Benzenesulfonic acid, 4-C10-13-sec-alkyl derivs., ammonium salts

Endpoint summary

Description of key information

Skin Sensitisation: Sensitising (Category 1B); OECD 429; A. Poole. (2018).

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 Jul - 15 Aug 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study was conducted in accordance with international guidelines and in accordance with GLP. All guideline validity criteria were met.

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

22 July 2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

(EC) No. 440/2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

2003

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese MAFF

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: n/a
- Specific activity: n/a
- Locations of the label: n/a
- Expiration date of radiochemical substance: n/a

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in the dark.
- Stability under test conditions: Assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: soluble in dimethyl formamide
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: No
- Preliminary purification step (if any): No
- Final dilution of a dissolved solid, stock liquid or gel: No
- Final preparation of a solid: No

FORM AS APPLIED IN THE TEST (if different from that of starting material): n/a

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS B.V., Inc., Horst, The Netherlands
- Females (if applicable) nulliparous and non-pregnant: Yes
- Microbiological status of animals, when known: Not reported
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 15 - 23 g
- Housing: The animals were housed in suspended solid floor polypropylene cages furnished with softwood woodflakes
- Diet (e.g. ad libitum): Free access to food (2014C Teklad Global Rodent diet supplied by Envigo RMS (UK) Limited, Oxon, UK)
- Water (e.g. ad libitum): Free access to mains tap water
- Acclimation period: At least 5 days
- Indication of any skin lesions: No

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25 ºC
- Humidity (%): 30 - 70 %
- Air changes (per hr): At least 15 changes/ hour
- Photoperiod (hrs dark / hrs light): 12 h: 12 h (light: dark)
- IN-LIFE DATES: Not reported

Vehicle:

dimethylformamide

Concentration:

5, 10 and 25 % (w/w)

No. of animals per dose:

5

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: in vehicle
- Irritation: Yes
- Systemic toxicity: Yes
- Ear thickness measurements: Yes
- Erythema scores: Yes

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: 2
- Criteria used to consider a positive response: a/a

TREATMENT PREPARATION AND ADMINISTRATION: The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local skin irritation at the highest suitable dose five mice were treated with the test item at , 5, 10 and 25 % (w/w) by daily application of 25 µL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). control group received similar concentration of vehicle only or, α‑Hexylcinnamaldehyde.
Clinical Observations: twice daily on Days 1, 2 and 3 and on a daily basis on Days 4, 5 and 6.
Body Weight: Recorded Day 1 (prior to dosing) and Day 6 (prior to termination).
Ear thickness and irritation: Measured on pre dose and post dose on Day 1, post dose on Days 2 and 3 and on Days 4 to 6.

Positive control substance(s):

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

Statistics:

Data was processed to give group mean values for disintegrations per minute and standard deviations where appropriate. Individual and group mean disintegrations per minute values were assessed for dose response relationships. Data was first assessed for suitability by analysis of normality and homogeneity of variance. If the assumptions that the data are both normally distributed and has homogeneity of variances, then parametric one way analysis of variance (ANOVA) and Dunnett’s multiple comparison procedure were used to determine statistical significance. If the assumptions were not met, non-parametric Kruskal-Wallis Rank Sum and Mann-Whitney U test procedures were used.

Probability values (p) are presented as follows:

P<0.001 ***
P<0.01 **
P<0.05 *
P>0.05 (not significant)

Positive control results:

Concurrent control was within the historical control data. The positive control α-Hexylcinnamaldehyde, tech., 85% gave a Stimulation Index of greater than 3 (5.60) when tested at a concentration of 25% v/v in dimethyl formamide, thus, demonstrating the sensitivity and reliability of the test system.The test item was considered to be a sensitiser under the conditions of the test.

Key result

Parameter:

SI

Value:

ca. 5.82

Test group / Remarks:

25%

Remarks on result:

other: Sensitiser

Key result

Parameter:

SI

Value:

ca. 3.7

Test group / Remarks:

10%

Remarks on result:

other: Sensitiser

Key result

Parameter:

SI

Value:

ca. 3.45

Test group / Remarks:

5%

Remarks on result:

other: Sensitiser

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA: Refer to data tables.

DETAILS ON STIMULATION INDEX CALCULATION: Mean treatment group dpm / mean vehicle dpm

EC3 CALCULATION: Not applicable, all stimulation indices were >3 therefore no EC3 was caculable.

CLINICAL OBSERVATIONS: There were no deaths. No signs of systemic toxicity were noted in teh test or control animals during the test. Ear thickness measurements presented an increase of 6.075 %, 9.677 % and 18.349 % for the 5 %, 10 % and 25 % treatment group respectively, compared to 4.734 % in the vehicle control.

BODY WEIGHTS: Body weight change of the test animals between Day 1 and Day 6 was comparable to that observed in the corresponding control group animals over the same period.

Individual Disintegrations per Minute and Stimulation Index during the Main Test

Treatment Group	Animal Number	dpm/ Animala	Mean dpm/Animal (Standard Deviation)	Stimulation Indexb	Result
Vehicle (dimethyl formamide)	1-1	1577.94	1051.77 (±362.82)	na	na
	1-2	629.00
	1-3	902.72
	1-4	920.16
	1-5	1229.04
Test Item 5% v/vin dimethyl formamide	2-1	2765.48	3625.53** (±609.81)	3.45	Positive
	2-2	3944.71
	2-3	3411.77
	2-4	4400.00
	2-5	3605.67
Test Item 10% v/vin dimethyl formamide	3-1	4416.16	3889.35** (±981.87)	3.70	Positive
	3-2	2595.48
	3-3	3784.73
	3-4	3455.20
	3-5	5195.19
Test Item 25% v/vin dimethyl formamide	4-1	5986.24	6124.84****** (±785.22)	5.82	Positive
	4-2	6989.50
	4-3	4944.16
	4-4	6670.04
	4-5	6034.27
Positive Control Item 25% v/vin dimethyl formamide	5-1	4995.44	5887.91*** (±1968.23)	5.60	Positive
	5-2	3011.45
	5-3	6088.73
	5-4	7770.11
	5-5	7573.81

dpm  = Disintegrations per minute

a     =  Total number of lymph nodes per animal is 2

b     = Stimulation Index of 3.0 or greater indicates a positive result

na    =  Not applicable

***   =  Significantly different from control group p<0.001

Measurement of Ear Thickness and Mean Ear Thickness Changes during teh Main Test

Treatment Group	Animal Number	Ear Thickness Measurement (mm)
		Day 1				Day 2		Day 3		Day 4		Day 5		Day 6
		pre‑dose		post‑dose
		left	right	left	right	left	right	left	right	left	right	left	right	left	right
Vehicle (dimethyl formamide)	1-1	0.20	0.21	0.21	0.21	0.20	0.23	0.22	0.22	0.23	0.21	0.22	0.21	0.20	0.22
	1-2	0.21	0.20	0.20	0.20	0.20	0.21	0.21	0.23	0.21	0.22	0.22	0.22	0.22	0.21
	1-3	0.23	0.20	0.23	0.21	0.22	0.21	0.21	0.22	0.21	0.22	0.21	0.21	0.23	0.23
	1-4	0.22	0.21	0.22	0.21	0.21	0.22	0.22	0.22	0.22	0.22	0.21	0.22	0.22	0.22
	1-5	0.22	0.20	0.20	0.21	0.22	0.20	0.23	0.21	0.23	0.22	0.23	0.21	0.22	0.22
overall mean (mm)		0.211		0.210		0.211		0.219		0.219		0.216		0.221
overall mean ear thickness change (%)		Not applicable		-0.592		0.000		3.550		3.550		2.367		4.734
Treatment Group	Animal Number	Ear Thickness Measurement (mm)
		Day 1				Day 2		Day 3		Day 4		Day 5		Day 6
		pre‑dose		post‑dose
		left	right	left	right	left	right	left	right	left	right	left	right	left	right
Test Item 5% v/vindimethyl formamide	2-1	0.23	0.23	0.23	0.23	0.20	0.23	0.21	0.22	0.22	0.22	0.22	0.23	0.23	0.24
	2-2	0.22	0.21	0.22	0.22	0.20	0.22	0.20	0.20	0.22	0.21	0.21	0.23	0.23	0.21
	2-3	0.23	0.20	0.22	0.21	0.22	0.22	0.21	0.22	0.21	0.23	0.22	0.23	0.25	0.24
	2-4	0.20	0.22	0.20	0.22	0.20	0.22	0.21	0.22	0.22	0.22	0.22	0.22	0.22	0.20
	2-5	0.20	0.20	0.20	0.22	0.19	0.21	0.20	0.21	0.21	0.21	0.21	0.21	0.22	0.23
overall mean (mm)		0.214		0.217		0.211		0.210		0.217		0.220		0.227
overall mean ear thickness change (%)		Not applicable		1.402		-1.402		-1.869		1.402		2.804		6.075

Treatment Group	Animal Number	Ear Thickness Measurement (mm)
		Day 1				Day 2		Day 3		Day 4		Day 5		Day 6
		pre‑dose		post‑dose
		left	right	left	right	left	right	left	right	left	right	left	right	left	right
Test Item 10% v/vin dimethyl formamide	3-1	0.21	0.23	0.20	0.23	0.23	0.20	0.22	0.20	0.21	0.21	0.22	0.23	0.24	0.25
	3-2	0.21	0.20	0.20	0.20	0.21	0.23	0.22	0.24	0.21	0.24	0.22	0.24	0.25	0.24
	3-3	0.21	0.22	0.20	0.20	0.22	0.23	0.21	0.23	0.22	0.23	0.24	0.23	0.24	0.22
	3-4	0.22	0.22	0.22	0.21	0.24	0.22	0.23	0.22	0.23	0.22	0.23	0.23	0.24	0.23
	3-5	0.23	0.22	0.22	0.22	0.22	0.21	0.21	0.22	0.21	0.23	0.23	0.25	0.25	0.22
overall mean (mm)		0.217		0.210		0.221		0.220		0.221		0.232		0.238
overall mean ear thickness change (%)		Not applicable		-3.226		1.843		1.382		1.843		6.912		9.677
Treatment Group	Animal Number	Ear Thickness Measurement (mm)
		Day 1				Day 2		Day 3		Day 4		Day 5		Day 6
		pre‑dose		post‑dose
		left	right	left	right	left	right	left	right	left	right	left	right	left	Right
Test Item 25%v/vin dimethyl formamide	4-1	0.24	0.21	0.24	0.21	0.24	0.22	0.24	0.22	0.25	0.24	0.25	0.25	0.28	0.25
	4-2	0.22	0.21	0.21	0.21	0.21	0.21	0.21	0.21	0.23	0.22	0.23	0.23	0.26	0.21
	4-3	0.21	0.21	0.21	0.21	0.23	0.21	0.24	0.23	0.25	0.23	0.25	0.24	0.28	0.27
	4-4	0.21	0.21	0.21	0.21	0.23	0.22	0.23	0.23	0.24	0.24	0.26	0.25	0.24	0.26
	4-5	0.23	0.23	0.23	0.23	0.24	0.21	0.24	0.22	0.24	0.23	0.24	0.25	0.28	0.25
overall mean (mm)		0.218		0.217		0.222		0.227		0.237		0.245		0.258
overall mean ear thickness change (%)		Not applicable		-0.459		1.835		4.128		8.716		12.385		18.349

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

The test item was considered to be a sensitiser (Category 1B) under the conditions of the test. Based on the condition of this study, the test item meet the criteria for classification according to the Globally Harmonized Classification System and Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures.

Executive summary:

OECD 429 (2017) - In a dermal sensitisation study with the test item, Benzenesulfonic acid, 4-C10-13-sec-alkyl derivs., ammonium salts in dimethyl fromamide was evaluated young female adult mice (CBA/Ca (CBA/CaOlaHsd)) using the Local Lymph Node Assay (LLNA)

 

Following a preliminary screening test in which no clinical signs of toxicity were noted at the maximum concentration tested, this concentration was selected as the highest dose investigated in the main test. Three groups, each of five animals, were treated with 50 μL (25 μL per ear) of the undiluted test item or the test item as a solution in the vehicle at concentrations 5, 10 and 25 % v/v. A further group of five animals was treated with the vehicle alone. A concurrent positive control test, using a group of five animals, was also performed with the known sensitiser, α-Hexylcinnamaldehyde tech., 85%, at a concentration of 25 % v/v in the vehicle.

 

There were no deaths.  No signs of systemic toxicity were noted in teh test or control animals during the test.  Ear thickness measurements presented an increase of 6.075 %, 9.677 % and 18.349 % for the 5 %, 10 % and 25 % treatment group respectively, compared to 4.734 % in the vehicle control.  Body weight change of the test animals between Day 1 and Day 6 was comparable to that observed in the corresponding control group animals over the same period. The Stimulation Index, expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group, was determined for each treatment group.

 

There was indication that the test item elicits a Stimulation Index ≥ 3 when tested up to  25 %, the test item was considered to be a sensitiser under the conditions of the test.

 

In this study, Benzenesulfonic acid, 4-C10-13-sec-alkyl derivs., ammonium salts was considered a dermal sensitiser.

 

Based on the condition of this study, the test item  met the criteria for classification  as a sensitiser (category 1B) according to the Globally Harmonized Classification System and Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

OECD 429 (2017) - In a dermal sensitisation study with the test item, Benzenesulfonic acid, 4-C10-13-sec-alkyl derivs., ammonium salts in dimethyl fromamide was evaluated young female adult mice (CBA/Ca (CBA/CaOlaHsd)) using the Local Lymph Node Assay (LLNA)

 

Following a preliminary screening test in which no clinical signs of toxicity were noted at the maximum concentration tested, this concentration was selected as the highest dose investigated in the main test. Three groups, each of five animals, were treated with 50 μL (25 μL per ear) of the undiluted test item or the test item as a solution in the vehicle at concentrations 5, 10 and 25 % v/v. A further group of five animals was treated with the vehicle alone. A concurrent positive control test, using a group of five animals, was also performed with the known sensitiser, α-Hexylcinnamaldehyde tech., 85%, at a concentration of 25 % v/v in the vehicle.

 

There were no deaths.  No signs of systemic toxicity were noted in teh test or control animals during the test.  Ear thickness measurements presented an increase of 6.075 %, 9.677 % and 18.349 % for the 5 %, 10 % and 25 % treatment group respectively, compared to 4.734 % in the vehicle control.  Body weight change of the test animals between Day 1 and Day 6 was comparable to that observed in the corresponding control group animals over the same period. The Stimulation Index, expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group, was determined for each treatment group.

 

There was indication that the test item elicits a Stimulation Index ≥ 3 when tested up to  25 %, the test item was considered to be a sensitiser under the conditions of the test.

 

In this study, Benzenesulfonic acid, 4-C10-13-sec-alkyl derivs., ammonium salts was considered a dermal sensitiser.

 

Based on the condition of this study, the test item  met the criteria for classification  as a sensitiser (category 1B) according to the Globally Harmonized Classification System and Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The test item  elicits a sensitisation index ≥ 3 in all three tested concentrations with SI of 5.82 observed at the highest concentration tested. Therefore, it met the criteria for classification as a  sensitiser (category 1B) according to the Globally Harmonized Classification System and Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures