Sodium sulphamidate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimenta; Starting Date: 07 Jul 2011, Experimental Completion Date; 12 Aug 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevant results.

Justification for type of information:

Please see attached justification for conducting a GPMT.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

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:

EU Method B.6 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

Please refer to the document attached in section "Justification for type of information"

Test material

Test material information

Constituent 1

Reference substance name:

Sodium Sulphamate

IUPAC Name:

Sodium Sulphamate

Details on test material:

Identification: Sodium SulphamateDescription: White solidPurity: 99.67 %Stability of Test Item: Stable under storage conditionsStability of the Test Item Dilution: Considered to be stable in water as the test item is manufactured in water.Expiry Date: 06-May-2013Storage Conditions: At room temperature (range of 20 ± 5 °C, provided by Harlan Laboratories Ltd.), light protected.

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

male

Details on test animals and environmental conditions:

TEST ANIMALS- Source: Albino Dunkin Hartley Guinea Pig, HsdPoc: DH, SPF from Harlan Laboratories B.V., The Netherlands- Age at study initiation: 4 weeks- Weight at study initiation: Bodweight at pretest start: Pretest Groups 327.7 - 356.0 gBody weight at beginning of acclimatisation period: Test and control groups: 311.0 - 373.0 g- Housing: In groups of up to 10 animals in stainless steel cages with standard softwood bedding- Diet (e.g. ad libitum): Teklad Global Guinea pig diet 2040C, ad libitum- Water (e.g. ad libitum): Community tap water in bottles, ad libitum.- Acclimation period: Twelve days for the test and control groups of the main test under standard laboratory conditions after health examination. No acclimatization for the animals of the pretest. Only animals without any visible signs of illness were used for the study.ENVIRONMENTAL CONDITIONS- Temperature (°C): continuously monitored environment with a room temperature of 22 ± 3 °C- Humidity (%): relative humidity between 30-70%,- Air changes (per hr): Air-conditioned with 10-15 air changes per hour.- Photoperiod (hrs dark / hrs light): automatically controlled light cycle of 12 hours light and 12 hours dark

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal and epicutaneous

Vehicle:

water

Remarks:

(see details on study design for justification).

Induction 2

Concentration / amount:

Main Study:Intradermal induction: 5% dilution of test item in purified water and in an emulsion of Freund's Complete Adjuvant (FCA)/physiological saline.Epidermal induction: 50% in purified water.Challenge - Epidermal application of the test item at 25% in purified water and purified water alone

Challenge

Challenge 1

Route:

epicutaneous, occlusive

Vehicle:

water

Remarks:

(see details on study design for justification).

Challenge 2

Concentration / amount:

Main Study:Intradermal induction: 5% dilution of test item in purified water and in an emulsion of Freund's Complete Adjuvant (FCA)/physiological saline.Epidermal induction: 50% in purified water.Challenge - Epidermal application of the test item at 25% in purified water and purified water alone

No. of animals per dose:

Main study:Test group: 10 malesControl group: 5 males

Details on study design:

VEHICLE:The vehicle was selected based on preliminary solubility testing, which was performed before the study initiation date. The test item was prepared at a concentration of 75% (w/w) in purified water, which was not entirely soluble and therefore not suitable for the application forms used in this study. The test item prepared at a concentration of 50% (w/w) was well soluble and resulted in a colorless liquid. Therefore, this concentration was used as highest concentration in the epidermal pretest.For the intradermal pretest, the test item was prepared at a concentration of 25% in FCA/physiological saline. This preparation went through a needle of 0.5 x 16 mm. Higher concentrations were not considered as feasible for administration through a needle. Therefore, this test item concentration in purified water was used as highest concentration in the intradermal pretest.AUXILIARY COMPOUNDS:FCA - Freund's Adjuvant - completePhysiological Saline - Natrium chloratum 0.9%PREPARATION OF DOSE FORMULATIONS:The test item was ground prior to test item preparation using a mortar and a pestle. The test item and vehicle or auxiliary compound were placed into a glass beaker on a tared Mettler balance and weight/weight dilutions were prepared. A magnetic stirrer was used to ensure homogeneous distribution of the test item preparation. The preparations were made immediately prior to each dosing.Homogeneity of the test item formulation was maintained during administration using a magnetic stirrer.SELECTION OF TEST ITEM CONCENTRATION FOR MAIN STUDY:Intradermal Induction:The test item prepared at 5% (used for the intradermal induction) did not cause any skin irritation while the test item concentrations of 10% and 25% in purified water did cause a moderate irritation (with/without blanching and edema) during the pretest. Therefore, the test item concentration of 5% in purified water was considered to be the suitable concentration selected for the main intradermal induction.Epidermal Induction:The test item prepared at 50% (used for the epidermal induction) was the highest technically applicable concentration and caused a mild skin irritation during the pretest. This concentration was considered to be appropriate for the main epidermal induction.Epidermal ChallengeThe test item prepared at 25% was the highest non-irritant concentration during the pretest. Therefore, this concentration was considered to be the suitable concentration for the challenge in the main test.To determine the different concentrations, intradermal and epidermal pretests were performed as described below.OBSERVATIONS:Viability / Mortality: Daily from delivery of the animals to the termination of the test.Clinical Signs: Daily from delivery of the animals to the termination of test.Local signs: Skin responses were graded during the pretest, induction and challenge periodBody Weights: At delivery/acclimatization start, at test item treatment day in the pretest, at the end of the pretest, at test day 1 (day of treatment) and at the termination of the study.PATHOLOGY:Necropsy:No necropsy was performed on all surviving animals. The control and test animals were euthanized at the end of the test period by intraperitoneal injection of pentobarbitone at a dose of 2.0 mL/kg of 162 mg/mL sodium pentobarbitone and discarded. The pretest animals were euthanized as described above at the treatment start of the main study.TREATMENT METHOD:The animal's fur was shaved with a fine clipper blade just prior to exposure. Intradermal injections or closed patches were applied to the animals as follows:0.1 mL/site for the intradermal administrations or 0.2 to 0.3 mL on a patch of filter paper for the epidermal administrations.The patch was covered by a strip of aluminium foil and firmly secured by an elastic plaster wrapped around the trunk of the animal and secured with impervious adhesive tape. The occlusive dressing was left in place for 24 (epidermal pretest and epidermal challenge) or 48 hours (epidermal induction).Identical patching method was used for the epidermal pretest, epidermal induction and epidermal challenge.PRETEST:The pretest was performed during the acclimatization period of the animals for the main test.The test item concentrations described below were selected during a preliminary solubility testing which was performed before the study initiation date.Intradermal injections:Four intradermal injections (0.1 mL/site) of a 1:1 (v/v) mixture of Freund's Complete Adjuvant/physiological saline were made into the shaved neck of one guinea pig (no. 73). Five days later, three intradermal injections (0.1 mL/site) were made into the clipped flank of the same guinea pig at concentrations of A = 25%, B = 10% and C = 5% of the test item in purified water.Dermal reactions were assessed 24 hours later.Based on the results, a test item concentration of 5% was selected for intradermal induction in the main test.Epidermal Applications:Four intradermal injections (0.1 mL/site) of a 1:1 (v/v) mixture of Freund's Complete Adjuvant/physiological saline were made into the shaved neck of two guinea pigs (nos. 74 - 75).Five days later, four patches of filter paper (3 x 3 cm) were saturated with the test item at D = 50% (technically the highest possible concentration to be applied sufficiently), E = 25%, F = 10% and G = 5% in purified water and applied to the shaved flanks of the same guinea pigs. The volume of test item preparation applied was approximately 0.2 mL. The occlusive dressings were left in place for 24 hours.The reaction sites were assessed 24 and 48 hours after removal of the bandage for erythema and oedema according to the method of Magnusson and Kligman.The allocation of the different test item dilutions to the sites (D, E, F, G) on the two animals was alternated in order to minimize site-to-site variation in responsiveness.Based on the results the test item concentration selected for epidermal induction and challenge in the main test was 50% and 25%, respectively.MAIN STUDY:INDUCTION:Intradermal injection performed on Test Day 1.An area of dorsal skin from the scapular region (approximately 6 x 8 cm) was clipped free of hair. Three pairs of intradermal injections (0.1 mL/site)were made just within the boundaries of a 4 x 6 cm area in the clipped region as follows: Test Group:1) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.2) The test item at 5% in purified water.3) The test item at 5% in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.Epidermal induction performed on Test Day 8.One week after the intradermal injections, the scapular area (approximately 6 x 8 cm) was again shaved prior to epidermal induction. A 2 x 4 cm patch of filter paper was saturated with the test item at 50% in purified water and placed over the injection sites of the test animals. The volume of test item preparation applied was approximately 0.3 mL. The patch was covered with aluminum foil and firmly secured by an elastic plaster wrapped around the trunk of the animal and secured with impervious adhesive tape. The occlusive dressings were left in place for 48 hours. The epidermal application procedure described ensured intensive contact of the test item.The reaction sites were assessed 24 and 48 hours after removal of the bandage for erythema and oedema according to the method of Magnusson and Kligman.CHALLENGEPerformed on Test Day 22The test and control guinea pigs were challenged two weeks after epidermal induction and treated in the same way.Two patches (3 x 3 cm) of filter paper were saturated with the test item at the highest tested non-irritating concentration of 25% (applied to the left flank) and the vehicle only (purified water applied to the right flank) using the same method as for the epidermal application. The volume of test item preparation and of vehicle applied was approximately 0.2 mL. The dressings were left in place for 24 hours.

Challenge controls:

Induction:Intradermal:Control Group: 1) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.2) Purified water.3) 1:1 (w/w) mixture of purified water in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.Epidermal:The guinea pigs were treated as for the test group with purified water only, applied at a volume of approximately 0.3 mL.Challenge:The test and control guinea pigs were challenged two weeks after epidermal induction and treated in the same way.Two patches (3 x 3 cm) of filter paper were saturated with the test item at the highest tested non-irritating concentration of 25% (applied to the left flank) and the vehicle only (purified water applied to the right flank) using the same method as for the epidermal application

Study design: in vivo (LLNA)

Positive control substance(s):

other: alpha-hexylcinnamaldehyde

Statistics:

Descriptive statistics (means and standard deviations) were calculated for body weights. No inferential statistics were used.

Results and discussion

Positive control results:

The results from positive control test confirm alpha-hexylcinnamaldehyde as skin sensitizer, as it produced allergic contact dermatitis in >30% of the test animals.

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

25% (test item in purified water)

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

None

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 25% (test item in purified water). No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: None.

Results 2

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

25% (test item in purified water)

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

None

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 25% (test item in purified water). No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: None.

Results 3

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

Purified water only

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

None

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: Purified water only. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: None.

Results 4

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

Purified water only

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

None

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: Purified water only. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: None.

Results 5

Reading:

1st reading

Hours after challenge:

24

Group:

other: Control group

Dose level:

25% (test item in purified water)

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

None

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: other: Control group. Dose level: 25% (test item in purified water). No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: None.

Results 6

Reading:

2nd reading

Hours after challenge:

48

Group:

other: Control group

Dose level:

25% (test item in purified water)

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

None

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: other: Control group. Dose level: 25% (test item in purified water). No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: None.

Results 7

Reading:

1st reading

Hours after challenge:

24

Group:

other: Control group

Dose level:

Purified water only

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

None

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: other: Control group. Dose level: Purified water only. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: None.

Results 8

Reading:

2nd reading

Hours after challenge:

48

Group:

other: Control group

Dose level:

Purified water only

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

None

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: other: Control group. Dose level: Purified water only. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: None.

Any other information on results incl. tables

Observations:

Viability/Mortality/Macroscopic Findings

No intercurrent deaths occurred during the course of the study, hence no necropsies were performed.

Clinical Signs

No clinical signs were recorded throughout the entire observation period.

Body Weights

The body weight of the animals was within the range commonly recorded for animals of this strain and age.

Skin Reactions in the Intradermal Induction

Expected common findings were observed in the test and control groups after the different injections using FCA intradermally. These findings consisted of erythema, oedema, necrotizing dermatitis, encrustation and exfoliation of encrustation.

No detailed description of the skin reactions is given in the report as these FCA effects are well known.

Skin Reactions in the Epidermal Induction

See Tables 1 and 2 (attached background material).

Control Group:

No erythematous or oedematous reaction was observed in the control animals treated with purified water only.

Test Group:

No erythematous or oedematous reaction was observed at any observation time in 9 out of 10 test animals after treatment with the test item prepared at 50% in purified water. One test animal (No. 84) was observed with a discrete/patchy erythema at the 24-hour reading which disappeared 24 hours later.

Skin Reactions in the Challenge

See Tables 3 to 6 (attached background material)

Control Group:

No local skin reactions were observed in the control animals when treated with purified water alone or when treated with the test item prepared at 25% in purified water.

Test Group:

No local skin reactions were observed in the test animals when treated with purified water alone or when treated with the test item prepared at 25% in purified water.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Based on the findings in an adjuvant sensitisation test (M&K-test) in guinea pigs and in accordance to Regulation (EC) No 1272/2008, Sodium Sulphamate, CAS 13845-18-6, NALCO TIS I0442 does not have to be classified and labeled as a skin sensitizer.

Executive summary:

In order to assess the cutaneous allergenic potential of Sodium Sulphamate, CAS 13845-18-6, NALCO TIS I0442, the Maximization-Test was performed in 15 (10 test and 5 control) male albino Dunkin Hartley guinea pigs, in accordance with OECD Guideline No. 406 and the Commission Regulation (EC) No 440/2008, B.6.

The intradermal induction of sensitisation in the test group was performed in the nuchal region with a 5% dilution of the test item in purified water and in an emulsion of Freund's Complete Adjuvant (FCA)/physiological saline. The epidermal induction of sensitisation was conducted for 48 hours under occlusion with the test item at 50% in purified water one week after the intradermal induction. The animals of the control group were intradermally induced with purified water and FCA/physiological saline and epidermally induced with purified water under occlusion.

Two weeks after epidermal induction the test and control animals were challenged by epidermal application of the test item at 25% in purified water and purified water alone under occlusive dressing.

Cutaneous reactions were evaluated at 24 and 48 hours after removal of the dressing.

Results - Skin Reactions after the Challenge Procedure

	After 24 hours Positive/Total % Positive of Total	After 48 hours Positive/Total % Positive of Total
Control Group
Sodium Sulphamate, 25% in purified water (left flank)	0/5 0%	0/5 0%
Purified water only (right flank)	0/5 0%	0/5 0%
Test Group
Sodium Sulphamate, 25% in purified water (left flank)	0/10 0%	0/10 0%
Purified water only (right flank)	0/10 0%	0/10 0%

No intercurrent deaths occurred during the course of the study.

No toxic signs were evident in the guinea pigs of the control or test group.

No local skin effects were observed in the guinea pigs of the control or test group.

Based on the findings in an adjuvant sensitisation test (M&K-test) in guinea pigs and in accordance to Regulation (EC) No 1272/2008, Sodium Sulphamate, CAS 13845-18-6, NALCO TIS I0442 does not have to be classified and labeled as a skin sensitizer.