI_DETA was classified as a Category 1A Skin Sensitiser. A guideline guinea pig maximisation test (NOTOX, 2010) is also available with the read across substance AAI_DETA; this study confirms the result obtained in the LLNA. As AAI_DETA is a major component in DTO_DETA, read across to the AAI_DETA dataset is justified and presents the worst case approach. Several negative studies (LLNAs, guinea pig maximisation tests and a Buehler assay) conducted with Rosins are provided as supporting information; these studies are provided to demonstrate that the Rosin component of DTO_DETA will not significantly increase the hazard profile of DTO_DETA with respect to skin sensitisation. A positive study conducted with Wood Rosin (CTL, 1997g) is available and is included in the dossier for information purposes, however the study was disregarded because the test substance sample was heavily oxidised and degraded.

Reason / purpose for cross-reference:

read-across source

Key result

Parameter:

EC3

Value:

0.3

Parameter:

SI

Value:

1

Test group / Remarks:

0.05% test item

Parameter:

SI

Value:

1.88

Test group / Remarks:

0.1% test item

Parameter:

SI

Value:

1.67

Test group / Remarks:

0.25% test item

Parameter:

SI

Value:

5.91

Test group / Remarks:

0.5% test item

Parameter:

SI

Value:

18.65

Test group / Remarks:

1% test item

Interpretation of results:

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

Conclusions:

Based on the results of the read across study, DTO_DETA is considered to be skin sensitiser. Classification according to CLP is required (Cat 1A Skin Sensitiser).

Executive summary:

A guideline (OECD 429) LLNA (CIT, 2007) is available for the read across (source) substance, AAI_DETA. Read across from AAI_DETA to the target, DTO_DETA, is considered appropriate because AAI_DETA is a major component of DTO_DETA. AAI_DETA was found to be to a strong skin sensitiser. Therefore, based on the results of this study, DTO_DETA should be classified as a Category 1A Skin Sensitiser according to the CLP Regulation. Several studies (LLNAs, guinea pig maximisation tests and a Buehler assay) are available for the Rosin data set, and are presented as supporting read across information to demonstrate that the Rosin component of DTO_DETA will not significantly increase the hazard profile of DTO_DETA with respect to skin sensitisation.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (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:

Fatty acids, C18 unsat, reaction products with diethylenetriamine

EC Number:

629-715-1

Cas Number:

1226892-43-8

Molecular formula:

UVCB substance - not applicable

IUPAC Name:

Fatty acids, C18 unsat, reaction products with diethylenetriamine

Specific details on test material used for the study:

Certificate of analysis included in report.
description: Fatty acids, C12-18 and C18 insaturated, reaction products with diethylenetriamine.
CASno:: 91001-82-0
Purity: 100%
Ration Imidazoline/amide: 50.24% imidazoline
Origin: CECA - Feuchy
Batch: pilote du 27/07/06
Arkema filing no: GRL 0055/06
Aspect: thick clear brown liquid
density: approx. 950 kg/m3 at 25°C
Expiry date: July 2007

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: approximately 9 weeks
- Weight at study initiation: mean body weight ± standard deviation of 20.9 ± 1.0 g.
- Housing: individually in disposable crystal polystyrene cages (22.00 cm x 8.50 cm x 8.00 cm). Each cage contained autoclaved sawdust (SICSA, Alfortville, France)
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 30 to 70%,
- Air changes (per hr): 12 cycles/hour of filtered, non-recycled air.
- Photoperiod (hrs dark / hrs light): 12 h/12 h,

IN-LIFE DATES: From: 20 Feb 2007 To: 28 Feb 2007

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

0, 0.05, 0.1, 0.25 and 1 % (v/v)

No. of animals per dose:

4 females/dose

Details on study design:

RANGE FINDING TESTS:
- Irritation: Erythema and crusts were noted on ears of all animals given the concentrations ≥ 2.5%. A significant increase in ear thickness was recorded at concentrations ≥ 2.5%, showing the irritant potential of the test item at these concentrations. The highest concentration retained for the main test was therefore 1%.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method:
- Criteria used to consider a positive response:

TREATMENT PREPARATION AND ADMINISTRATION:

Positive control substance(s):

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

Statistics:

SI = dpm of treated group / dpm of control group
skin sensitizer when the SI for a dose group is ≥ 3.
EC3 derived by linear interpolation of points on the dose-response curve, immediately above and below the 3-fold threshold.

Results and discussion

Positive control results:

HCA 25%, SI = 14.38. The study was therefore considered valid.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

EC3

Value:

0.3

Results 2

Parameter:

SI

Value:

1

Test group / Remarks:

0.05% test item

Results 3

Parameter:

SI

Value:

1.88

Test group / Remarks:

0.1% test item

Results 4

Parameter:

SI

Value:

1.67

Test group / Remarks:

0.25% test item

Results 5

Parameter:

SI

Value:

5.91

Test group / Remarks:

0.5% test item

Results 6

Parameter:

SI

Value:

18.65

Test group / Remarks:

1% test item

Any other information on results incl. tables

No mortality and no clinical signs were observed during the study.

The body weight change of treated animals was similar to that of control animals.

Local irritation:

A dryness of the skin of the ears was noted on day 6 in animals given the concentrations of 0.5 and 1%.

Increases in ear thickness were noted at concentrations of 0.5% (19.19% increase) and 1% (30.10% increase).

Proliferation assay:

The quantity of cells obtained in each group was satisfactory and the cellularity correlated with incorporation of 3H-TdR. The cell viability was higher than 70% in each group.

In the positive control group given HCA at the concentration of 25%, a moderate increase in cellularity and a stimulation index exceeding the threshold value of 3 (SI = 14.38) were noted. The study was therefore considered valid.

A dose-related increase in the SI was recorded and the threshold positive value of 3 was exceeded at 0.5%.

In the absence of an excessive local irritation at the concentration of 0.5% (increase in ear thickness < 30%), the significant lymphoproliferative response observed was attributed to delayed contact hypersensitivity.

The EC3 value for the test item IMIDAZOLINE 4900 is equal to 0.3%.

Study results

Group	Treatment concentration	Cell count		viable %	amount of cells (x 10^6)	Cellularity index	Number of nodes per group	dpm per group	dpm per node	Stimulation Index (SI)	Increase in ear thickness (% between day 1 and day 6)	Irritation level	EC3 value (%)
		viable	dead
1	Vehicle	42	8	84.00	4.20		8	375.97	47.00		1.98
2	Test Item 0.05%	46	9	83.64	4.60	1.10	8	375.95	47.12	1.00	0.98	I
3	Test Item 0.1%	66	10	86.84	6.60	1.57	8	707.84	88.48	1.88	1.00	I	0.3
4	Test Item 0.25%	67	8	89.33	6.70	1.60	8	629.60	78.70	1.67	8.16	I
5	Test Item 0.5%	154	40	79.38	15.40	3.67	8	2221.13	277.64	5.91	19.19	II
6	Test Item 1%	258	52	83.23	25.80	6.14	8	7012.20	876.53	18.65	30.10	III
7	HCA 0.05%	250	32	88.65	25.00	5.95	8	5406.58	675.82	14.38

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

Test item should be considered as a strong sensitizer. CLP skin sensitiser Cat. 1A

Executive summary:

The aim of this study was to evaluate the potential of the test item IMIDAZOLINE 4900 (batch No. pilote du 27/07/06) to induce delayed contact hypersensitivity using the murine Local Lymph Node Assay (LLNA). Evaluation of local irritation was also carried out in parallel.

This study was conducted in compliance with the principles of Good Laboratory Practice Regulations.

 

Methods

A preliminary test was first performed in order to define the concentrations of test item to be used in the main test.

In the main test, twenty-eight female CBA/J mice were allocated to seven groups:

- five treated groups of four animals receiving the test item IMIDAZOLINE 4900 at the concentration of 0.05, 0.1, 0.25, 0.5 or 1%,

- one negative control group of four animals receiving the vehicle (mixture acetone/olive oil (4/1, v/v)),

- one positive control group of four animals receiving the reference item,α-hexylcinnamaldehyde (HCA), a moderate sensitizer, at the concentration of 25%.

During the induction phase, the test item, vehicle or reference item was applied over the ears (25 μL per ear) for 3 consecutive days (days 1, 2 and 3). After 2 days of resting, the proliferation of lymphocytes in the lymph node draining the application site was measured by incorporation of tritiated methyl thymidine (day 6). The obtained values were used to calculate stimulation indices (SI).

 

The irritant potential of the test item was assessed in parallel by measurement of ear thickness on days 1, 2, 3 and 6.

 

Results

The test item was not soluble in any of the five recommended vehicles, consequently, an homogenous suspension, obtained in acetone/olive oil (4/1, v/v), was used for the treatment.

The maximum practicable concentration in this vehicle was 50%. Consequently, the concentrations selected for the first preliminary test were 10, 25, 50 and 100%.

Since the test item was irritant at all these concentrations, a complementary preliminary test was undertaken at lower concentrations: 0.5, 1, 2.5 and 5%.

Since the test item was irritant in the complementary preliminary test at the concentrations of 2.5 and 5%, the highest concentration retained for the main test was 1%.

 

Systemic clinical signs and mortality

No mortality and no clinical signs were observed during the study.

 

Local irritation

A dryness of the skin of the ears was noted on day 6 in animals given the concentrations of 0.5 and 1%.

Increases in ear thickness were noted at concentrations of 0.5% (19.19% increase) and 1% (30.10% increase).

Proliferation assay

A significant lymphoproliferation was noted in the positive control group given HCA, the study was therefore considered as valid.

A dose-related increase in the SI was recorded and the threshold positive value of 3 was exceeded at 0.5%.

The results are presented in the following table:

Treatment	Concentration (%)	Irritation level	Stimulation Index (SI)
Test item	0.05	non-irritant	1
Test item	0.1	non-irritant	1.88
Test item	0.25	non-irritant	1.67
Test item	0.5	slightly-irritant	5.91
Test item	1	irritant	18.65
HCA	25	-	14.38

   

In the absence of an excessive local irritation at 0.5% (increase in ear thickness < 30%), the significant lymphoproliferative response observed at this concentration was attributed to delayed contact hypersensitivity.

The EC3value for the test item IMIDAZOLINE 4900 is equal to 0.3%.

 

Conclusion

Under our experimental conditions, the test item IMIDAZOLINE 4900 (batch No. pilote du 27/07/06) induced delayed contact hypersensitivity in the murine Local Lymph Node Assay.

According to the EC3 value obtained in this experiment, the test item IMIDAZOLINE 4900 should be considered as a strong sensitizer.

These results lead to classification according to CLP (ATP 2): skin sensitiser Cat.1A (EC 3 ≤ 2%)