Disodium 7-[[4,6-bis[(2-hydroxyethyl)amino]-1,3,5-triazin-2-yl]amino]-4-hydroxy-3-[[4-[(4-sulphonatophenyl)azo]phenyl]azo]naphthalene-2-sulphonate

Endpoint summary

Description of key information

Not skin sensitizing

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

other: experimental results on same main component, with a different counter ion. The different salificaton which does not influence the characteristics related to the specific end-point

Adequacy of study:

key study

Study period:

From January 8th to February 14th, 1990

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

adopted May 12, 1981

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

GLP compliance:

yes

Type of study:

Buehler test

Justification for non-LLNA method:

A LLNA study has not been conducted because a adequate data from a previously conducted experiment already exists.

Species:

guinea pig

Strain:

other: Ibm: GOHI, SPF-quality (synonym: Himalayan spotted)

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: BRL, Biological Research Laboratories Ltd.
- Age at delivery: 7 - 8 weeks
- Body weight at start of acclimatization: males 316 - 377 g, females 310 - 332 g
- Identification: by unique cage number and corresponding individual number of animals and restrainer.
- Randomization: randomly selected at time of delivery.
- Housing: individually in Makrolon type-3 cages with standard softwood bedding.
- Diet: pelleted standard Kliba 342, guinea pig breeding/ maintenance diet, ad libitum.
- Water : community tap water from Itingen, ad libitum.
- Acclimation period: 7 days under test conditions after veterinary examination.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 40 - 70 %
- Air changes: 10-15 air changes per hour.
- Photoperiod: 12 hours artificial fluorescent light/12 hours dark, music/light period.

Route:

epicutaneous, occlusive

Vehicle:

water

Remarks:

bi-distilled water

Concentration / amount:

Induction: 25 % test item in bi-distilled water
Challenge: 25 % test item in bi-distilled water

Route:

epicutaneous, occlusive

Vehicle:

water

Remarks:

bi-distilled water

Concentration / amount:

Challenge: 25 % test item in bi-distilled water

No. of animals per dose:

Main test: 10 males and 10 females.
Pretest: 2 males and 2 females for induction and 2 males and 2 females for challenge.

Details on study design:

RANGE FINDING TESTS
4 guinea pigs (2 males and 2 females) were used for pretest for induction and further 4 animals (2 males and 2 females) were used for pretest for challenge. The concentrations used for irritation screen for induction were 5, 10, 15 and 25 % of test item in bi-distilled water.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: three inductions.
- Exposure period: about 6 hours (± 15 minutes) later, patches were removed.
- Test groups: 10 males and 10 females.
- Control group: untreated control animals.
- Site: fur was shaved the day before exposure. The area shaved was approximately one-fourth of the animals' back and side.
- Frequency: the patch application was repeated at the same site once a week for the next two weeks for a total of three 6-hour exposures.
- Evaluation: the responses were graded at 24 hours (± 2 hours) after patch removal.

After the last induction exposure, the animals were left untreated for two weeks before primary challenge.

B. CHALLENGE EXPOSURE
- Test groups: the animals previously exposed during the induction period (i.e. test group) were challenged two weeks after the last induction exposure using the dose as prescribed.
- Control group: 5 males and 5 females used for the first challenge treatment with test item. Further 5 males and 5 females,. due to the unequivocal findings observed after the first challenge no second challenge was performed. Cntrol animals were graded before the test animals. Grading of all animals was done by positioning the animal under true-light.
- Site: the challenge fur was clipped from the left posterior quadrant of the side and back of the animal.
- Evaluation: 18 to 22 hours after patches had been removed, all animals were depilated using an approved depilatory cream . The depilatory was placed on the patch sites and surrounding areas, and left on for 15 minutes or less. It was then thoroughly washed off with a stream of warm, running water. The animals were then dried with a disposable towel, and returned to their cages. A minimum of 2 hours after depilation (24 hour grade) the test sites were graded.

OBSERVATIONS
- Mortality/Viability: daily.
- Body weights: at acclimatization start, at day one (induction start) and at termination of the test.
- Symptoms (local/systemic): daily, including irritation effects during induction period.

PATHOLOGY
No necropsy was performed in the animals killed at termination of observation. All animals were killed at the end of the test period with an intra-peritoneal injection of T61 (Hoechst AG) and discarded.

Challenge controls:

Control group for 1st challenge: 5 / sex / dose
Control group for 2nd challene: 5 / sex / dose

Positive control substance(s):

yes

Remarks:

Formaldehyde solution

Positive control results:

In a separate test (between November and December, 1989), the incidence to allergenic reactions of the guinea pig strain used, was tested with a known allergen (the positive control). Significant positive results were obtained in this experiment.

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

25 %

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

None, except for staining around the epidermal induction sites, application area

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

25 %

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

None, except for staining around the epidermal induction sites, application area

Induction

Induction week 1: no erythema or edema was recorded in all 20 animals in the test group.

Induction week 2: slight confluent, or moderate but patchy erythema was observed in 3 female rabbits. No edema was recorded in all guinea pigs.

Induction week 3: slight confluent, or moderate but patchy erythema was observed in 1 male rabbit. No edema was observed in all guinea pigs.

Skin reactions observed during induction (25 % test article)

Animal N.	Sex	Induction week 1		Induction week 2		Induction week 3
		Erythema	Oedema	Erythema	Oedema	Erythema	Oedema
241	M	0	0	0	0	0	0
242	M	0	0	0	0	0	0
243	M	0	0	0	0	0	0
244	M	0	0	0	0	0	0
245	M	0	0	0	0	0	0
246	M	0	0	0	0	0	0
247	M	0	0	0	0	0	0
248	M	0	0	0	0	0	0
249	M	0	0	0	0	0	0
250	M	0	0	0	0	1	0
261	F	0	0	0	0	0	0
262	F	0	0	1	0	0	0
263	F	0	0	0	0	0	0
264	F	0	0	0	0	0	0
265	F	0	0	1	0	0	0
266	F	0	0	0	0	0	0
267	F	0	0	1	0	0	0
268	F	0	0	0	0	0	0
269	F	0	0	0	0	0	0
270	F	0	0	0	0	0	0

Challenge

First challenge control group: no erythema or edema occured after 24 h (± 2 h) or 48 h (± 2 h) in all rabbits.

First challenge test group: no erythema or edema occured after 24 h (± 2 h) or 48 h (± 2 h) in all rabbits.

Second challenge: due to unequivocal findings observed after the first challenge, no second challenge was performed.

	Test group (20 animals)		Control group (10 animals)
Score	24 hrs	48 hrs	24 hrs	48 hrs
0	20	20	10	10
0*	0	0	0	0
1	0	0	0	0
2	0	0	0	0
3	0	0	0	0
N. with grades ≥ 1	0	0	0	0
N. tested	20	20	10	10
Total	0		0

*The total is the number of animals showing a grade ≥ 1 at either 24 or 48 hours.

OBSERVATION

Viability / Mortality: no death occurred during the study.

Bodyweights: the body weight gain of the animals was not affected during the study.

Local symptoms in the test group: application area around the epidermal induction sites was found to show staining from day 2 to 26.

Systemic symptoms: no systemic symptoms were observed in any animal during the study.

Interpretation of results:

not sensitising

Remarks:

according to the CLP Regulation (EC 1272/2008)

Conclusions:

Not sensitising

Executive summary:

The allergenic potential of the test substance was examined in a modified Buehler test according to OECD test guideline 406. 2 animals per sex were used in an irritation screen for induction and irritation screen for challenge. 4 test substance concentrations were applied at different sites on each animal: 25 %, 15 % , 10 % and 5 %. A concentration of 25 % was chosen for induction and challenge based on the results of the irritation screen for induction. In the main study, the control group consisted of 5 animals per sex per dose and the test group consisted of 10 animals per sex per dose. The exposure period was 6 h in the irritation screens, induction and challenge tests. Erythema and edema was scored at 24 h and 48 h. Mortality/viability, bodyweights and local/systemic symptoms were observed during the study. No necropsy was performed in the animals killed at termination of observation. All animals were killed at the end of the test period with an intra-peritoneal injection of T61 (Hoechst AG) and discarded.

No mortality occurred during the study. Bodyweight gain of all animals was unaffected during the study. The application area around the epidermal induction sites showed staining around day 2 to day 26. No systemic symptoms were observed in any animal in the study. No skin reactions were observed in the irritation screen for induction and irritation screen for challenge. 25 % was determined to be the highest non-irritating concentration. Each animal was exposed to the test substance once a week for 3 weeks in induction. In the first induction week, no skin reaction was observed. In the second induction week, no edema was observed and 3 female guinea pigs showed slight erythema or moderate but patchy erythema. In the third induction week, no edema was observed and 1 male guinea pig showed slight erythema or moderate but patchy erythema.

Challenge took place 2 weeks after the last induction exposure. In the first challenge, no skin reaction occurred at 24 h or 48 h in all animals in the control group and test group. No second challenge was performed because of unequivocal findings from the first challenge.

Conclusion

The test substance is not a skin sensitiser.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The allergenic potential was examined in a modified Buehler test according to OECD test guideline 406. The experiment was conducted on Direct Red 253 sodium and triethanolammonium salt, which has the same main component of the substance under registration (i.e. Direct Red 253) with a different counter ion. The information can be considered as suitable and appropriated because the difference in salification is expected to not influence the characteristics related to the specific end-point.

A concentration of 25 % was chosen for induction and challenge based on the results of the irritation screen for induction. In the main study, the control group consisted of 5 animals per sex per dose and the test group consisted of 10 animals per sex per dose. The exposure period was 6 h in the irritation screens, induction and challenge tests. Mortality/viability, bodyweights and local/systemic symptoms were observed during the study. No necropsy was performed in the animals killed at termination of observation.

No mortality occurred during the study. Bodyweight gain of all animals was unaffected during the study. The application area around the epidermal induction sites showed staining around day 2 to day 26. No systemic symptoms were observed in any animal in the study. No skin reactions were observed in the irritation screen for induction and irritation screen for challenge. 25 % was determined to be the highest non-irritating concentration. Each animal was exposed to the test substance once a week for 3 weeks in induction. In the first induction week, no skin reaction was observed. In the second induction week, no edema was observed and 3 female guinea pigs showed slight erythema or moderate but patchy erythema. In the third induction week, no edema was observed and 1 male guinea pig showed slight erythema or moderate but patchy erythema.

Challenge took place 2 weeks after the last induction exposure. In the first challenge, no skin reaction occurred at 24 h or 48 h in all animals in the control group and test group. No second challenge was performed because of unequivocal findings from the first challenge (Ullmann, 1990).

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

According to the 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.

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

Based on the read across approach, Direct Red 253 (DR253) can be considered as non skin sensitizer.

In conclusion, the substance does not meet the criteria to be classified as skin sensitizer, according to the CLP Regulation (EC 1272/2008).