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Description of key information

Key study: Test method according to OECD 429. GLP study. The substance does not have sensitisation potential (non-sensitizer) in the Local Lymph Node Assay.

Key study data waiving: Study IN VITRO is not necessary because the study IN VIVO already exists.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
3 September 2014 - 23 September 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
Details on test material
- Name of test material (as cited in study report): 4-amino-5-ethylsulfonyl-2-methoxybenzoic acid
- Physical state: Solid
- Analytical purity: 99.81%
- Lot/batch No.: MP1032.31
- Expiration date of the lot/batch: 16 June 2017

Species:
mouse
Strain:
CBA
Remarks:
CBA/J Rj mice
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: ELEVAGE JANVIER, Route des Chènes Secs B.P. 4105, 53940 LE GENEST-ST-ISLE, France
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 9 weeks old
- Weight at study initiation: 20.3-21.9 g (the weight variation in animals in the study did not exceed ± 20% of the mean weight)
- Housing: Group caging / mice were provided with glass tunnel-tubes. Cage Type II. polypropylene / polycarbonate.
- Diet (e.g. ad libitum): ad libitum, ssniff® SM Rat/Mouse – “Breeding & Maintenance, 15 mm, autoclavable Complete diet for rats/mice”
- Water (e.g. ad libitum): ad libitum, tap water.
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.3-26.8 °C
- Humidity (%): 33-79 %
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Vehicle:
dimethylformamide
Remarks:
According to the recommendation of the relevant OECD guideline, DMF was selected for vehicle of the study.
Concentration:
0 (control), 10, 25, 50 %
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:
- Compatibility test: The following solvents were analysed: Acetone: Olive oil 4:1 (v/v) mixture (AOO), N,N-Dimethyl formamide (DMF), Methyl ethyl ketone (MEK), Propylene glycol (PG), Dimethyl sulfoxide (DMSO) and 1% aqueous Pluronic PE9200 (1% Pluronic) as vehicles.The maximum achievable concentration of test substance was 50 % (w/v) in DMF according the OECD guideline.

- Preliminary Irritation/Toxicity Test: Two animals(13 week of age and 24.1-24.5 grams) per dose were exposed to 50 and 25 % (w/v) test item in DMF. Animals were treated as in the main test but it was terminated on Day 6 with a body weight measurement. All mice were observed daily for clinical signs of systemic toxicity or local irritation at the application site. Both ears of each mouse were observed for erythema. Ear thickness was measured using a thickness gauge on Day 1 (pre-dose), Day 3 (48 hours after the first dose) and Day 6. Additional quantification of the ear thickness was performed by ear punch weight determination after the euthanasia. No mortality or signs of systemic toxicity were observed. No marked body weight loss was observed. Test item precipitate was observed in the 50% (w/v) dose group on Days 1-4 and in the 25% (w/v) dose group on Days 2-3. No significant increase was observed in the ear thickness. The draining auricular lymph nodes of the animals had a normal appearance. Based on these results, 50 and 25% (w/v) doses were considered to be acceptable for the main test.

MAIN STUDY
TREATMENT PREPARATION AND ADMINISTRATION:
- Criteria used to consider a positive response: The test item is regarded as a sensitizer if both of the following criteria are fulfilled:
*That exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in
control mice, as indicated by the stimulation index.
*The data are compatible with a conventional dose response, although allowance must be made for either local toxicity or immunological suppression.

TREATMENT PREPARATION AND ADMINISTRATION:
Animals were topically dosed with 25 μL on the dorsal surface of each ear once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on
Days 4, 5 and 6. On day 6, each mouse received an injection of 250μl of sterile PBS (phosphate buffered saline) containing aprox. 20 μCi of 3HTdR. Five hours
later, the mice were euthanized and the auricular lymph nodes were extracted from the animals. A single cell suspension (SCS) of pooled lymph node cells
(LNCs) was prepared and the samples were prepared to be examined in a β-scintillation counter.

OBSERVATIONS:
During the study (Day 1 to Day 6) each animal was observed daily for clinical signs, local irritation and systemic toxicity. Clinical observations were performed
twice a day (before and after treatments) on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Individual body weights were recorded on Day 1 (beginning of
the test) and on Day 6 (prior to 3HTdR injection).

EVALUATION OF RESULTS:
Radioactive disintegrations per minute (DPM) was measured for each pooled group of nodes and corrected with the background DPM value. The results were
expressed as “DPN” (DPM divided by the number of lymph nodes). Stimulation index (SI = DPN value of a treated group divided by the DPN value of the
negative control group) for each treatment group was also calculated.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
- Criteria used to consider a positive response:
The test item is regarded as a sensitizer if both of the following criteria are fulfilled:
*That exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index.
*The data are compatible with a conventional dose response, although allowance must be made for either local toxicity or immunological suppression.
Positive control results:
The positive control substance was examined at a concentration of 25% (w/v) in the relevant vehicle (DMF) using CBA/J Rj mice. No mortality, cutaneous reactions or signs of toxicity were observed for the positive control substance in the study. A significant lymphoproliferative response (stimulation index value of 4.8) was noted for HCA in the main experiment. This value was considered to confirm the appropriate performance of the assay.
Key result
Parameter:
SI
Value:
ca. 0.7
Test group / Remarks:
Tets item (50% (w/v) in DMF)
Remarks on result:
other: No indication of skin sensitisation
Key result
Parameter:
SI
Value:
ca. 0.6
Test group / Remarks:
Test item (25% (w/v) in DMF)
Remarks on result:
other: No indication of skin sensitisation
Key result
Parameter:
SI
Value:
ca. 1
Test group / Remarks:
Test item (10% (w/v) in DMF)
Remarks on result:
other: No indication of skin sensitisation
Parameter:
other: EC 1.6
Remarks on result:
not determinable
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA: See : Any other information on results below.

DETAILS ON STIMULATION INDEX CALCULATION :SI (Stimulation Index) = DPN of a treated group divided by DPN of the appropriate control group.

DPN (Disintegrations Per Node) = DPM (Disintegrations Per Minute) divided by the number of lymph nodes.
In case of individual approach, SI values were calculated from the mean DPN values of the group

CLINICAL OBSERVATIONS:No mortality or signs of systemic toxicity were observed during the study. Test item precipitate was observed in the 50 and 25% (w/v) dose groups on Days 1-3. There were no Indications of any irritancy at the site of application.

BODY WEIGHTS:No treatment related effects were observed on body weights. No marked body weight loss (>5%) was detected for any experimental animals.

Disintegrations per minute (DPM)

Test item 50 % (w/v) in DMF: 3162.5 DPM

Test item 25 % (w/v) in DMF: 2880.5 DPM

Test item 10 % (w/v) in DMF: 4242.5 DPM

Negative (vehicle) control (DMF): 4441.5 DPM

Positive control (25% (w/v) HCA in DMF): 21332.5 DPM

Table No.4: Individual Body Weights for all Animals with Group Means

Identity

Number

Animal

Number

Test Group

Name

Initial

Body

Weight

(g)

Terminal

Body

Weight*

(g)

Change#

(%)

6961

1

Negative (vehicle) control

DMF

20.3

21.6

6.4

6974

2

20.9

21.7

3.8

6952

3

21.2

22.9

8.0

6963

4

21.8

22.6

3.7

 

 

MEAN

21.1

22.2

5.5

6968

5

Test item in 50%(w/v) in DMF

20.4

22.6

10.8

6953

6

20.7

21.1

1.9

6985

7

21.1

21.9

3.8

6958

8

21.7

22.8

5.1

 

 

MEAN

21.0

22.1

5.4

6972

9

Test item in 25%(w/v) in DMF

21.7

22.3

2.8

6982

10

20.5

20.8

1.5

6957

11

20.3

20.7

2.0

6967

12

21.8

22.7

4.1

 

 

MEAN

21.1

21.6

2.6

6976

13

Test item in 10%(w/v) in DMF

20.4

20.4

0.0

6989

14

20.9

22.3

6.7

6965

15

21.1

21.3

0.9

6977

16

21.9

22.3

1.8

 

 

MEAN

21.1

21.6

2.4

6970

17

Positive control

(25% (w/v) HCA

in DMF)

20.9

22.0

5.3

6979

18

20.7

21.3

2.9

6971

19

21.8

23.6

8.3

6981

20

21.3

21.2

-0.5

 

 

MEAN

21.2

22.0

4.0

*: Terminal body weights were measured on Day 6.

#: = (Terminal Body Weight – Initial Body Weight) / Initial Body Weight x 100

Table No.5: DPM, DPN and Stimulation Index Values for all Groups

Proliferation assay:

Test Group Name

Measured DPM / group

DPM

Number
lymph nodes

DPN

Stimulation Index

Background (5 % (w/v) TCA)

74-31

-

-

-

-

(-) control (DMF)

4494

4441.5

8

555.2

1.0

Test item 50 % (w/v) in DMF

3215

3162.55

8

395.3

0.7

Test item 25 % (w/v) in DMF

2933

2880.5

8

360.1

0.6

Test item 10 % (w/v) in DMF

4295

4242.5

8

530

1.0

(+) control (25 % (w/v) HCA

21385

21332.5

8

2666.6

4.8

The appearance of the lymph nodes was normal in the negative (vehicle) control group and in all test item treated groups. Larger than normal lymph nodes were observed in the positive control group.

The DPN values observed for the vehicle and positive control substance in this experiment were within the historical control range.

No mortality, cutaneous reactions or signs of toxicity were observed for the positive control substance in the study. A significant lymphoproliferative response (stimulation index value of 4.8) was noted for HCA in the main experiment. This value was considered to confirm the appropriate performance of the assay.

The DPN value of the negative control group was slightly above the general historical control range of vehicles, but this fact was considered not to adversely affect the results of the study. The DPN value of the positive control substance in this experiment was within the historical control range.

Table No.6: Summarized Clinical Observations

Group

Animal No.

                                                             CLINICAL OBSERVATIONS

DAY 1

DAY 2

DAY 3

DAY 4

DAY 5

DAY 6

Negative control

(DMF)

1

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

Symptom free

Symptom free

Symptom free

2

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

Symptom free

Symptom free

Symptom free

3

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

Symptom free

Symptom free

Symptom free

4

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

Symptom free

Symptom free

Symptom free

4-amino-5-

ethylsulfonyl-2-

methoxybenzoic

acid

(50% (w/v)

in DMF)

5

BT: symptom-free

AT: symptom-free*

BT: symptom-free

AT: symptom-free*

BT: symptom-free

AT: symptom-free*

Symptom free

Symptom free

Symptom free

6

BT: symptom-free

AT: symptom-free*

BT: symptom-free

AT: symptom-free*

BT: symptom-free

AT: symptom-free*

Symptom free

Symptom free

Symptom free

7

BT: symptom-free

AT: symptom-free*

BT: symptom-free

AT: symptom-free*

BT: symptom-free

AT: symptom-free*

Symptom free

Symptom free

Symptom free

8

BT: symptom-free

AT: symptom-free*

BT: symptom-free

AT: symptom-free*

BT: symptom-free

AT: symptom-free*

Symptom free

Symptom free

Symptom free

4-amino-5-

ethylsulfonyl-2-

methoxybenzoic

acid

(25% (w/v)

in DMF)

9

BT: symptom-free

AT: symptom-free*

BT: symptom-free

AT: symptom-free*

BT: symptom-free

AT: symptom-free*

Symptom free

Symptom free

Symptom free

10

BT: symptom-free

AT: symptom-free*

BT: symptom-free

AT: symptom-free*

BT: symptom-free

AT: symptom-free*

Symptom free

Symptom free

Symptom free

11

BT: symptom-free

AT: symptom-free*

BT: symptom-free

AT: symptom-free*

BT: symptom-free

AT: symptom-free*

Symptom free

Symptom free

Symptom free

12

BT: symptom-free

AT: symptom-free*

BT: symptom-free

AT: symptom-free*

BT: symptom-free

AT: symptom-free*

Symptom free

Symptom free

Symptom free

4-amino-5-

ethylsulfonyl-2-

methoxybenzoic

acid

(10% (w/v)

in DMF)

13

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

Symptom free

Symptom free

Symptom free

14

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

Symptom free

Symptom free

Symptom free

15

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

Symptom free

Symptom free

Symptom free

16

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

Symptom free

Symptom free

Symptom free

Positive control

(25% (w/v) HCA

in DMF)

17

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

Symptom free

Symptom free

Symptom free

18

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

Symptom free

Symptom free

Symptom free

19

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

Symptom free

Symptom free

Symptom free

20

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

Symptom free

Symptom free

Symptom free

1. BT: before treatment, AT: after treatment

2. *: test item precipitate

Interpretation of results:
GHS criteria not met
Remarks:
STIMULATION INDEX IS < 3 (non sensitizer)
Conclusions:
The stimulation index values were 0.7, 0.6 and 1.0 at concentrations of 50, 25 and 10% (w/v), respectively. The substance does not have sensitisation potential (non-sensitizer) in the Local Lymph Node Assay.
Executive summary:

The skin sensitisation test following dermal exposure was performed according to OECD Guideline 429 and EU method B.42. Based on the results of the Preliminary Compatibility Test, the test item was formulated in N,N-dimethylformamide (DMF) at a highest achievable concentration of 50 % (w/v). The Preliminary Irritation / Toxicity Test was performed in CBA/J Rj mice using two doses: 50 and 25 % (w/v) in DMF. The 50 % (w/v) was selected as top dose for the main test. Based on these results, four female CBA/J Rj mice per group received 50, 25 and 10 % (w/v) of tes item, DMF (vehicle, negative control) and 25 % (w/v) HCA (dissolved in DMF, positive control) in the main test.. The test item solutions were applied on the dorsal surface of ears of experimental animals (25 μL/ear) for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. On Day 6, the cell proliferation in the local lymph nodes was measured by incorporation of tritiated methyl thymidine (3HTdR) and the values obtained were used to calculate stimulation indices (SI). No mortality or signs of systemic toxicity were observed during the study. Test item precipitate was observed in the 50 and 25% (w/v) dose groups on Days 1-3. No treatment related body weight loss was observed in the test item treated animals. There were no indications of any irritancy at the site of application. The stimulation index values were 0.7, 0.6 and 1.0 at concentrations of 50, 25 and 10% (w/v), respectively. All validity criteria were fulfilled. In conclusion, the test item was shown to have no sensitisation potential (non-sensitizer) in the Local Lymph Node Assay.

Endpoint:
skin sensitisation: in vitro
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Justification for type of information:
JUSTIFICATION FOR DATA WAIVING
Study scientifically not necessary / other information available: Study IN VITRO is not necessary because the study IN VIVO already exist.
Reason / purpose:
reference to same study
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

Key study: The skin sensitisation test following dermal exposure was performed according to OECD Guideline 429 and EU method B.42. Based on the results of the Preliminary Compatibility Test, the test item was formulated in N,N-dimethylformamide (DMF) at a highest achievable concentration of 50 % (w/v). No mortality or signs of systemic toxicity were observed during the study. Test item precipitate was observed in the 50 and 25% (w/v) dose groups on Days 1-3. No treatment related body weight loss was observed in the test item treated animals. There were no indications of any irritancy at the site of application. The stimulation index values were 0.7, 0.6 and 1.0 at concentrations of 50, 25 and 10% (w/v), respectively. In conclusion, the test item was shown to have no sensitisation potential (non-sensitizer) in the Local Lymph Node Assay.

Key study data waiving: Study IN VITRO is not necessary because the study IN VIVO already exists.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the available data, the substance is not classified according to CLP Regulation (EC) no. 1272/2008.