Methyl N-[4-[(2-bromo-6-chloro-4-nitrophenyl)azo]phenyl]-N-(3-methoxy-3-oxopropyl)-β-alaninate

Endpoint summary

Description of key information

The test substance has no skin sensitising effects.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From May 13, 2002 to May 21, 2002

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)

Deviations:

yes

Remarks:

male mice instead of female mice were used; groupe housing instead of single housing; no body weight measurement

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA/Ca

Sex:

male

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan, Interfauna UK Limited, Blackthorne, Bicester, Oxon, UK
- Sex: male
- Age at study initiation: young adults
- Weight at study initiation:
- Housing: 4/cage
- Diet (e.g. ad libitum): RM1 ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: at least 5 d
- Indication of any skin lesions:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-3
- Humidity (%): 30-70
- Air changes (per hr): minimum 15
- Photoperiod (hrs dark / hrs light): 12/12
- IN-LIFE DATES: The main study was initiated on 13 May 2002. The experimental phase started on 14 May 2002 and was completed on 21 May 2002.
For the positive control study, the experimental phase started on 18 September 2001 and was completed on 25 September 2001.

Vehicle:

other: acetone

Concentration:

3%, 10% and 50% w/v

No. of animals per dose:

groups of 4 males

Details on study design:

Groups of four male mice were used for this study. Approximately 25 µL of a 3%, 10% or 30% w/v preparation of the test substance in acetone was applied, using a variable volume micro-pipette, to the dorsal surface of each ear. A vehicle control group was similarly treated using acetone alone. The procedure was repeated daily for 3 consecutive days.
Three days after the third application, all animals were injected, via the tail, with approximately 250 µL of PBS containing about 20 µCi of a 2.0Ci/mmol specific activity 3H-methyl thymidine (for beta-scintillation counting using a Packard Tri-Carb 2500TR Liquid Scintillation Counter). After 5 d, the animals were sacrificed. The draining auricular lymph nodes were removed from each animal and, together with the nodes from the other animals in the group, were placed in a container of PBS.

The results are expressed as a counts per minute (cpm) value per lymph node for each group. The stimulation index for each test group is then calculated by dividing the cpm value per lymph node by the equivalent value for the control (vehicle only) group.
The criterion for a positive response is that one or more concentrations of the test substance should elicit a 3-fold or greater increase in isotope incorporation relative to the vehicle control group. The assay is able to identify those materials that elicit responses in standard guinea pig tests for skin sensitisation (Kimber et al 1994). Consequently, a test substance which does not fulfil the above criterion is designated as unlikely to be a sensitiser.

Positive control substance(s):

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

Positive control results:

The application of hexylcinnamaldehyde at concentrations of 1, 3 and 10% w/v in acetone resulted in a greater than 3-fold increase in isotope incorporation at all three concentrations.

The validity of the protocol was confirmed.

Key result

Parameter:

SI

Value:

0.83

Test group / Remarks:

3% of test substance

Key result

Parameter:

SI

Value:

1.19

Test group / Remarks:

10% of test substance

Key result

Parameter:

SI

Value:

1.06

Test group / Remarks:

30% of test substance

Cellular proliferation data / Observations:

The application of the test substance at concentrations of 3%, 10% and 30% w/v in acetone resulted in an increase in isotope incorporation which was less than 3-fold at all three concentrations.

Concentration of test substance (% w/v)	Number of lymph nodes assayed	Counts per minute (cpm)	cpm per lymph node (x10^-2)	Test control ratio
0 (vehicle only)	8	1092	1.37	N/A
3	8	913	1.14	0.83
10	8	1303	1.63	1.19
30	8	1160	1.45	1.06

Interpretation of results:

GHS criteria not met

Conclusions:

Under the study conditions, the test substance was considered to be not sensitizing to mouse skin (LLNA).

Executive summary:

A study was conducted to determine the skin sensitisation potential of the test substance according to OECD Guideline 429 (local lymph node assay), in compliance with GLP. Male CBA/Ca mice were exposed to the test substance at concentrations of 0, 3, 10 and 30% in acetone. Isotope (3H-methyl thymidine) incorporation was measured in lymph nodes. The negative (vehicle alone) and positive (hexylcinnamaldehyde at concentrations of 1, 3, and 10% w/v in acetone) controls were valid. The isotope incorporation was increased by less than a threefold at all concentrations of the test substance. Under the study conditions, the test substance was considered to be not sensitizing to mouse skin (Johnson, 2002).

Reference 2

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

December 1978

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

GLP compliance:

no

Remarks:

Study pre-dates GLP

Type of study:

not specified

Justification for non-LLNA method:

Study available is over 12 years old.

Species:

guinea pig

Strain:

Hartley

Sex:

female

Details on test animals and environmental conditions:

Ten young female Hartley-Dunkin albino guinea pigs obtained from Redfern Animal Breeders, Jasons Farm, Old Hay, Brenchley, Kent were used for this study. They were conditioned to the laboratory environment for not less than 6 days and on the day before initial treatment they were weighed. At this time the body weights of the guinea pigs were within the range 300 - 430 g. Three animals were marginally over the body weight range required by the protocol.

Diet
The animals were allowed free access to amins water and food (Guinea pig Diet-Standard supplemented with Vit. C., BP Nutrition (U.K.) Ltd., Stepfield, Witham, Essex).

Environment
All animals were housed in a single air-conditioned room maintained at a temperature of 22 + 3°C, relative humidity 50 + 10% and exposed to natural lighting conditions. They were caged in groups of 2 in grid floor polypropylene boxes.

Route:

epicutaneous, open

Vehicle:

N,N-dimethylformamide

Concentration / amount:

0.1 ml of a 10% w/v solution

Day(s)/duration:

3 days

Adequacy of induction:

not specified

No.:

#1

Route:

epicutaneous, open

Vehicle:

N,N-dimethylformamide

Concentration / amount:

0.2 ml
10% w/v, 1% w/v and 0.1% w/v

Day(s)/duration:

1 day

No. of animals per dose:

6 animals

Details on study design:

The test article (0.1 ml of a 10% w/v solution in dimethylformamjde) was applied daily by means of a glass syringe to the outer surface of the ears of 6 guinea pigs (animal numbers 1 - 6) for three days
(days 1, 2 and 3). On Day 8, 0.2 ml of the challenge solutions (10% w/v, 1% w/v and 0.1% w/v in dimethylformamide) was applied topically to 1 cm diameter circular areas on the clipped flanks of each of the same 6 animals. Solutions of test article were also applied in the same way on Day 8 to the clipped flanks of control animals (animal numbers 7 - 10) which had no previous treatment on the ears. The-applications were made on both flanks of all 10 guinea pigs, with each concentration being applied to each flank. The highest concentration was applied closest to the posterior end of the animal while the lowest concentration was applied nearest the anterior end.
The erythema produced on each site was assessed 24 hours later (Day 9) and graded on a 6 point scale.

Challenge controls:

The highest concentration was applied closest to the posterior end of the animal while the lowest concentration was applied nearest the anterior end.

Positive control substance(s):

not specified

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

10% w/v

No. with + reactions:

0

Total no. in group:

6

Clinical observations:

None specified

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

1% w/v

No. with + reactions:

0

Total no. in group:

6

Clinical observations:

None specified

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

0.1% w/v

No. with + reactions:

0

Total no. in group:

6

Clinical observations:

None specified

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

10% w/v

No. with + reactions:

0

Total no. in group:

4

Clinical observations:

None specified

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

1.0% w/v

No. with + reactions:

0

Total no. in group:

4

Clinical observations:

None specified

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

0.1% w/v

No. with + reactions:

0

Total no. in group:

4

Clinical observations:

None specified

Remarks on result:

no indication of skin sensitisation

Skin reactions at challenge phase

Test article: Disperse Brown 19

Vehicle: dimethylformamide

Concentration: 10% w/v, 1% w/v and 0.1% w/v

Animal number and sex	Weight (g)	Skin reactions assessed at 24 hours after application
		10% w/v	1% w/v	0.1% w/v
1 2 3 4 5 6 7 control 8 control 9 control 10 control	410 410 430 321 308 318 340 355 344 351	0 0 0 0 0 0 0 0 0 0	0 0 0 0 0 0 0 0 0 0	0 0 0 0 0 0 0 0 0 0

 

Interpretation of results:

GHS criteria not met

Conclusions:

No erythema was noted in any of the test or control animals. The test article was not considered to be a strong sensitiser.

Executive summary:

A study was carried out to determine the skin-sensitising potential of test article in the albino guinea pig. The study was performed in accordance with Standard Operating Procedures.

 

Ten young female Hartley-Dunkin albino guinea pigs were used for this study. They were conditioned to the laboratory environment for not less than 6 days and on the day before initial treatment they were weighed. At this time the body weights of the guinea pigs were within the range 300 - 430 g. Three animals were marginally over the body weight range required by the protocol.

 

The test article (0.1 ml of a 10% w/v solution in dimethylformamjde) was applied daily by means of a glass syringe to the outer surface of the ears of 6 guinea pigs (animal numbers 1 - 6) for three days (days 1, 2 and 3).

On Day 8, 0.2 ml of the challenge solutions (10% w/v, 1% w/v and 0.1% w/v in dimethylformamide) was applied topically to 1 cm diameter circular areas on the clipped flanks of each of the same 6 animals. Solutions of test article were also applied in the same way on Day 8 to the clipped flanks of control animals (animal numbers 7 - 10) which had no previous treatment on the ears. The applications were made on both flanks of all 10 guinea pigs, with each concentration being applied to each flank.

The highest concentration was applied closest to the posterior end of the animal while the lowest concentration was applied nearest the anterior end.

The erythema produced on each site was assessed 24 hours later (Day 9).

 

Results

No erythema was noted in any of the test or control animals. The test article was not considered to be a strong sensitiser.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

A study was carried out to determine the skin-sensitising potential of test article in the albino guinea pig. The study was performed in accordance with Standard Operating Procedures.

Ten young female Hartley-Dunkin albino guinea pigs were used for this study. They were conditioned to the laboratory environment for not less than 6 days and on the day before initial treatment they were weighed. At this time the body weights of the guinea pigs were within the range 300 - 430 g. Three animals were marginally over the body weight range required by the protocol.

The test article (0.1 ml of a 10% w/v solution in dimethylformamjde) was applied daily by means of a glass syringe to the outer surface of the ears of 6 guinea pigs (animal numbers 1 - 6) for three days (days 1, 2 and 3).

On Day 8, 0.2 ml of the challenge solutions (10% w/v, 1% w/v and 0.1% w/v in dimethylformamide) was applied topically to 1 cm diameter circular areas on the clipped flanks of each of the same 6 animals. Solutions of test article were also applied in the same way on Day 8 to the clipped flanks of control animals (animal numbers 7 - 10) which had no previous treatment on the ears. The applications were made on both flanks of all 10 guinea pigs, with each concentration being applied to each flank.

The highest concentration was applied closest to the posterior end of the animal while the lowest concentration was applied nearest the anterior end. The erythema produced on each site was assessed 24 hours later (Day 9).

No erythema was noted in any of the test or control animals. The test article was considered not to be a strong sensitiser.

A study was conducted to determine the skin sensitisation potential of the structural analogue according to OECD Guideline 429 (local lymph node assay) in compliance with GLP. Male CBA/Ca mice were exposed to the test substance at concentrations of 0, 3, 10 and 30% in acetone. Isotope (3H-methyl thymidine) incorporation was measured in lymph nodes. The negative (vehicle alone) and positive (hexylcinnamaldehyde at concentrations of 1, 3, and 10% w/v in acetone) controls were valid. The isotope incorporation was increased by less than a threefold at all concentrations of the test substance. Under the study conditions, the test substance was considered to be not sensitizing to mouse skin.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The results of the above studies do not trigger a classification for skin sensitisation according to CLP (EC 1272/2008) criteria.