Reaction products of 4,4'-methylenebis(2,6-dimethylaniline) and 4-aminobenzenesulfonic acid with 2,4,6-trichloro-1,3,5-triazine, diazotised and subsequently coupled with N-(2-methoxyphenyl)-3-oxobutanamide

Endpoint summary

Description of key information

Skin Sensitisation Key Study: Wang-Fan (2006)

Under the conditions of the study an EC3 value of 4.8 % was derived and the test material was therefore found to be a skin sensitiser.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 February 2006 to 09 March 2006

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

2003

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA/Ca

Remarks:

CBA/CaHsdRcc(SPF)

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: 8 - 12 weeks (beginning of acclimatisation).
- Weight at study initiation: 16 g - 24 g (ordered).
- Housing: Individual in Makrolon type-2 cages with standard softwood bedding.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: Under test conditions after health examination.
- Indication of any skin lesions: Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): Relative humidity 30 - 70 %
- Air changes (per hr): 10 - 15 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 h light and 12 h darkness

Vehicle:

dimethylformamide

Concentration:

5 % was the highest technically achievable concentration in the chosen vehicle. Concentration selection was based on results of a non-GLP local toxicity pre-test

No. of animals per dose:

Four

Details on study design:

PRE-SCREEN TESTS:
- In non-GLP solubility pre-test, the test material was tested in different vehicles: acetone:olive oil (4:1 v/v), ethanol:water (7:3 v/v) and DMF. DMF was found to be a suitable vehicle and was selected and used in the main test. 5 % was the highest technically applicable concentration in the chosen vehicle.
- A non-GLP local toxicity pre-test was performed for determination of concentrations for the main test. Three single animals were each treated with one of three different concentrations: 1 %, 2.5 % and 5 %, in both ears on three consecutive days. One day after the second and the third topical application, the residual test material was found at all the dosing sites. 5 % was the highest dosing concentration selected for the main tests.

MAIN STUDY
TOPICAL APPLICATI ON:
- Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with the test material at concentrations of 1 %, 2.5 % and 5 % in DMF. The application volume (25 µL) was spread over the entire dorsal surface (Ø ~ 8 mm) of each ear lobe once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals).

ADMINISTRATION OF ³HTDR:
- Five days after the first topical application, all mice were administered with 250 µL of PBS containing 77.22 µCi/mL 3HTdR (equal to 19.3 µCi ³HTdR) by intravenous injection via a tail vein.

DETERMINATION OF INCORPORATED ³HTDR:
Approximately five hours after treatment with ³HTdR all mice were euthanized by inhalation of CO2 (dry ice).
The draining lymph nodes were rapidly excised and pooled for each experimental group (8 nodes per group). Single cell suspensions (phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 µm mesh size). After washing twice with phosphate buffered saline (approx. 10 mL) the lymph node cells were re-suspended in 5 % trichloroacetic acid (approx. 3 mL) and incubated at approximately 4 °C for at least 18 hours for precipitation of macromolecules. The precipitates were then re-suspended in 5 % trichloroacetic acid (1 mL) and transferred to glass scintillation vials with 10 mL of 'lrga-Safe Plus' scintillation liquid and thoroughly mixed.
The level of ³HTdR incorporation was then measured on a ß-scintillation counter. Similarly, background ³HTdR levels were also measured in two 1 mL aliquots of 5 % trichloroacetic acid. The ß-scintillation counter expresses ³HTdR incorporation as the number of radioactive disintegrations per minute (dpm).

OBSERVATIONS
In addition to the sensitising reactions the following observations and data were recorded during the test and observation period:
- Mortality / viability: Once daily from acclimatisation start to the termination of in-life phase.
- Body weights: On the test day 1 (prior to the 1st application) and on the test day 6.
- Clinical signs (local / systemic): Once daily on the day of animals delivery and from test day 1 (after the 1st application) to the termination of in-life phase.
- Size of the draining lymph nodes: Immediately after the excision.

ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: The proliferative response of lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (dpm/node) and as the ratio of ³HTdR incorporated into lymph node cells of test group relative to that recorded for control group (S.I.). Before dpm/node values were determined, mean scintillation-background dpm was subtracted from test and control raw data.
A test material is regarded as a sensitiser in the LLNA if the following criteria are fulfilled:
First, exposure to at least one concentration of the test material resulted in an incorporation of ³HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the S.I.
Second, the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

TREATMENT PREPARATION AND ADMINISTRATION:
The test material was placed into a volumetric flask on a tared Mettler balance and the vehicle DMF was quantitatively added. The weight/volume (w/v) dilutions were prepared individually using a magnetic stirrer as homogeniser.
Test material formulations were made freshly before each dosing occasion and no more than 4 h prior to application to the ears.
Homogeneity of the test material in the vehicle was maintained until start of treatment using an appropriate homogeniser.
The test material in the main study was assayed at three consecutive. The top dose was the highest technically applicable concentration in the chosen vehicle.
Concentrations were in terms of material as supplied.

Positive control substance(s):

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

Statistics:

The mean values and standard deviations were calculated in the body weight tables.
A statistical analysis was conducted for assessment of the dose-response relationship, and the EC3 value was calculated according to the equation:

EC3 = (a-c) [(3-d)/(b-d)] + c

Where EC3 is the estimated concentration of the test material required to produce a 3-fold increase in draining lymph node cell proliferative activity; (a, b) and (c, d) are respectively the co-ordinates of the two pair of data lying immediately below and above the S.I. value of 3 on the local lymph node assay dose response plot.

Positive control results:

In order to study a possible contact allergenic potential of αhexylcinnamaldehyde, three groups each of four female mice were treated daily with the test material at concentrations of 5 %, 10 % and 25 % in acetone:olive oil, 4:1 (v/v) by topical application to the dorsum of each ear lobe (left and right) for three consecutive days. A control group of four mice was treated with the vehicle acetone:olive oil, 4:1 (v/v) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (³H methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of ³H-methylthymidine measured in a (ß-scintillation counter).
All treated animals survived the scheduled study period.
Neither clinical / local signs nor other findings were observed in any animals of the control group. On the second application day, slight to severe ear swelling was observed at both dosing sites in all mice of Groups 2 - 4, persisting for a total of four days or for the remainder of the in-life phase of the study, respectively. In addition, on the second application day, slight to severe ear erythema was also noted at both dosing sites in all mice of Groups 3 - 4, persisting for a total of four days or for the remainder of the in-life phase of the study, respectively.
The estimated concentration of test material required to produce a S.I. of 3 is referred to as the EC3 value.
5 % w/v test material SI = 1.8
10 % w/v test material SI = 2.9*
25 % w/v test material SI = 6.2*
* This value was used in calculation of EC3.
EC 3 = 10.5 %
A clear dose-response relationship was observed.

Parameter:

SI

Value:

1.2

Test group / Remarks:

1 % w/v test material

Parameter:

SI

Value:

1.8

Test group / Remarks:

2.5 % w/v test material

Parameter:

SI

Value:

3.1

Test group / Remarks:

5 % w/v test material

Parameter:

EC3

Value:

4.8

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
- The proliferative capacity of pooled lymph node cells was determined by the incorporation of ³HTdR measured on a ß-scintillation counter.

DETAILS ON STIMULATION INDEX CALCULATION
- Group 2 (1 % w/v test material): SI = 1.2
- Group 3 (2.5 % w/v test material): SI = 1.8
- Group 4 (5 % w/v test material): SI = 3.1

EC3 CALCULATION
- The values from Groups 3 and 4 were used in calculation of the EC3.
- The EC3 = 4.8 %.
- A dose-response relationship was observed.

CLINICAL OBSERVATIONS:
- No deaths occurred during the study period.
- Neither clinical / local signs nor other findings were observed in any animals of the control group. One day after the first topical application, slight to moderate ear erythema was observed at both dosing sites in all mice of Group 4 (5 %), persisting for a total of three days. On the third application day or after the first application, the residual test material was found at all the dosing sites in all mice of Group 2 (1 %), Group 3 (2.5 %) and Group 4 (5 %), persisting for the remainder of the in-life phase of the study.
- No findings were observed on the size of the draining lymph nodes.

BODY WEIGHTS
- The body weight of the animals, recorded prior to the first application and prior to necropsy, was within the range commonly recorded for animals of the strain and age.

Calculation of Results

Test material concentration % (w/v)		Measurement dpm	Calculation			Result
			dpm – BG*	Number of lymph nodes	Dpm per lymph node**	S.I
-	BG1	28	-	-	-	-
-	BG2	33	-	-	-	-
-	CG1	5247	5216	8	652	-
1	TG1	6512	6481	8	810	1.2
2.5	TG2	9317	9286	8	1161	1.8
5	TG3	16032	16001	8	2000	3.1

BG: Background (1 mL 5 % trichloroacetic acid) in duplicate.

CG: Control group

TG: Test group

S.I.: Simulation Index

* The mean BG was calculated from BG 1 and BG 2

** Since the lymph nodes of the animals of a dose group were pooled, dpm/node was determined by dividing the measured value by the number of lymph nodes pooled.

 

	Test material concentration % (w/v)	S.I.
Group 3	2.5 (a)	1.8 (b)
Group 4	5 (c)	3.1 (d)
EC 3 = (a - c) [(3 - d) / (b - c)] + c = 4.8 %

EC3 = Estimated concentration for a simulation index of 3

A, b, c, d = Coordinates of the two pair of data lying immediately below and above the S.I. value of 3 on the LLNA dose response plot.

Interpretation of results:

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

Conclusions:

Under the conditions of the study an EC3 value of 4.8 % was derived and the test material was therefore found to be a skin sensitiser.

Executive summary:

In order to study a possible contact allergenic potential of the test material a study was conducted according to OECD Test Guideline 429 and EU Method B.42 using a LLNA method in compliance with GLP.

During the study, three groups each of four female mice were treated daily with the test material at concentrations of 1 %, 2.5 % and 5 % in N,N-Dimethylformamide (DMF) by topical application to the dorsum of each ear lobe (left and right) for three consecutive days. 5 % was the highest technically achievable concentration in the chosen vehicle. A control group of four mice was treated with the vehicle DMF only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (³H-methyl thymidine, ³HTdR). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of ³HTdR measured in a ẞ-scintillation counter.

All treated animals survived the scheduled study period.

Neither clinical / local signs nor other findings were observed in any animals of the control group. One day after the first topical application, slight to moderate ear erythema was observed at both dosing sites in all mice of Group 4 (5 %), persisting for a total of three days. On the third application day or after the first application, the residual test material was found at all the dosing sites in all mice of Group 2 (1 %), Group 3 (2.5 %) and Group 4 (5 %), persisting for the remainder of the in-life phase of the study.

The estimated concentration of test material required to produce a S.I. of 3 is referred to as the EC3 value.

Group 2 (1 % w/v test material): SI = 1.2

Group 3 (2.5 % w/v test material): SI = 1.8

Group 4 (5 % w/v test material): SI = 3.1

The values from Groups 3 and 4 were used in calculation of the EC3.

The EC3 = 4.8 %. A dose-response relationship was observed.

A test material is regarded as a sensitiser in the LLNA if the exposure to one or more test concentrations resulted in 3-fold or greater increase in incorporation of ³HTdR compared with concurrent controls, as indicated by the S.I.

In this study S.I. of 1.2, 1.8 and 3.1 were determined with the test material at concentrations of 1 %, 2.5 % and 5 %, respectively, in DMF.

Under the conditions of the study an EC3 value of 4.8 % was derived and the test material was therefore found to be a skin sensitiser.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

In order to study a possible contact allergenic potential of the test material a study was conducted according to OECD Test Guideline 429 and EU Method B.42 using a LLNA method in compliance with GLP. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

During the study, three groups each of four female mice were treated daily with the test material at concentrations of 1 %, 2.5 % and 5 % in N,N-Dimethylformamide (DMF) by topical application to the dorsum of each ear lobe (left and right) for three consecutive days. 5 % was the highest technically achievable concentration in the chosen vehicle. A control group of four mice was treated with the vehicle DMF only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (³H-methyl thymidine, ³HTdR). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of ³HTdR measured in a ẞ-scintillation counter.

All treated animals survived the scheduled study period.

Neither clinical / local signs nor other findings were observed in any animals of the control group. One day after the first topical application, slight to moderate ear erythema was observed at both dosing sites in all mice of Group 4 (5 %), persisting for a total of three days. On the third application day or after the first application, the residual test material was found at all the dosing sites in all mice of Group 2 (1 %), Group 3 (2.5 %) and Group 4 (5 %), persisting for the remainder of the in-life phase of the study.

The estimated concentration of test material required to produce a S.I. of 3 is referred to as the EC3 value.

Group 2 (1 % w/v test material): SI = 1.2

Group 3 (2.5 % w/v test material): SI = 1.8

Group 4 (5 % w/v test material): SI = 3.1

The values from Groups 3 and 4 were used in calculation of the EC3.

The EC3 = 4.8 %. A dose-response relationship was observed.

A test material is regarded as a sensitiser in the LLNA if the exposure to one or more test concentrations resulted in 3-fold or greater increase in incorporation of ³HTdR compared with concurrent controls, as indicated by the S.I.

In this study S.I. of 1.2, 1.8 and 3.1 were determined with the test material at concentrations of 1 %, 2.5 % and 5 %, respectively, in DMF.

Under the conditions of the study an EC3 value of 4.8 % was derived and the test material was therefore found to be a skin sensitiser.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance requires classification with respect to skin sensitisation.