Disodium 4,4'-bis[[4-anilino-6-[(2-carbamoylethyl)(2-hydroxyethyl)amino]-1,3,5,-triazin-2-yl]amino]stilbene-2,2'-disulphonate

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From December 21, 1988 to January 14, 1989.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The test procedures are well documented and scientifically acceptable.

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Principles of method if other than guideline:

Intradermal injection and topical skin application (Magnusson-Kligman), protocol P-252.

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

The test was performed before the validation of LLNA OECD method and is considered to be valid and acceptable for the assessment.

Species:

guinea pig

Strain:

Hartley

Sex:

male

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Davidson's Mill Farm, NJ.
- Number of animals: 15.
-Weight: 250-320 g.
- Acclimation period: 9 days.
- Housing: in cage, five per suspended stainless steel gang cages with mesh floors.
- Diet: ad libitum, Purina Guinea Pig.
- Water: ad libitum.
- Identification: ear tag bearing, with an unique identification number.

ENVIRONMENTAL CONDITIONS
- Temperature: 65-74 F.

Route:

intradermal and epicutaneous

Vehicle:

other: water and Freunds Complete Adjuvant

Concentration / amount:

A. INDUCTION EXPOSURE
0.05 ml in water (5 %).
0.05 % in 50 % aqueous Freunds Complete Adjuvant.
0.05 % in 50 % aqueous Freunds Complete Adjuvant at multiple site.

B. CHALLENGE
0.1 ml.

Route:

epicutaneous, open

Vehicle:

other: water and Freunds Complete Adjuvant

Concentration / amount:

A. INDUCTION EXPOSURE
0.05 ml in water (5 %).
0.05 % in 50 % aqueous Freunds Complete Adjuvant.
0.05 % in 50 % aqueous Freunds Complete Adjuvant at multiple site.

B. CHALLENGE
0.1 ml.

No. of animals per dose:

10 animals.

Details on study design:

SITE
A. INDUCTION EXPOSURE
Intradermal injection
- Test site: anterior left and right shoulder.
- Frequency of application: single application.
- Type of application: intradermal injection.
- Date of application: December 21, 1988.

Topical Induction (Booster)
- Test site: anterior left and right shoulder.
- Frequency of application: 7 days after first application on shoulders, by Hilltop Chamber.
- Type of application: intradermal injection.
- Date of the first topical application: December 28, 1988.
- Exposure period: 48 hours for the first application.

B. CHALLENGE
- Test site: shaved flanks.
- Frequency of application: 14 days after the induction.
- Type of application: topical application by Hilltop Chamber.
- Date of the challenge dose: January 11, 1989.
- Exposure period: 24 hours for the second application.

Challenge controls:

Evaluation:
- at 72 and 72 hours after the initial topical application;
- at 48 and 72 hours after challenge.

SCORING:
No evidence of any effect: 0
(Barely Perceptible) - minimal faint (light pink) uniform or spotty erythema: 0.5
(Mild) - Pink uniform erythema covering most of contact site: 1
(Moderate) - Pink-red erythema uniform in entire contact area: 2
(Marked) - Bright red erythema with accompanying edema, petachiae or papules: 3
(Severe) - Deep red erythema with vesiculation or weeping, with or without edema: 4

The sensitization potential (Sensitization Rate) of the test product was determined by determining the ratio of positive responses with scores greater than 0.5, 48 hours after patch removal to the total number of animals tested.
The degree of sensitization (erythema index) was calculated by adding the scores of 1 or more and dividing by the number of animals exhibiting a positive response.
See table 2 for further details.

Positive control substance(s):

yes

Positive control results:

CHALLENGE SCORE
0/0.

Reading:

other: Sensitization Rate

Hours after challenge:

48

Group:

test chemical

Dose level:

0.1 ml

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: other: Sensitization Rate. . Hours after challenge: 48.0. Group: test group. Dose level: 0.1 ml. No with. + reactions: 0.0. Total no. in groups: 10.0.

Reading:

other: Sensitization Rate

Hours after challenge:

72

Group:

test chemical

Dose level:

0.1 ml

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: other: Sensitization Rate. . Hours after challenge: 72.0. Group: test group. Dose level: 0.1 ml. No with. + reactions: 0.0. Total no. in groups: 10.0.

Reading:

other: Erythema Index

Hours after challenge:

48

Group:

test chemical

Dose level:

0.1 ml

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: other: Erythema Index. . Hours after challenge: 48.0. Group: test group. Dose level: 0.1 ml. No with. + reactions: 0.0. Total no. in groups: 10.0.

Reading:

other: Erythema Index

Hours after challenge:

72

Group:

test chemical

Dose level:

0.1 ml

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: other: Erythema Index. . Hours after challenge: 72.0. Group: test group. Dose level: 0.1 ml. No with. + reactions: 0.0. Total no. in groups: 10.0.

Reading:

other: all parameters

Group:

negative control

Remarks on result:

not measured/tested

Reading:

other: all parameters

Group:

positive control

Remarks on result:

not measured/tested

RESULTS

General: apart from the slight irritation observed after the topical booster application, all guinea pigs appeared active and healthy throughout the test period. There were no signs of gross toxicity, adverse pharmacologic effects or abnormal behaviour. All animals gained weight.

Induction: barely perceptible to mild erythema was observed at all sites 24 hours after the topical booster application. By 48 hours all sites were clear.

Challenge: there was no evidence of erythema at any sites on any of the test animals 24 or 48 hours after challenge. Similarly, no irritation occurred on any of the dosed sites on the naive Guinea Pigs.

Erythema after induction and challenge

ANIMAL N.	INDUCTION SCORE		CHALLENGE SCORE
Test animal	24 h	48 h	24 h	48 h
3359	0.5/0	0/0	0/0	0/0
3360	0/0.5	0/0	0/0	0/0
3361	0.5/0.5	0/0	0/0	0/0
3362	0.5/0.5	0/0	0/0	0/0
3363	0.5/0.5	0/0	0/0	0/0
3364	1/0	0/0	0/0	0/0
3365	1/0.5	0/0	0/0	0/0
3366	0.5/0.5	0/0	0/0	0/0
3367	0.5/1	0/0	0/0	0/0
3368	0.5/1	0/0	0/0	0/0
NAIVE CONTROL ANIMALS
3369			0/0	0/0
3370			0/0	0/0
3371			0/0	0/0
3372			0/0	0/0
3373			0/0	0/0

Interpretation of results:

not sensitising

Remarks:

Migrated information According to the CLP Regulation. Criteria used for interpretation of results: EU

Conclusions:

Non sensitising.

Executive summary:

Method

The potential of the test material to provoke delayed skin sensitization reactions was assessed after intradermal injection and topical skin insult applications of the test item.

Induction: after acclimating to the laboratory environment, 10 healthy, male, Hartley Strain, guinea pigs were injected ID with approximately 0.05 ml of test product in water, 0.05 ml 5 % test material in 50 % aqueous Freunds Complete Adjuvant and 0.05 ml 50 % aqueous Freunds Complete Adjuvant at multiple sites in the shaven anterior left and right shoulder areas. 

Topical induction (booster): 7 days later, a Hilltop Chamber containing 0.1 ml of test material was placed adjacent to each previously injected area for 48 hours at which time the chambers were removed. The sites were scored for erythema 24 and 48 hours after patch removal (72 and 96 hours after initial topical application). 

Challenge: 14 later, both flanks of each guinea pig were shaved and each received a topical application of 0.1 ml test material per site on a Hilltop Chamber. The chamber was secured in place for 24 hours, then removed. 24 and 48 hours later (48 and 72 hours after challenge), the sites were scored for a sensitization response.

Results

General: apart from the slight irritation observed after the topical booster application, all guinea pigs appeared active and healthy throughout the test period. There were no signs of gross toxicity, adverse pharmacologic effects or abnormal behaviour. All animals gained weight.

Induction: barely perceptible to mild erythema was observed at all sites 24 hours after the topical booster application. By 48 hours all sites were clear.

Challenge: there was no evidence of erythema at any sites on any of the test animals 24 or 48 hours after challenge. Similarly, no irritation occurred on any of the dosed sites on the naive Guinea Pigs.

Discussion and conclusion

According to the CLP Regulation, a substance is classified as skin sensitiser when in the Guinea pig maximisation test the response of at least 30 % of the animals is considered as positive.

There are not positive results reported in this appropriate animal test.

In conclusion, the test item can be classified as NON sensitising, according to the CLP (EC 1278/2008) Regulation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The potential of the test material to provoke delayed skin sensitization reactions was assessed after intradermal injection and topical skin insult applications of the test item. After acclimating to the laboratory environment, 10 healthy, male, Hartley Strain, guinea pigs were injected ID with approximately 0.05 ml of test product in water, 0.05 ml 5 % test material in 50 % aqueous Freunds Complete Adjuvant and 0.05 ml 50 % aqueous Freunds Complete Adjuvant at multiple sites in the shaven anterior left and right shoulder areas.  Topical induction (booster): 7 days later, a Hilltop Chamber containing 0.1 ml of test material was placed adjacent to each previously injected area for 48 hours at which time the chambers were removed. The sites were scored for erythema 24 and 48 hours after patch removal (72 and 96 hours after initial topical application). 

14 days later, both flanks of each guinea pig were shaved and each received a topical application of 0.1 ml test material per site on a Hilltop Chamber. The chamber was secured in place for 24 hours, then removed. 24 and 48 hours later (48 and 72 hours after challenge), the sites were scored for a sensitization response. Apart from the slight irritation observed after the topical booster application, all guinea pigs appeared active and healthy throughout the test period. There were no signs of gross toxicity, adverse pharmacologic effects or abnormal behaviour. All animals gained weight. Induction: barely perceptible to mild erythema was observed at all sites 24 hours after the topical booster application. By 48 hours all sites were clear (Ventura and Shapiro, 1989).

In a second study on the substance the potential of the test material to cause photosensitization reactions was assessed.

After acclimating to the laboratory environment for 10 days, 20 male guinea pigs were clipped free of hair along the flanks and dorsal surface and subsequently depilated. Approximately 2 hours after clipping or depilation the animals were dosed with 0.1 ml of either the test products 2 % TCSA in DAB (positive control) or 100 % DAB (vehicle control). To induce sensitization, 6 induction doses were applied on 3 alternate days for each of 2 consecutive weeks. Prior to the third and sixth induction, Freunds Adjuvant was injected intradermally around the perimeter of each induction sits. UV exposure: approximately 1 hour after dosing the animals were exposed to UVA (inductions 1 and 2) and UVA followed by UVB (inductions 3-6). In all cases the light source was kept at a distance of approximately 10 cm from the dorsal surface of the animals. The duration of exposure was 10 minutes for UVA and 3 minutes for UVB. Light intensities were measured and recorded.

Two weeks after the induction phase, the animals were challenged with either 0.1 ml 5 % test product in DAE, 0.01, 0.1 or 1.0 % TCSA in DAE or 100% DAR only and then exposed to UVA light at 10 cm for 10 minutes. In addition to these exposed done sites, each animal had an untreated exposed site and a covered treated site to respectively evaluate the erythemic response caused by light exposure only and to determine whether the product caused a topical sensitization response without UVA activation. All animals were active and healthy throughout the entire test period. Other than the erythemic responses in the positive control animals, there were no signs of gross toxicity, adverse pharmacologic effects or abnormal behaviour. After challenge none of the animals treated with the test material or those treated with the vehicle control (DAB) exhibited an erythemic response. All scores were zero. On the other hand, the positive control animals exhibited a weak to moderate sensitization response when dosed with 0.1 % TCSA and a moderate to strong response when dosed with 1.0 % TSCA. All positive control animals were responsive. None of the test animals or vehicle control animals had erythema at untreated sites exposed to UVA or at unexposed (covered) treated sites. Several animals treated with 0.1 or 1.0 % TCSA exhibited a very weak sensitization response from topical application only (i.e. treated, covered sites) suggesting weak topical sensitization potential at these concentrations.

The scoring system criteria used to record of the skin photosensitization followed in the current test defined the test item as not photosensitizer for the skin (Ventura and Shapiro, 1989).

There are not positive results reported in this appropriate animal test.

As a conclusion, the substance is considered as non sensitizing.

Migrated from Short description of key information:

Not skin sensitising.

Justification for selection of skin sensitisation endpoint:

The test procedures are well documented and scientifically acceptable.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

According to CLP Regulation (EC 1272/2008), 3.4 Respiratory or skin sensitisation section, skin sensitizer means a substance that will lead to an allergic response following skin contact.

The criteria to classify a substance as skin sensitizer, on the basis of results from the Guinea pig Maximization test, are reported into the second adaptation to technical progress*. A substance in considered a skin sensitizer when:

- an adjuvant type test method for skin sensitisation is used and a response of at least 30 % of the animals is considered as positive;

- for a non-adjuvant Guinea pig test method a response of at least 15 % of the animals is considered positive;

- a stimulation index of three or more is considered a positive response in the local lymph node assay.

Therefore, in the case of the Guinea pig maximisation test a substance is classify as sensitising if the results indicate that a number of animals ≥ 30 % to < 60 % responding at concentrations > 0.1 % to ≤ 1 % of intradermal induction dose or a number of animals ≥ 30 % responding at > 1 % intradermal induction dose.

Less than the 30 % of animals showed a reaction, therefore the substance can be considered as non sensitising.

In conclusion, the available experimental data is adequate for classification and labelling and the test substance is not classified as skin sensitizing according to CLP Regulation (EC 1272/2008).

*Commission Regulation (EU) No 286/2011 of 10 March 2011, amending, for the purposes of its adaptation to technical and scientific progress, Regulation (EC) No 1272/2008 of the European Parliament and of the Council on classification, labelling and packaging of substances and mixtures