2,4,6-trimethylbenzene-1,3-diamine

Endpoint summary

Description of key information

Under the present test conditions, test item revealed no sensitising properties in guinea pigs in a skin sensitisation test according to E. V. BUEHLER (LPT, 2016).

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:

2016-10-31 to 2016-12-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

adopted July 17, 1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Version / remarks:

published in the Official Journal of the European Union L142, dated May 30, 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

Buehler test

Justification for non-LLNA method:

Due to high restrictions and infeasibility of the LLNA for testing of irritating substances and the fact that this test often achieved false positive results, a skin sensitisation test according to OECD 406 (Buehler test) was conducted.

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

male

Details on test animals and environmental conditions:

TEST ANIMALS: 
- Strain: Dunkin-Hartley
- Sex: male
- Breeder: Charles River Laboratories, France
- Supplier: Charles River Laboratories, Research Models and Services Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age: 29 days
- Weight at study initiation: 299  - 356 g, positive control group: 300 - 347 g
- Housing: max 5 animals per cage
- Diet (e.g. ad libitum): Commercial diet ssniff7 Ms-H V2233 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany,
- Water (e.g. ad libitum): tap water
- Acclimation period: at least 5 days
- Controls: 10 animals; treatment: vehicle

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 +/- 3°C
- Humidity (%): 55 +/- 15 %
- Photoperiod (hrs dark / hrs light): 12 hour light/dark rhythm

Route:

epicutaneous, occlusive

Vehicle:

other: sesame oil

Concentration / amount:

6 hour occlusive patch (left side) with 0.5 g of a 75% concentration in sesam oil ; then removal of  residual test material

Day(s)/duration:

day 1/8/15 for 6 hours

Adequacy of induction:

highest technically applicable concentration used

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

other: sesame oil

Concentration / amount:

two 6 hour occlusive patches (right side), 0.5 mL vehicle control or 0.5 g test item, was applied at the 75% concentration (maximum feasible and non-irritant)  subsequent removal of residual test material

Day(s)/duration:

day 29 for 6 hours

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

10 animals control group
20 animals test material
20 animals positive control

Details on study design:

RANGE FINDING TESTS:
A preliminary test was performed with 2 animals employing a range of concentrations of test item (as a 10, 25, 50 or 75% solutions in sesame oil).
Test item was a yellow powder. A 75% concentration in form of a wet powder was the highest feasible concentration that could be prepared for dermal application.
The flank regions of the animals were cleared of hair (closely-clipped) one day of before application.
The test item-concentrations of 50% and 75% were wet powders and could not be applied by volume. The application weight was set at 0.5 g.
The test patch system, a square cotton pad of approximately 4 - 6 cm2, was fully loaded with 0.5 mL or 0.5 g of the test item in sesame oil. The test patch system was applied to the test area and was held in contact with the skin by an occlusive patch and a suitable dressing for 6 hours. The test patch system was occlusive. The patch and the occlusive dressing were secured with adhesive strips (exposure time: 6 hours each).
For each induction stage of the main study, the concentration of test item was, if possible, adjusted to the highest level that could be well tolerated systemically and which, for irritant substances, produces mild to moderate irritation in the majority of test animals. The challenge concentration should be the maximum which produces no evidence of skin irritation in non-sensitised animals. As no changes of the skin were observed in this preliminary test at any concentration the 75% concentration of the test item in sesame oil was chosen for the induction and the challenge of the main experiment.


ADMINISTRATION/EXPOSURE 

- Induction - Topical application
Day 1 - Treated Group
One day before application the left side of the flank region of the animals were cleared of hair (closely-clipped).
The test patch system, a square cotton pad of approximately 4 - 6 cm2 was fully loaded with 0.5 g of a 75% concentration of test item in sesame oil.
The test patch system was applied to the test area and was held in contact with the skin by an occlusive patch and a dressing for 6 hours.
The test patch system was occlusive. The patch and the occlusive dressing were secured with adhesive strips (exposure time: 6 hours each).

Day 1 - Control Group
One day before application the left side of the flank region of the animals were cleared of hair (closely-clipped).
The control animals were treated in a similar manner to that used for the treated group, but received sesame oil instead of test item.
The test patch system was applied to the test area and was held in contact with the skin by an occlusive patch and a suitable dressing for 6 hours.

Days 8 and 15 - treated and control groups
The same application as on day 1 was carried out on the same test area (cleared of hair one day before application) on the same flank region on day 8 and again on day 15.

- Challenge
Day 29 - Treated and Control Groups:   
One day before application the untreated right flank of treated and control animals was cleared of hair (closely-clipped).
An occlusive patch containing the appropriate amount of test item (0.5 mL vehicle control or 0.5 g test item) was applied at the 75% concentration (maximum feasible and non-irritant) to the posterior untreated flank of treated and control animals.
The patches were held in contact with the skin by a suitable dressing for 6 hours.

- Positive control:   alpha-hexylcinnamaldehyde (CAS No. 101-86-0) (not concurrent)  
The animals of the positive control group were treated with 0.5 mL of undiluted a-hexyl cinnamaldehyde (HCA, 85%) on test days 1, 8 and 15 in the same manner as the animals of the negative control and the test item groups.
For the challenge on test day 29, HCA was used undiluted, too. The exposure time was 6 hours.
The sensitivity of the strain of guinea pigs employed is checked at regular intervals of approximately 6 months.


EXAMINATIONS
Skin observations and scoring
Days 30, 31 and 32: 24, 48 and 72 hours after removing of the patch the skin reaction was observed and recorded.
In addition, the skin condition was evaluated immediately and 18 hours after the end of induction exposures (6 and 24 hours after start of treatment).
- Grading system:
The skin reactions were graded as follows as given in the OECD and EC guidelines:

Grading scale for the evaluation of challenge patch test reactions Grade
no visible change 0
discrete or patchy erythema 1
moderate and confluent erythema 2
intense erythema and swelling 3

Maximum possible: 3



OTHER OBSERVATIONS
Mortality: daily during the observation period
Clinical signs: daily during the observation period
Body weight: before the test commences and at study termination
Records were maintained on all additional and standard observations

Challenge controls:

sesame oil

Positive control substance(s):

yes

Remarks:

alpha-hexylcinnamaldehyde (CAS No. 101-86-0) (HCA 85%)   Bühler test with 20 test and 10 control animals   Occlusive epicutaneous induction, undiluted   Occlusive epicutaneous challenge undiluted too

Positive control results:

The positive control alpha-hexyl cinnamaldehyde in sesame oil revealed pronounced sensitising properties.

Key result

Reading:

1st reading

Hours after challenge:

30

Group:

test chemical

Dose level:

75%

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

no

Key result

Reading:

2nd reading

Hours after challenge:

54

Group:

test chemical

Dose level:

75%

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

no

Key result

Reading:

other: 3nd reading

Hours after challenge:

78

Group:

test chemical

Dose level:

75%

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

no

Key result

Reading:

1st reading

Hours after challenge:

30

Group:

negative control

Dose level:

100%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

no

Key result

Reading:

2nd reading

Hours after challenge:

54

Group:

negative control

Dose level:

100%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

no

Key result

Reading:

other: 3nd reading

Hours after challenge:

78

Group:

negative control

Dose level:

100%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

no

RESULTS OF PILOT STUDY: 

Four concentrations of test item in sesame oil (10, 25, 50 and 75%) were tested by dermal application to guinea pigs.

Test item was a yellow powder. A 75% concentration in form of a wet powder was the highest feasible concentration that could be prepared for dermal application.

No skin reactions were observed up to the concentration of 75%.

RESULTS OF TEST

A 75% concentration of test item in sesame oil was used for the induction and for the challenge.

Sesame oil served as negative control.

Test item did not cause any skin reactions at any of the 4 stages.

The positive control a-hexyl cinnamaldehyde in sesame oil revealed pronounced sensitising properties.

No deaths or clinical signs were observed.

The body weight gain of the animals was not influenced.

Conclusions:

Under the present test conditions, test item revealed no sensitising properties in guinea pigs in a skin sensitisation test according to E. V. BUEHLER.

Executive summary:

The purpose of this study was to determine the sensitising potential of test item in a skin sensitisation test in guinea pigs according to E. V. BUEHLER. 

A 75% concentration of test item in sesame oil was used for the induction stages and for the challenge.

Test item was a yellow powder. A 75% concentration in form of a wet powder was the highest feasible concentration that could be prepared for dermal application.

Sesame oil served as negative control.

Test item did not cause any skin reactions at any of the 4 stages.

The positive control a-hexyl cinnamaldehyde solution revealed sensitising properties in all 20 animals employed.

Conclusion

Under the present test conditions, test item revealed no sensitising properties in guinea pigs in a skin sensitisation test according to E. V. BUEHLER.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

Assessment of the test substance concerning respiratory sensitisation is not possible because no data are available for this toxicological endpoint.

Justification for classification or non-classification

Based on the results of the in vivo skin sensitization test and according to the criteria of EC Directive 67/548/EEC and EC Regulation 1272/2008 the test item is not classified as sensitizing to skin.