Benzene-1,4-diammonium sulphate

Endpoint summary

Description of key information

Data available for the structurally and functionally similar read across chemicals has been reviewed to determine the skin sensitization potential of the test chemical Benzene-1,4-diammonium sulphate (CAS No:16245-77-5). The studies are as mentioned below:

 

The skin sensitization study of read across chemical was conducted on mouse by using local lymph nodes assay. The objective of the study was to determine if the test substance is a contact skin sensitiser in CBA/J mouse as measured by cell proliferation in the draining lymph nodes after topical application on the pinnae. Morbidity/mortality checks were performed twice daily. Clinical examinations were performed daily. Individual body weights were recorded on days –1 and 5. Ear swelling measurement was performed once pretest (on day 0) and on day 5. All animals were sacrificed on day 5 for assessment of cell viability, cellularity and cell proliferation. The positive control hexyl cinnamic aldehyde induced a positive response in the local lymph node assay, as there was at least a 3-fold increase in isotope incorporation in the draining auricular lymph node relative to the vehicle. The mean stimulation index was 27.8 for the positive control at the concentration of 50%. The test substance induced a positive response in the local lymph node assay, as there was at least a 3-fold increase in isotope incorporation in the draining auricular lymph node relative to the vehicle, with a calculated EC3 value of 0.06%. The mean stimulation indices were 2.6, 10.4, and 16.1 at the concentrations of 0.05, 0.25, and 1.25%, respectively. The test substance did not increase ear thickness following topical application, whereas there was a 38.8% increase of thickness in the positive control group. In the absence of evidence of local irritation, the lymphoproliferative responses observed with the test substance were attributed to delayed contact hypersensitivity. The cellularity index was 1.17, 2.36, and 2.46 at 0.05, 0.25, and 1.25% of the test substance, respectively, and 5.10 for the positive control. The irritation class of the test substance is 1 (increase in ear thickness ≤10) at the three tested concentrations, whereas the irritation class of hexyl cinnamic aldehyde is 3 (increase in ear thickness ≥ 30%). On the basis of observed effects, the test substance was considered as skin sensitizer.

 

The above result was further supported by another Mouse Local Lymphnode Assay conducted for similar read across chemical. The LLNA was conducted on groups of CBA mice (7-12 weeks of age) by mean of topical application of chemical on the dorsum of both ears at a dose of 25µl or to an equal volume of relevant vehicle (Acetic acid in olive oil (4:1))only. Treatment was performed daily for 3 consecutive days. Five days after initiation of exposure all mice were injected via the tail vein with 250µl of PBS containing 20µCi of tritiatied thymidine. The mice were sacrificed 5 hours later, and draining the auricular lymph nodes were excised and pooled for each experimental group or each individual animal. The incorporation of tritiated thymidine measured by β-scintillation counting and was reported in disintegrations /minute. An SI was calculated for each chemical group as the ratio of disintegrations/minute of the treated group to the disintegrations/minute of the concurrent vehicle control group. A substance was classified skin sensitizer, if at one or more than one concentrations, it induced a three-fold or greater increase in local lymph node proliferative activity when treated with the concurrent vehicle treated controls (SI ≥3). The approach to estimation of the relative skin sensitization potential is based on the mathematical estimation of the concentration of chemical necessary to obtain a threshold positive response (SI = 3); this is termed as the EC3 value. The relative potency index of was estimated to be 0.49. Based on the relative potency index, the test chemical was considered as skin sensitizer.

 

Based on the above summarized studies for target chemical Benzene-1,4-diammonium sulphate (CAS No:16245-77-5) and its structurally and functionally similar read across substances,it can be concluded that the testchemical is able to cause skin sensitization and considered as skin sensitizer. Comparing the above annotations with the criteria of CLP regulation, it can be classified under the category “Skin Sensitizer”.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

experimental data of read across substances

Justification for type of information:

Data for the target chemical is summarized based on the structurally and functionally similar read across chemicals

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: as mentioned below

Principles of method if other than guideline:

WoE report is based on 2 skin sensitization studies as- WoE-2 and WoE-3. Skin sensitization of test chemical was determined by performing Mouse Local Lymphnode Assay

GLP compliance:

not specified

Type of study:

mouse local lymph node assay (LLNA)

Justification for non-LLNA method:

not specified

Specific details on test material used for the study:

- Name of test material (as cited in study report): Benzene-1,4-diammonium sulphate- Molecular formula: C6H8N2.H2O4S- Molecular weight: 206.221 g/mol- Smiles notation: S(=O)(=O)(O)O.c1(ccc(cc1)N)N - InChl : 1S/C6H8N2.H2O4S/c7-5-1-2-6(8)4-3-5;1-5(2,3)4/h1-4H,7-8H2;(H2,1,2,3,4)- Substance type: organic- Physical state: Solid

Species:

mouse

Strain:

other: 1.CBA/J 2.CBA

Sex:

female

Details on test animals and environmental conditions:

1.TEST ANIMALS- Age at study initiation: 9 weeks - Weight at study initiation: 19-28 grams2.TEST ANIMALSSource: No dataAge at study initiation: 7-12 weeksENVIRONMENTAL CONDITIONS: No data- Housing: Animals were housed in groups of 5 of the same dose group in plastic cages (265 x 160 x 140 mm)- Diet (e.g. ad libitum): ad libitum- Water (e.g. ad libitum): ad libitum- Acclimation period: 8 daysENVIRONMENTAL CONDITIONS- Temperature (°C): 22± 2 °C- Humidity (%): 55 ± 15%- Air changes (per hr): minimum 15 air changes per hour- Photoperiod (hrs dark / hrs light):12-hour light/dark cycle 2.

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

1.0% (Vehicle Control), 0.05%, 0.25%, 1.25%2.25 µl of 2.5,5.0 and 10%

No. of animals per dose:

1.5 females mice per dose2.Not specified

Details on study design:

1.MORBIDITY/MORTALITY: All animals were observed twice daily.CLINICAL SIGNS: Animals were observed daily. On treatment days, animals were examined before and at least once after dosing to detect any clinical signs or reaction to treatment (especially at the treatment sites).2.Treatment was performed daily for 3 consecutive days. Five days after initiation of exposure all mice were injected via the tail vein with 250µl of PBS containing 20µCi of tritiatied thymidine. The mice were sacrificed 5 hours later, and draining the auricular lymph nodes were excised and pooled for each experimental group or each individual animal. The incorporation of tritiated thymidine measured by β-scintillation counting and was reported in disintegrations /minute. An SI was calculated for each chemical group as the ratio of disintegrations/minute of the treated group to the disintegrations/minute of the concurrent vehicle control group. A substance was classified skin sensitizer , if at one or more than one concentrations, it induced a three-fold or greater increase in local lymph node proliferative activity when treated with the concurrent vehicle treated controls (SI ≥3) BODY WEIGHT: All animals were weighed on days –1 and 5.EAR SWELLING MEASUREMENT: For each animal, ear thickness was measured once pretest (on day 0) and on day 5 using a constant pressure micrometer.EVALUATION OF THE CELL PROLIFERATION: Method and material: all mice received an intravenous injection via the tail vein (using an infusion pump) of 250 μL of phosphate buffered saline (PBS) containing 21.4 μCi of [3H] methyl thymidine (specific activity of 25 Ci/mmol). Five hours later the mice were sacrificed by carbon dioxide inhalation and the draining auricular lymph nodes were excised. A single cell suspension was prepared for each animal. Cells were washed twice with PBS. Cell viability and cellularity were assessed using the Trypan blue exclusion test. Cells were precipitated with ice cold 5% trichloro-acetic acid (TCA). Approximately 18 hours later, the pellets were resuspended in 1 mL of TCA and transferred into the scintillation cocktail. [3H]-methyl-thymidine incorporation was measured by liquid scintillation counting in a TRI-CARB 2700TR counter.2.

Positive control substance(s):

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

Statistics:

1.Body weights on days -1 and 5, body weight gains over days -1 to 5, ear thickness changes over days 0 to 5, cell proliferation and cellularity were analysed statistically using a SAS softare package. Levene’s test was used to test the equality of variance across groups and Shapiro-Wilk’s test was used to assess the normality of the data distribution in each group. Data with homogeneous variances and normal distribution in all groups were analysed using Anova followed by Dunnett’s test. Data showing non homogeneous variances or a non normal distribution in at least one group were analysed using Kruskal-Wallis test followed by the Wilcoxon’s rank sum test.2.The approach to estimation of the relative skin sensitization potential is based on the mathematical estimation of the concentration of chemical necessary to obtain a threshold positive response (SI = 3); this is termed as the EC3 value

Positive control results:

1.The positive control hexyl cinnamic aldehyde was shown to be a skin sensitiser and thus validated the experimental conditions.2.Not specified

Parameter:

other: 1.SI

Remarks on result:

other: 2.6, 10.4, and 16.1 at 0.05, 0.25, and 1.25%, respectively.

Parameter:

other: 1.disintegrations per minute (DPM)

Remarks on result:

other: 273.0, 1101.0, and 1710.0 at 0.05, 0.25, and 1.25%, respectively.

Parameter:

other: EC3

Value:

0.49

Remarks on result:

other: The relative potency index of of 3-aminophenol was estimated to be 0.49.

Cellular proliferation data / Observations:

1.The mean stimulation indices were 2.6, 10.4, and 16.1 at the concentrations of 0.05, 0.25, and 1.25%, respectively.2.The relative potency index of of 3-aminophenol was estimated to be 0.49.

1.

Table 2 : Data Summary

Treatments and Concentrations	DPM (per node)	Stimulation Index	Cellularity index	Irritation Class
Negative Controls (AOO)	106.0	-	-	-
0.05%	273.0	2.6	1.17	1
0.25%	1101.0	10.4	2.36	1
1.25%	1710.0	16.1	2.46	1
Positive Control (HCA 50%)	2939.5	27.8	5.10	3

2.

Table 2:

Test chemical	Vehicle	LLNA%	LLNA%	LLNA%	LLNA SI	LLNA SI	LLNA SI	LLNA EC3	Relative Potency
	AOO	2.5	5.0	10.0	11.7	15.4	19.2	0.49	Strong

Interpretation of results:

other: sensitising

Conclusions:

The test chemical Benzene-1,4-diammonium sulphate (CAS No:16245-77-5) was considered to be sensitising.

Executive summary:

Data available for the structurally and functionally similar read across chemicals has been reviewed to determine the skin sensitization potential of the test chemical Benzene-1,4-diammonium sulphate (CAS No:16245-77-5). The studies are as mentioned below:

 

The skin sensitization study of read across chemical was conducted on mouse by using local lymph nodes assay. The objective of the study was to determine if the test substance is a contact skin sensitiser in CBA/J mouse as measured by cell proliferation in the draining lymph nodes after topical application on the pinnae. Morbidity/mortality checks were performed twice daily. Clinical examinations were performed daily. Individual body weights were recorded on days –1 and 5. Ear swelling measurement was performed once pretest (on day 0) and on day 5. All animals were sacrificed on day 5 for assessment of cell viability, cellularity and cell proliferation. The positive control hexyl cinnamic aldehyde induced a positive response in the local lymph node assay, as there was at least a 3-fold increase in isotope incorporation in the draining auricular lymph node relative to the vehicle. The mean stimulation index was 27.8 for the positive control at the concentration of 50%. The test substance induced a positive response in the local lymph node assay, as there was at least a 3-fold increase in isotope incorporation in the draining auricular lymph node relative to the vehicle, with a calculated EC3 value of 0.06%. The mean stimulation indices were 2.6, 10.4, and 16.1 at the concentrations of 0.05, 0.25, and 1.25%, respectively. The test substance did not increase ear thickness following topical application, whereas there was a 38.8% increase of thickness in the positive control group. In the absence of evidence of local irritation, the lymphoproliferative responses observed with the test substance were attributed to delayed contact hypersensitivity. The cellularity index was 1.17, 2.36, and 2.46 at 0.05, 0.25, and 1.25% of the test substance, respectively, and 5.10 for the positive control. The irritation class of the test substance is 1 (increase in ear thickness ≤10) at the three tested concentrations, whereas the irritation class of hexyl cinnamic aldehyde is 3 (increase in ear thickness ≥ 30%). On the basis of observed effects, the test substance was considered as skin sensitizer.

 

The above result was further supported by another Mouse Local Lymphnode Assay conducted for similar read across chemical. The LLNA was conducted on groups of CBA mice (7-12 weeks of age) by mean of topical application of chemical on the dorsum of both ears at a dose of 25µl or to an equal volume of relevant vehicle (Acetic acid in olive oil (4:1))only. Treatment was performed daily for 3 consecutive days. Five days after initiation of exposure all mice were injected via the tail vein with 250µl of PBS containing 20µCi of tritiatied thymidine. The mice were sacrificed 5 hours later, and draining the auricular lymph nodes were excised and pooled for each experimental group or each individual animal. The incorporation of tritiated thymidine measured by β-scintillation counting and was reported in disintegrations /minute. An SI was calculated for each chemical group as the ratio of disintegrations/minute of the treated group to the disintegrations/minute of the concurrent vehicle control group. A substance was classified skin sensitizer, if at one or more than one concentrations, it induced a three-fold or greater increase in local lymph node proliferative activity when treated with the concurrent vehicle treated controls (SI ≥3). The approach to estimation of the relative skin sensitization potential is based on the mathematical estimation of the concentration of chemical necessary to obtain a threshold positive response (SI = 3); this is termed as the EC3 value. The relative potency index of was estimated to be 0.49. Based on the relative potency index, the test chemical was considered as skin sensitizer.

 

Based on the above summarized studies for target chemical Benzene-1,4-diammonium sulphate (CAS No:16245-77-5) and its structurally and functionally similar read across substances,it can be concluded that the testchemical is able to cause skin sensitization and considered as skin sensitizer. Comparing the above annotations with the criteria of CLP regulation, it can be classified under the category “Skin Sensitizer”.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Data available for the structurally and functionally similar read across chemicals has been reviewed to determine the skin sensitization potential of the test chemical Benzene-1,4-diammonium sulphate (CAS No:16245-77-5). The studies are as mentioned below:

 

The skin sensitization study of read across chemical was conducted on mouse by using local lymph nodes assay. The objective of the study was to determine if the test substance is a contact skin sensitiser in CBA/J mouse as measured by cell proliferation in the draining lymph nodes after topical application on the pinnae. Morbidity/mortality checks were performed twice daily. Clinical examinations were performed daily. Individual body weights were recorded on days –1 and 5. Ear swelling measurement was performed once pretest (on day 0) and on day 5. All animals were sacrificed on day 5 for assessment of cell viability, cellularity and cell proliferation. The positive control hexyl cinnamic aldehyde induced a positive response in the local lymph node assay, as there was at least a 3-fold increase in isotope incorporation in the draining auricular lymph node relative to the vehicle. The mean stimulation index was 27.8 for the positive control at the concentration of 50%. The test substance induced a positive response in the local lymph node assay, as there was at least a 3-fold increase in isotope incorporation in the draining auricular lymph node relative to the vehicle, with a calculated EC3 value of 0.06%. The mean stimulation indices were 2.6, 10.4, and 16.1 at the concentrations of 0.05, 0.25, and 1.25%, respectively. The test substance did not increase ear thickness following topical application, whereas there was a 38.8% increase of thickness in the positive control group. In the absence of evidence of local irritation, the lymphoproliferative responses observed with the test substance were attributed to delayed contact hypersensitivity. The cellularity index was 1.17, 2.36, and 2.46 at 0.05, 0.25, and 1.25% of the test substance, respectively, and 5.10 for the positive control. The irritation class of the test substance is 1 (increase in ear thickness ≤10) at the three tested concentrations, whereas the irritation class of hexyl cinnamic aldehyde is 3 (increase in ear thickness ≥ 30%). On the basis of observed effects, the test substance was considered as skin sensitizer.

 

The above result was further supported by another Mouse Local Lymphnode Assay conducted for similar read across chemical. The LLNA was conducted on groups of CBA mice (7-12 weeks of age) by mean of topical application of chemical on the dorsum of both ears at a dose of 25µl or to an equal volume of relevant vehicle (Acetic acid in olive oil (4:1))only. Treatment was performed daily for 3 consecutive days. Five days after initiation of exposure all mice were injected via the tail vein with 250µl of PBS containing 20µCi of tritiatied thymidine. The mice were sacrificed 5 hours later, and draining the auricular lymph nodes were excised and pooled for each experimental group or each individual animal. The incorporation of tritiated thymidine measured by β-scintillation counting and was reported in disintegrations /minute. An SI was calculated for each chemical group as the ratio of disintegrations/minute of the treated group to the disintegrations/minute of the concurrent vehicle control group. A substance was classified skin sensitizer, if at one or more than one concentrations, it induced a three-fold or greater increase in local lymph node proliferative activity when treated with the concurrent vehicle treated controls (SI ≥3). The approach to estimation of the relative skin sensitization potential is based on the mathematical estimation of the concentration of chemical necessary to obtain a threshold positive response (SI = 3); this is termed as the EC3 value. The relative potency index of was estimated to be 0.49. Based on the relative potency index, the test chemical was considered as skin sensitizer.

 

Based on the above summarized studies for target chemical Benzene-1,4-diammonium sulphate (CAS No:16245-77-5) and its structurally and functionally similar read across substances,it can be concluded that the testchemical is able to cause skin sensitization and considered as skin sensitizer. Comparing the above annotations with the criteria of CLP regulation, it can be classified under the category “Skin Sensitizer”.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The skin sensitization potential of test substance Benzene-1,4-diammonium sulphate (CAS No:16245-77-5) and its structurally and functionally similar read across substanceswere observed in various studies. From the results obtained from these studies it is concluded that the chemical Benzene-1,4-diammonium sulphate (CAS No:16245-77-5) is likely to cause skin sensitization and hence can be classified as “Skin Sensitizer”.