Reaction product of naphthalene, propan-2-ol, sulfonated and neutralized by caustic soda

Endpoint summary

Description of key information

LLNA (GLP and OECD 429 guideline study): ambigous (SI = 3.19 at the highest tested concentration but irritating effects are also observed)
SENS-IS test (non GLP study): ambigous. The test item was found irritant to skin and weakly sensitizing.
OECD QSAR Toolbox: negative: No alerts found for any of the 3 isomers of the registered substance or their predicted metabolites based on a profiling approach.

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:

weight of evidence

Study period:

from 26 March 2012 to 27 June 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was performed according to EU / OECD guidelines and GLP

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

N° 2011/40

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: 8 weeks (main test); 9 weeks (preliminary test)
- Weight (mean) at study initiation: 18.9 g +/- 0.8 g
- Fasting period before study: No
- Housing: The animals were housed by group of two (preliminary test) or four (main test) in polycarbonate cages (Techniplast 1145T, 435 cm2, 36.9 x 15.6 x 13.2 cm) containing autoclaved sawdust (SICSA, Alfortville, France). Each cage contained two enrichments (mouse hut and cocoon). In the main test, on day 6 before the 3H-TdR injections, the animals were individually housed in disposable crystal polystyrene cages (22.0 cm x 8.5 cm x 8.0 cm).
- Diet (ad libitum): SSNIFF R/M-H pelleted diet, batch No. 5776558 (SSNIFF Spezialdiäten GmbH, Soest, Germany)
- Water (ad libitum): tap water (filtered with a 0.22 µm filter)
- Acclimation period: 6 or 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air.
- Photoperiod (hrs dark / hrs light): 12h/12h (7:00 - 19:00)

IN-LIFE DATES (Main test): From: 05 April 2012 to 10 April 2012

Vehicle:

propylene glycol

Concentration:

0, 5, 10 and 25% (w/v) in propylene glycol

No. of animals per dose:

4 females/dose

Details on study design:

RANGE FINDING TESTS:
- Irritation:
A preliminary test was performed in four animals to assess the irritant potential of the test item (through ear thickness measurement). The tested concentrations were 2.5, 5, 10 and 25%. Dryness of the skin was observed on day 6 in right ear of both females treated at 25%. No notable increase in ear thickness was observed at any tested concentrations. Therefore, in the main test, concentrations applied were 5, 10 and 25%.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response:
The proliferation of lymphocytes in the lymph node draining the application site was measured by incorporation of 3H-TdR. The results were expressed as disintegrations per minute (dpm) per group and as dpm/node. The obtained values were used to calculate Stimulation Indices (SI). The test item is considered as a skin sensitizer when the SI for a dose group is > or = 3 together with consideration of a dose-response relationship. Other relevant criteria such as radioactivity levels and ear thickness are also taken into account to evaluate the data.

TREATMENT PREPARATION AND ADMINISTRATION:
On days 1, 2 and 3, a dose-volume of 25 µL of the vehicle control or dosage form preparations was applied to the dorsal surface of both ears, using an adjustable pipette fitted with a plastic tip. In order to avoid licking and to ensure an optimized application of the test materials, the animals were placed under light isoflurane anesthezia during the administration. No massage was performed but the tip was used to spread the preparation over the application site. No rinsing was performed.
Auricular Lymph Node Cell (ALNC) proliferation assay:
Lymph node cell proliferative response was measured as described by Kimber and Dearman (1). On day 6 of the main test, all animals of all groups received a single intravenous injection of 250 µL of 0.9% NaCl containing 20 µCi of 3H-TdR (specific activity of 20 Ci/mmol) via the tail vein.
Approximately 5 hours later, the main test animals were sacrificed by an intraperitoneal injection of pentobarbital sodium followed by a cervical dislocation and the auricular lymph nodes were excised. The lymph nodes were pooled for each experimental group. A single cell suspension of ALNC was prepared by mechanical disaggregation in Petri dishes with the plunger of a syringe.
Cell suspensions were washed once with 15 mL of 0.9% NaCl. Each cell suspension was then centrifuged and pellets were precipitated with 3 mL of 5% (w/v) trichloroacetic (TCA) at +4°C overnight. After a last centrifugation, the pellets were precipitated with 1 mL of 5% TCA. Three milliliters of Ultima GoldxR scintillation fluid (Packard) were added in order to measure incorporation of 3H-TdR using  scintillation counting.
The results were expressed as disintegrations per minute (dpm) per group and as dpm/node. Stimulation Indices (SI) were calculated according to the following formula: SI = dpm per node of the treated group / dpm per node of the vehicle control group.

Positive control substance(s):

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

Statistics:

According to the OECD guideline 429, a statistical analysis of the data is not mandatory.

Positive control results:

The threshold positive value of 3 for the SI was reached in the positive control group (SI = 13.90).

Key result

Parameter:

SI

Value:

0.92

Test group / Remarks:

5%

Key result

Parameter:

SI

Value:

1.14

Test group / Remarks:

10%

Key result

Parameter:

SI

Value:

3.14

Test group / Remarks:

25%

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: DPM per lymph node: 264.38 for control 243.63 at 5% 301.63 at 10% 831.00 at 25%

Summary results of the proliferation assay:

Treatment and concentrations	Number of nodes per group	dpm per group	dpm per node	Stimulation index (SI)	Increase in ear thickness (% between day 1 and day 6)	Irritation level	ECs value
Vehicle	8	2115	264.38	/	-4.90	/	23.95%
Test item 5%	8	1949	243.63	0.92	-0.99	I
Test item 10%	8	2413	301.63	1.14	2.94	I
Test item 25%	8	6648	831.00	3.14	30.77	II
HCA 25%	8	29405	3675.63	13.90	/	/

 

dpm = disintegrations per minute

I = non-irritant (increase in ear thickness < 10%)

III = irritant (increase in ear thickness > 25%)

ECs value = theoretical concentration resulting in a SI value of 3

HCA = alpha-hexylcinnamaldehyde

Other observations:

No unscheduled deaths occurred during the main test. 

No clinical signs were observed in any animals

The body weight change of test item-treated animals was similar to that of control animals.

Residual test item was observed on day 3 in 1/4 females treated at 25%.

On day 6, erythema and dryness of ear skin were noted in all females treated at 25%. Erythema was also observed in 1/4 females treated at 10%.

A mean increase in ear thickness of 31% was observed in females treated at 25%.

Interpretation of results:

ambiguous

Remarks:

Migrated information Criteria used for interpretation of results: expert judgment

Conclusions:

In this study the stimulation indices (S.I.) of 0.92, 1.14 and 3.14 were determined with the test item at concentrations of 5, 10 and 25% in propylene glycol.
As a significant lymphoproliferation (SI > 3) was noted at the concentration of 25%, the EC3 value was determined (23.95%).
Based on the positive SI value observed at the concentration of 25%, the test material may be considered as a skin sensitiser. However, irritation was also observed at this concentration and is known to be potentially involved in false positive lymphoproliferation responses.
Therefore, the test item may not be a skin sensitizer for the following reasons: an increase in ear thickness was above the limit of 25% in two out of four mice (Nos. 14 and 15); the SI cut-off of 3 was only just exceeded (SI = 3.14). Therefore it is highly likely that this weak lymphoproliferation was the consequence of the local irritation clearly observed in 2/4 animals of this group.

Based on the results of this study, it was not possible to definitively reject the impact of irritation on the stimulation index observed at the highest concentration, therefore it was not possible to clearly conclude on the potential of reaction product of naphthalene, propan-2-ol, sulfonated and neutralized by caustic soda to induce delayed contact hypersensitivity.

Executive summary:

In order to study a possible contact allergenic potential of reaction product of naphthalene, propan-2-ol, sulfonated and neutralized by caustic soda, a local lymph node assay (LLNA) was performed according to the OECD Guideline 429 and under GLP regulations.

For this purpose, three groups of four female mice received the test item by topical route to the dorsal surface of both ears on days 1, 2 and 3 at concentrations of 5, 10 or 25% (maximum technically applicable concentration) under a dose‑volume of 25 µL. One negative control group of four females received the vehicle (Propylene Glycol) under the same experimental conditions. Additionally, one positive control group of 4 females received the positive control,α‑hexylcinnamaldehyde (HCA), at 25% in a mixture acetone/olive oil (4/1; v/v) under the same experimental conditions.

From day 1 to day 3 then on day 6, the thickness of the left ear of each animal was measured, except in animals of the positive control group and the local reactions were recorded. Each animal was observed at least once a day for mortality and clinical signs. Body weight was recorded on days 1 and 6. After 2 days of resting, on day 6, the animals received a single intravenous injection of tritiated methyl thymidine (³H-TdR). Approximately 5 hours later, the animals were sacrificed and the auricular lymph nodes were excised. The proliferation of lymphocytes in the lymph node draining the application site was measured by incorporation of³H-TdR. The results were expressed as disintegrations per minute (dpm) per group and as dpm/node. The obtained values were used to calculate Stimulation Indices (SI). A test item is regarded as a sensitizer in the LLNA if the exposure to one or more test concentrations resulted in 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the stimulation index (S.I.).

 

No unscheduled deaths occurred and no clinical signs were observed in any animals throughout the study. On day 6, erythema and dryness of ear skin were noted in all females treated at 25%. Erythema was also observed in 1/4 females treated at 10%. An increase in ear thickness of 31% was observed in females treated at 25%.

In this study the stimulation indices (S.I.) of 0.92, 1.14 and 3.14 were determined with the test item at concentrations of 5, 10 and 25% in propylene glycol.

As a significant lymphoproliferation (SI > 3) was noted at the concentration of 25%, the EC3 value was determined (23.95%).

Based on the positive SI value observed at the concentration of 25%, the test material may be considered as a skin sensitiser. However, irritation was also observed at this concentration and is known to be potentially involved in false positive lymphoproliferation responses.

Therefore, the test item may not be a skin sensitizer for the following reasons: an increase in ear thickness was above the limit of 25% in two out of four mice (Nos. 14 and 15); the SI cut-off of 3 was only just exceeded (SI = 3.14). Therefore it is highly likely that this weak lymphoproliferation was the consequence of the local irritation clearly observed in 2/4 animals of this group.

Based on the results of this study, it was not possible to definitively reject the impact of irritation on the stimulation index observed at the highest concentration, therefore it was not possible to clearly conclude on the potential of reaction product of naphthalene, propan-2-ol, sulfonated and neutralized by caustic soda to induce delayed contact hypersensitivity.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

from 26 September 2012 to 20 February 2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study was performed neither in compliance with the GLP nor in accordance with existing testing guideline but the study is well documented and meets acceptable scientific principles.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

no guideline followed

Principles of method if other than guideline:

The study assessed the potential of the test product to specifically induce the expression of irritation and/or sensitisation biomarkers in an in vitro 3D skin model.
Application of irritant and/or sensitizer onto the skin induces the reversible destruction of tissue as well as the activation of the innate and adaptative immune
system. These biological changes are induced by the specific expression (or repression) of distinct set of genes with a kinetic dependent of the type of reaction
observed.
The SENS-IS test is based on the analysis by RTPCR (reverse transcription polymerase chain reaction) of two set of genes, one specifically reflecting the irritant potential of a chemical after application to the skin (“IRRITATION” set of genes) and the other set of genes correlating with the sensitization potential (splited in 2 subsets of genes namely “SENS_IS”and “REDOX”). By measuring the level of expression of these two separate sets of genes at a given time point after application and by comparison with internal negative, irritant and sensitizer positive control, the SENS-IS test can determine the irritant and sensitizer potential of a
chemical.

GLP compliance:

no

Remarks:

No GLP approval for this specific laboratory

Type of study:

other: SENS-IS test

Positive control results:

The number of gene over expressed for the different positive control (irritation and sensitisation) were higher than 7 in experiments BM87 and BM88. Therefore the test was considered as valid. See details in Tables 7.4.1/1 and 7.4.1/2.

Remarks on result:

other: Supragil WP was found irritant to skin and weakly sensitizing.

Three independent experiments were performed: BM86, BM87 and BM88. The IC50 value for Episkin was 1.2 mg/ml, 1.4 mg/ml and 1.4 mg/ml respectively for experiment BM86, BM87 and BM 88. As in BM86 the IC50 (1.2 mg/ml) corresponds to the limit of the acceptance criteria and DMSO (negative control) was detected positive for sensitization this experiment was considered as invalid (data not shown).

Table 7.4.1/1 :Results of experiment BM87

	Positive control for irritation	Positive controls for sensitisation		Negative control	Test item
Number of overexpressed genes	SLS 5%	HCA 50%	TNBS 1 %	DMSO 100%	50 %	10 %	1 %	0.1 %
IRRITATION	21	16	7	7	23	20	11	8
SENS_IS	3	9	3	3	14	10	7	5
REDOX	2	13	13	2	9	4	6	5
CONCLUSION
IRRITATION	POSITIVE	POSITIVE	WEAK	WEAK	Too irritant	POSITIVE	POSITIVE	POSITIVE
SENSITIZATION	NEGATIVE	POSITIVE	POSITIVE	NEGATIVE	NC	POSITIVE	POSITIVE	NEGATIVE

  NC : non-conclusive

Table 7.4.1/2 :Results of experiment BM88

	Positive control for irritation	Positive controls for sensitisation		Negative control	Test item
Number of overexpressed genes	SLS 5%	HCA 50%	TNBS 1 %	DMSO 100%	50 %	10 %	1 %	0.1 %
IRRITATION	19	14	5	2	21	16	4	7
SENS_IS	1	2	4	3	13	8	4	4
REDOX	1	12	11	0	8	4	3	5
CONCLUSION
IRRITATION	POSITIVE	POSITIVE	NEGATIVE	NEGATIVE	Too irritant	POSITIVE	POSITIVE	POSITIVE
SENSITIZATION	NEGATIVE	POSITIVE	POSITIVE	NEGATIVE	NC	POSITIVE	POSITIVE	NEGATIVE

  NC : non-conclusive

At a dose of 50%, SUPRAGIL WP was too irritant onto the EPISKIN model in both experiments to fulfill the acceptance criteria (number of genes in the «IRRITATION “ group >20).

At 10% although the number of “IRRITATION” genes was still high it remained within the acceptance criteria. At that dose SUPRAGIL WP induced a number of genes in the SENS-IS group above 7 in both experiments. The level of genes induced in the REDOX group was below 7.

At 1% and 0.1% the test item was slightly irritant.

At 1% the number of gene induced in the SENS-IS group was equal to 7 in experiment BM87. However, a sensitizing potential at this dose level is unlikely since this number of genes induced corresponds to the lower limit for considering a positive response (≥ 7) and it was not confirmed in experiment BM88.

Interpretation of results:

ambiguous

Remarks:

Migrated information Criteria used for interpretation of results: expert judgment

Conclusions:

Under the experimental conditions of this study, the test product, Supragil WP, was found irritant to skin and weakly sensitizing. However, the mean number of “irritation” genes overexpressed at 10% (n=20 in experiment #2 and 16 in experiment #3) was close to the limit of 20 genes above which no conclusion can be made owing to cell stress. Therefore, it cannot be entirely excluded that the overexpression of 10 and 8 SENS-IS genes in experiments n° 2 and 3, respectively at the dose of 10% could be the consequence of excessive irritation.

Executive summary:

An in vitro test was performed in order to assess the irritation and/or skin sensitisation potential (SENS-Is test) of Reaction product of naphthalene, propan-2-ol, sulfonated and neutralized by caustic soda. This test was not GLP complian and no test guideline is available for this in vitro test.

The SENS-IS test is based on the analysis by RT-PCR (reverse transcription polymerase chain reaction) of two set of genes, one specifically reflecting the irritant potential of a chemical after application to the skin (“IRRITATION” set of genes) and the other set of genes correlating with the sensitization potential (splited in 2 subsets of genes namely “SENS_IS”and “REDOX”). By measuring the level of expression of these two separate sets of genes at a given time point after application and by comparison with internal negative, irritant and sensitizer positive control, the SENS-IS test can determine the irritant and sensitizer potential of a chemical.

The test item solubilized in phosphate buffer saline (PBS) at the concentrations of 50%; 10%; 1% and 0.1% was applied on the top of each reconstituted epidermis (Episkin model). The test product was gently spread on the epidermis surfaces to ensure it covers all the surface. After 15 mn exposure, the Episkin were rinsed with PBS. Epidermis were then incubated at 37°C for 6 hours.

After incubation, the complete epidermis was collected, placed in a RNAzol solution and the total RNA was isolated by homogenization of the skin. After reverse transcription, quantitative gene expression was measured by RT-PCR. For each analysis three negative controls (PBS, olive oil and DMSO treated skins), a positive irritation control (5% SLS) and two positive sensitization controls (HCA at 50% and TNBS at 1%) were performed. The test product and the controls were tested in at 3 experiments using different batches of Episkin models. However the results of one experiment were considered invalid as some acceptance criteria were not met.

The test item used at 50% was too irritant to the episkin model, therefore the result at this concentration were not taken into account. At 10% although the number of “IRRITATION” genes was still high it remained within the acceptance criteria (<20). At that dose the test item induced a number of genes in the SENS-IS group above 7 in both experiments (7 being the lower limit for considering a positive response). The level of genes induced in the REDOX group was below 7 in both experiments. At 1% and 0.1% the test item was slightly irritant. At 1% the number of gene induced in the SENS-IS group was equal to 7 in one experiment only. However, a sensitizing potential at this dose level is unlikely since this number of genes induced corresponds to the lower limit for considering a positive response (≥ 7) and it was not confirmed in the second experiment. At 0.1%, the number of genes in the SENS-IS and REDOX groups was below 7 in both experiments.

In conclusion, under the experimental conditions of this study, the test item was found irritant to skin and weakly sensitizing. However, the mean number of “irritation” genes overexpressed at 10% (n=20 in experiment #2 and 16 in experiment #3) was close to the limit of 20 genes above which no conclusion can be made owing to cell stress. Therefore, it cannot be entirely excluded that the overexpression of 10 and 8 SENS-IS genes in experiments n° 2 and 3, respectively at the dose of 10% could be the consequence of excessive irritation.

Reference 3

Endpoint:

skin sensitisation

Remarks:

other: QSAR

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Analysis conducted using the OECD QSAR Toolbox ( v3.0.0.995)

Justification for type of information:

QSAR prediction: migrated from IUCLID 5.6

Guideline:

other: QSAR using OECD Toolbox (v3.0.0.995)

Principles of method if other than guideline:

QSAR model including algorithms to predict the skin sensitization potential

GLP compliance:

no

Type of study:

other: QSAR

Remarks on result:

other: no quantitative results (QSAR oecd Toolbox)

To check the skin sensitization potential of the registered substance, the 3 isomers Sodium mono-, di- and triisopropylnaphthalene sulphonate were profiled using the QSAR Toolbox (v3.0.0.995 OECD, 2012). Prior to the analysis, it was verified that the isomers fall within the applicability domain of the QSAR model. The results are presented in the following table:

 

Name (proportion in the registered substance)	Sodium monoisopropyl naphthalene sulphonate (19.6%)	Sodium diisopropyl naphthalene sulphonate (40.8%)	Sodium triisopropyl naphthalene sulphonate (14.7%)
CAS no.	28348-64-3	1322-93-6	1323-19-9
Substance without metabolism
Structural description
Organic functional groups	Aryl; Fused carbocyclic aromatic; Isopropyl; Naphtalene; Sulfonic acid
General mechanistic
DPRA Cysteine peptide depletion	Not possible to classify according to these rules
DPRA Lysine peptide depletion	Not possible to classify according to these rules
Protein binding by OASIS v1.1	No alert found
Protein binding by OECD	No alert found
Protein binding potency	Not possible to classify according to these rules (GSH)
Endpoint specific
Keratinocyte gene expression	Not possible to classify according to these rules
Protein binding alerts for skin sensitization by OASIS v1.1	No alert found
Skin metabolism simulator
Number of Metabolites found	5 metabolites	5 metabolites	7 metabolites
Structural description
Organic functional groups	1xAlcohol; 4xAryl; 4xFused carbocyclic aromatic; 3xIsopropyl; 4xNaphtalene; 1xNo functional group found; 2xPhenol; 4xSulfonic acid	2xAlcohol; 4xAryl; 4xFused carbocyclic aromatic; 4xIsopropyl; 4xNaphtalene; 1xNo functional group found; 1xPhenol; 4xSulfonic acid	3xAlcohol; 6xAryl; 6xFused carbocyclic aromatic; 6xIsopropyl; 6xNaphtalene; 1xNo functional group found; 2xPhenol; 6xSulfonic acid
General mechanistic
DPRA Cysteine peptide depletion	5 x Not possible to classify according to these rules	5 x Not possible to classify according to these rules	7 x Not possible to classify according to these rules
DPRA Lysine peptide depletion	5 x Not possible to classify according to these rules	5 x Not possible to classify according to these rules	7 x Not possible to classify according to these rules
Protein binding by OASIS v1.1	5 x No alert found	5 x No alert found	7 x No alert found
Protein binding by OECD	5 x No alert found	5 x No alert found	7 x No alert found
Protein binding potency	5 x Not possible to classify according to these rules (GSH)	5 x Not possible to classify according to these rules (GSH)	7 x Not possible to classify according to these rules (GSH)
Endpoint specific
Keratinocyte gene expression	5 x Not possible to classify according to these rules	5 x Not possible to classify according to these rules	7 x Not possible to classify according to these rules
Protein binding alerts for skin sensitization by OASIS v1.1	5 x No alert found	5 x No alert found	7 x No alert found

 

 

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: expert judgment

Conclusions:

To check the skin sensitization potential of the registered substance, the 3 isomers namely Sodium mono-, di- and triisopropylnaphthalene sulphonate were profiled using the QSAR Toolbox (v3.0.0.995 OECD, 2012). No alerts were found for any isomers or their predicted metabolites based on three Protein binding algorithms namely "Protein binding by OASIS v1.1", "Protein binding by OECD" and "Protein binding alerts for skin sensitization by OASIS v1.1".

Executive summary:

To check the skin sensitization potential of the registered substance, the 3 isomers namely Sodium mono-, di- and triisopropylnaphthalene sulphonate were profiled using the QSAR Toolbox (v3.0.0.995 OECD, 2012). No alerts were found for any isomers or their predicted metabolites based on three Protein binding algorithms namely "Protein binding by OASIS v1.1", "Protein binding by OECD" and "Protein binding alerts for skin sensitization by OASIS v1.1".

Besides the OECD Toolbox, the above structures were also tested using the Skin Sensitization model CAESAR (v 2.1.5) and the Danish EPA QSAR Database (for skin sensitization) but no prediction could be performed because the structures are out of the applicability domain of these models (data not shown).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

One in vivo study (LLNA), one in vitro assay (SENS-IS test) and a QSAR analysis were conducted and considered in a weight of evidence approach to assess the skin sensitisation potential of the registered substance.

First an in vivo test (LLNA) was performed which gave ambiguous results. The local lymph node assay (LLNA) was performed according to the OECD Guideline 429 and under GLP regulations. For this purpose, three groups of four female mice received the test item by topical route to the dorsal surface of both ears on days 1, 2 and 3 at concentrations of 5, 10 or 25% (maximum technically applicable concentration) under a dose‑volume of 25 µL. No unscheduled deaths occurred and no clinical signs were observed in any animals throughout the study.On day 6, erythema and dryness of ear skin were noted in all females treated at 25%. Erythema was also observed in 1/4 females treated at 10%. An increase in ear thickness of 31% was observed in females treated at 25%. In this study the stimulation indices (S.I.) of 0.92, 1.14 and 3.14 were determined with the test item at concentrations of 5, 10 and 25% in propylene glycol. As a significant lymphoproliferation (SI > 3) was noted at the concentration of 25%, the EC3 value was determined (23.95%). Based on the positive SI value observed at the concentration of 25%, the test material may be considered as a skin sensitiser. However, irritation was also observed at this concentration and is known to be potentially involved in false positive lymphoproliferation responses. Therefore, the test item may not be a skin sensitizer for the following reasons: an increase in ear thickness was above the limit of 25% in two out of four mice; the SI cut-off of 3 was only just exceeded (SI = 3.14). Therefore it is highly likely that this weak lymphoproliferation was the consequence of the local irritation clearly observed in 2/4 animals of this group.

 Based on the results of this study, it was not possible to definitively reject the impact of irritation on the stimulation index observed at the highest concentration, therefore it was not possible to clearly conclude on the potential of reaction product of naphthalene, propan-2-ol, sulfonated and neutralized by caustic soda to induce delayed contact hypersensitivity.

 

LLNA tends to overestimate the sensitisation potential of surfactants (Kreiling et al. 2008; Ball et al., 2011; ICCVAM 2011) which are often skin irritant and therefore interfere with the induction phase of the sensitisation. In other words, some surfactants can give false positive results in the LLNA. Indeed, inflammatory processes in the skin due to irritation phenomenon may lead to non-specific activation of dendritic cells, cell migration and non-specific proliferation of lymph node cells (Hans-Werner and Jürgen, 2005). Although not classified for skin irritation, the registered substance was slightly irritating to the rabbit skin in the OCDE 405 study (1994) which was confirmed in the LLNA as described above.Therefore the weak lymphoproliferation seen in the LLNA is likely the consequence of the local irritationseen in some animals. 

In order to discriminate the sensitization potential from the irritating response, an in vitro test (SENS-IS test) was performed on the Reaction product of naphthalene, propan-2-ol, sulfonated and neutralized by caustic soda.

The SENS-IS test is based on the analysis by RT-PCR (reverse transcription polymerase chain reaction) of two set of genes, one specifically reflecting the irritant potential of a chemical after application to the skin (“IRRITATION” set of genes) and the other set of genes correlating with the sensitization potential (splited in 2 subsets of genes namely “SENS_IS” and “REDOX”). By measuring the level of expression of these two separate sets of genes at a given time point after application and by comparison with internal negative, irritant and sensitizer positive control, the SENS-IS test can determine the irritant and sensitizer potential of a chemical.The test item solubilized in phosphate buffer saline (PBS) at the concentrations of 50%; 10%; 1% and 0.1% was applied on the top of each reconstituted epidermis (Episkin model). The test product was gently spread on the epidermis surfaces to ensure it covers all the surface. After 15 mn exposure, the Episkin were rinsed with PBS. Epidermis were then incubated at 37°C for 6 hours.After incubation, the complete epidermis was collected, placed in a RNAzol solution and the total RNA was isolated by homogenization of the skin. After reverse transcription, quantitative gene expression was measured by RT-PCR. For each analysis three negative controls (PBS, olive oil and DMSO treated skins), a positive irritation control (5% SLS) and two positive sensitization controls (HCA at 50% and TNBS at 1%) were performed. The test product and the controls were tested in at 3 experiments using different batches of Episkin models. However the results of one experiment were considered invalid as some acceptance criteria were not met.The test item used at 50% was too irritant to the episkin model, therefore the result at this concentration were not taken into account. At 10% although the number of “IRRITATION” genes was still high it remained within the acceptance criteria (<20). At that dose the test item induced a number of genes in the SENS-IS group above 7 in both experiments (7 being the lower limit for considering a positive response). The level of genes induced in the REDOX group was below 7 in both experiments. At 1% and 0.1% the test item was slightly irritant. At 1% the number of gene induced in the SENS-IS group was equal to 7 in one experiment only. However, a sensitizing potential at this dose level is unlikely since this number of genes induced corresponds to the lower limit for considering a positive response (≥ 7) and it was not confirmed in the second experiment. At 0.1%, the number of genes in the SENS-IS and REDOX groups was below 7 in both experiments.

In conclusion, under the experimental conditions of this study, the test item was found irritant to skin and weakly sensitizing. However, the mean number of “irritation” genes overexpressed at 10% (n=20 in experiment #2 and 16 in experiment #3) was close to the limit of 20 genes above which no conclusion can be made owing to cell stress. Therefore, it cannot be entirely excluded that the overexpression of 10 and 8 SENS-IS genes in experiments n° 2 and 3, respectively at the dose of 10% could be the consequence of excessive irritation.

 

Since both the LLNA and SENS-IS assay concluded on ambiguous results, the skin sensitization potential of the three isomers of the registered substance (Sodium mono-, di- and triisopropylnaphthalene sulphonate) were profiled using the QSAR Toolbox (v3.0.0.995 OECD, 2012). No alerts were found for any isomers or their predicted metabolites based on three Protein binding algorithms namely "Protein binding by OASIS v1.1", "Protein binding by OECD" and "Protein binding alerts for skin sensitization by OASIS v1.1".

Taken together, the LLNA, SENS-IS and QSAR studies support the conclusion that the registered substance does not present any skin sensitizing potential. The ambiguous results obtained in the LLNA and SENS-IS studies are considered to be secondary to the slight irritating potential of the substance.

 

References:

- Ball N,Cagen S,Carrillo JC,Certa H,Eigler D,Emter R,Faulhammer F,Garcia C,Graham C,Haux C,Kolle SN,Kreiling R,Natsch A,Mehling A., 2011.Evaluating the sensitization potential of surfactants: integrating data from the local lymph node assay, guinea pig maximization test, and in vitro methods in a weight-of-evidence approach.Regul Toxicol Pharmacol.2011 Aug;60(3):389-400

- Hans-Werner V, Jürgen AH, 2005. The local lymph node assay being too sensitive?Arch Toxicol. ; 79(12):721-8.

- ICCVAM 2011, NIH Publication Number 11-7709, ICCVAM Test Method Evaluation Report: Usefulness and Limitations of the Murine Local Lymph Node Assay for Potency Categorization of Chemicals Causing Allergic Contact Dermatitis in Humans

Migrated from Short description of key information:
LLNA (GLP and OECD 429 guideline study): ambigous (SI = 3.19 at the highest tested concentration but irritating effects are also observed)
SENS-IS test (non GLP study): ambigous. The test item was found irritant to skin and weakly sensitizing.
OECD QSAR Toolbox: negative: No alerts found for any of the 3 isomers of the registered substance or their predicted metabolites based on a profiling approach.

Justification for selection of skin sensitisation endpoint:
Weight of evidence approach based on one in vivo study (LLNA), one in vitro assay (SENS-IS test) and a QSAR analysis

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Harmonized classification:

No harmonized classification is available according to the Regulation (EC) No 1272/2008.

Self classification:

Based on a expert judgment of the in vivo and in vitro studies and the QSAR analysis available, Reaction product of naphthalene, propan-2-ol, sulfonated and neutralized by caustic soda is not classified as a skin sensitiser according to the Regulation (EC) No. 1272/2008 (CLP) and GHS UN.