Benzene, mono-C10-13-alkyl derivs., distn. residues, sulfonated, barium salts

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 October 2015 to 29 October 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes

Remarks:

United States Code of Federal Regulations, Title 40, Parts 792

Type of study:

Buehler test

Justification for non-LLNA method:

According to OECD 429 guideline, „despite the advantages of the LLNA over TG 406, it should be recognised that there are certain limitations that may necessitate the use of TG 406 (13) (e.g. false negative findings in the LLNA with certain metals, false positive findings with certain skin irritants [such as some surfactant type chemicals]…” Basketer & Kimber (2011) and Ball et al. (2011) conducted a battery of in vivo and in vitro tests with several exemplary surfactant and confirmed that the LLNA tends to overestimate the sensitization potential of surfactants. Even though the registered substance barium sulfonate has no surfactant properties, the read across substances are in several cases, and the Buehler tests (OECD TG 406) conducted with the calcium sulfonate read-across substances are more appropriate to differentiate between the skin sensitization potential of low and high TBN calcium sulfonates. Calcium sulfonates with a large excess of calcium carbonate are referred to as high overbased or high total base number (TBN) calcium sulfonates, whereas calcium sulfonates with small amounts of added calcium carbonate are called low overbased or low TBN calcium sulfonates. The results of numerous animal studies and human repeat insult patch tests clearly showed that low TBN calcium sulfonates (TBN < 300) are skin sensitisers with a specific concentration limit (SCL) of 10% and that high TBN calcium sulfonates (TBN ≥ 300) are not skin sensitisers. Thus, to confirm this assumption Buehler test was chosen.

Test material

Test material information

1

Reference substance name:

Benzene, mono-C10-13-alkyl derivs., distn. residues, sulfonated, barium salts

EC Number:

947-582-0

Molecular formula:

Too complex

IUPAC Name:

Benzene, mono-C10-13-alkyl derivs., distn. residues, sulfonated, barium salts

Test material form:

liquid: viscous

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Kept in a controlled temperature area

In vivo test system

Test animals

Species:

guinea pig

Strain:

Hartley

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: Male range finding animals - 5 weeks. Female range finding animals - 6 weeks. Male main phase animals - 6 weeks. Female main phase animals - 7 weeks. Male second range finding animals - 9 weeks. Female second range finding - 10 weeks.
- Weight at study initiation: Male range finding animals - 348-380 g. Female range finding animals - 326-330 g. Male main phase animals - 365-474 g. Female main phase animals - 349-433 g. Male second range finding animals - 564-597 g. Female second range finding - 463-477 g.
- Housing: Pair housing (same sex and dosing group. Polycarbonate cages containing direct bedding material.
- Diet: ad libitum (except during procedures)
- Water: Municipal tap water treated by reverse osmosis and UV irradiation available ad libitum
- Acclimation period: At least 7 days
- Indication of any skin lesions: Only healthy animals were used during the test

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-22 °C
- Humidity (%): 45 - 54 %
- Air changes (per hr): Ten or more changes per hour with 100 % fresh air (no recirculation)
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle
- IN-LIFE DATES: From: To: 25-27 November 2015

Study design: in vivo (non-LLNA)

Induction

Route:

epicutaneous, occlusive

Vehicle:

unchanged (no vehicle)

Concentration / amount:

100 % (0.3 mL)

Day(s)/duration:

6 hours on days 1, 7 and 14

Adequacy of induction:

highest technically applicable concentration used

Challenge

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

other: mineral oil

Concentration / amount:

25 % (0.3 mL)

Day(s)/duration:

6 hours on Day 28

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

Test group:
10 animals per sex per dose
Challenge control:
5 animals per sex per dose
HCA Test:
5 animals per sex per dose (2.5 % and 1 % HCA in ethanol)
HCA Control:
5 animals per sex per dose (2.5 % and 1 % HCA in ethanol)

Details on study design:

RANGE FINDING TESTS:
1st Range finder:
2 animals per sex per dose (concentrations 100, 75, 50 and 25 %)
2nd Range finder:
2 animals per sex per dose (concentrations 75 and 50 %)

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 3
- Exposure period: Days 1, 7 and 14
- Test groups: Test group
- Control group: Positive control group
- Site: 1
- Frequency of applications: 1 application
- Duration: 6 hours
- Concentrations: 100 % test group, 5 % in ethanol HCA control group

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: Day 28
- Exposure period: 6 hours
- Test groups: Test and Challenge groups
- Control group: HCA test and Control group
- Site: Test and Challenge control group - site 2. HCA test and Control - Site 2 at 2.5 % and Site 4 at 1 %
- Concentrations: Test and Challenge control - 25 % in mineral oil. HCA test and Control - 1 and 2.5 %
- Evaluation (hr after challenge): 24 and 48 hours

Challenge controls:

Yes, challenge control performed with 25 % test material in mineral oil on day 28.

Positive control substance(s):

yes

Remarks:

α-Hexylcinnamaldehyde (HCA) (in ethanol)

Results and discussion

Positive control results:

Following challenge with 2.5% w/v HCA in acetone, dermal scores of 1 or 2 were noted in 10/10 HCA test animals at the 24-hour and 48-hour scoring intervals. Dermal reactions in the HCA control animals were limited to scores of 0. Group mean dermal scores were higher in the HCA test animals (1.8 to 1.9) compared to the HCA control animals (0.0). Following challenge with 1.0% w/v HCA in acetone, dermal scores of 1 or 2 were noted in 9/10 HCA test animals at the 24-hour scoring interval and in 8/10 HCA test animals at the 48-hour scoring interval. Dermal reactions in the remaining HCA test animal and HCA control animals were limited to scores of 0 or ±. Group mean dermal scores were higher in the HCA test animals (1.0 to 1.1) compared to the HCA control animals (0.0).

In vivo (non-LLNA)

Results

Results 1

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

25 %

No. with + reactions:

17

Total no. in group:

20

Clinical observations:

Group mean dermal scores 1.2 to 1.3. No effects on bodyweight were observed.

Remarks on result:

positive indication of skin sensitisation

Results 2

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

25 %

No. with + reactions:

19

Total no. in group:

20

Clinical observations:

Group mean dermal scores 1.2 to 1.3. No effects on bodyweight were observed.

Remarks on result:

positive indication of skin sensitisation

Results 3

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

25 % (challenge control)

No. with + reactions:

1

Total no. in group:

10

Clinical observations:

Group mean dermal scores 0.2

Remarks on result:

no indication of skin sensitisation

Results 4

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

25 % (challenge control)

No. with + reactions:

1

Total no. in group:

10

Clinical observations:

Group mean dermal scores 0.2

Remarks on result:

no indication of skin sensitisation

Results 5

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

2.5 %

No. with + reactions:

10

Total no. in group:

10

Remarks on result:

positive indication of skin sensitisation

Results 6

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

1 %

No. with + reactions:

9

Total no. in group:

10

Remarks on result:

positive indication of skin sensitisation

Results 7

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

2.5 %

No. with + reactions:

10

Total no. in group:

10

Remarks on result:

positive indication of skin sensitisation

Results 8

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

1 %

No. with + reactions:

8

Total no. in group:

10

Remarks on result:

positive indication of skin sensitisation

Any other information on results incl. tables

Range finding phase:

Based on the initial range-finding phase, the 100% as received concentration was considered appropriate for conducting the induction phase as no dermal irritation was present at concentrations of 100%, 75%, 50%, or 25%. During induction, irritation was observed at the 100% concentration, therefore, prior to challenge, a second range-finding phase was conducted by repeating the 75% and 50% concentration. The second range-finding showed a progressive decrease in response with concentration. Therefore, the weight-of-evidence indicated that it was appropriate to consider a 25% concentration as the highest non-irritating concentration.

Induction phase:

During the induction phase, dermal scores of ± (slight patchy erythema), 1 (slight, but confluent or moderate patchy erythema), and 2 (moderate, confluent erythema) were noted for the test animals.

Table 2: Dermal Scores - Results of challenge readings

Group	Sex	Animal	Dermal Scores (25 %)
			24	48 Hours
Test	Male	6005	1	1
		6006	2	2
		6007	1	1
		6008	1	1
		6009	2 (ED-1)	2
		6010	2	2
		6011	1	1
		6012	2	2
		6013	1	1
		6014	1	1
	Female	6049	1	2
		6050	2	2
		6051	1	1
		6052	2	1
		6053	0	0
		6054	1	1
		6055	1	1
		6056	±	1
		6057	2	2
		6058	0	1
Mean Test			1.2	1.3
Challenge control	Male	6015	0	0
		6016	0	0
		6017	±	±
		6018	0	0
		6019	0	0
	Female	6059	±	±
		6060	1	1
		6061	0	0
		6062	0	0
		6063	0	0
Mean Challenge control			0.2	0.2

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

Based on the results of this study, the test material is considered to be a contact sensitizer in guinea pigs. The criterion for sensitization (dermal scores ≥ 1 in at least 15% of the test animals) was met at challenge, even with a single naïve animal responding with a dermal score of 1, as 35% of the test animals responded with a dermal score of 2. There was a clear demonstration of sensitization potential in the guinea pig based on increasing irritation during induction with a similar to higher dermal response at challenge, as would be expected in the guinea pig when sensitized to a test substance. The results of the HCA positive control study demonstrated that a valid test was performed and indicated that the test design would detect potential contact sensitizers.

Executive summary:

SUMMARY

The dermal sensitization potential of the test material was evaluated in Hartley-derived albino guinea pigs. In the induction phase, 10 male and 10 female guinea pigs were topically treated with the test material, as received, once per week, for 3 consecutive weeks.

Following a 2-week rest period, a challenge was performed whereby the 20 test and 10 previously untreated (naïve) challenge control guinea pigs were topically treated with 25% of the test material in mineral oil.

Anα-Hexylcinnamaldehyde (HCA) positive control group consisting of 10 HCA test and 10 HCA control guinea pigs was included in this study. The animals were treated as above with

the HCA test animals receiving 5% w/v HCA in ethanol for induction and 2.5% and 1.0% w/v HCA in acetone for challenge.

 

Test Material

Following challenge with 25% test material in mineral oil, dermal scores of 1 or 2 were noted in 17/20 test animals and 1/10 challenge control animals at the 24-hour scoring interval. At the 48-hour scoring interval, dermal scores of 1 or 2 were noted in 19/20 test animals and 1/10 challenge control animals. Dermal reactions in the remaining test and challenge control animals were scores of 0 or ±. Group mean dermal scores were higher in the test animals (1.2 to 1.3) as compared to challenge control animals (0.2 to 0.2).

 

 α-Hexylcinnamaldehyde (HCA) (Positive control)

Following challenge with 2.5% w/v HCA in acetone, dermal scores of 1 or 2 were noted in 10/10 HCA test animals at the 24-hour and 48-hour scoring intervals. Dermal reactions in the HCA control animals were limited to scores of 0. Group mean dermal scores were higher in the HCA test animals (1.8 to 1.9) compared to the HCA control animals (0.0).

Following challenge with 1.0% w/v HCA in acetone, dermal scores of 1 or 2 were noted in 9/10 HCA test animals at the 24-hour scoring interval and in 8/10 HCA test animals at the 48-hour scoring interval. Dermal reactions in the remaining HCA test animal and HCA control animals were limited to scores of 0 or ±. Group mean dermal scores were higher in the HCA test animals (1.0 to 1.1) compared to the HCA control animals (0.0).

Conclusion

Based on the results of this study, the test material is considered to be a contact sensitizer in guinea pigs. The criterion for sensitization (dermal scores≥1 in at least 15% of the test animals) was met at challenge, even with a single naïve animal responding with a dermal score of 1, as 35% of the test animals responded with a dermal score of 2. There was a clear demonstration of sensitization potential in the guinea pig based on increasing irritation during induction with a similar to higher dermal response at challenge, as would be expected in the guinea pig when sensitized to a test substance. The results of the HCA positive control study demonstrated that a valid test was performed and indicated that the test design would detect potential contact sensitizers.