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Description of key information

A negative local lymph node assay (LLNA) performed to GLP and OECD 442B is available for the submission substance.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA): BrdU-ELISA
Specific details on test material used for the study:
D-Glucose, reaction products with nitric acid and sodium nitrite (1:1), sodium salts. Sodium glucarate (40-50%) + Other Sodium Salts of Sugar Acids (25-30%), + other ingredients, Impurities (2-5%). 100.0% purity of solids; 52.9% Solids in water.
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Jackson Laboratories
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 9-10 weeks
- Weight at study initiation: 17.9-23.3 g
- Housing: group housed (at least 3/cage)
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 22 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26
- Humidity (%): 30-70
- Air changes (per hr): minmum 10
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
25, 50, 100%
No. of animals per dose:
Five females
Details on study design:
Concentrations used in the main study were based on the results of a screening study in which no local irritation was observed at concentrations of up to 100%.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Numerical data collected during the course of the study were subjected to calculation of group means and standard deviations. Statistical analysis of numerical data was done by either PDS-built in statistical functionalities or SigmaStat. The data were analyzed for homogeneity of variance and for normality. Homogeneous data were analyzed using the Analysis of Variance (ANOVA; p ≤0.05). The mean SI and standard deviation for each treatment group was calculated. An ANOVA followed by Dunnett’s method at p<0.05 was performed to statistically compare each group to the negative control group.
Positive control results:
A mean stimulation index of 2.191 is reported for the positive control group, thereby confirming the sensitivity of the study.
Parameter:
SI
Value:
1
Test group / Remarks:
Control
Key result
Parameter:
SI
Value:
1.332
Test group / Remarks:
25%
Remarks on result:
other: does not meet the criterion for a positive response
Key result
Parameter:
SI
Value:
1.266
Test group / Remarks:
50%
Remarks on result:
other: does not meet the criterion for a positive response
Key result
Parameter:
SI
Value:
1.147
Test group / Remarks:
100%
Remarks on result:
other: does not meet the criterion for a positive response
Parameter:
SI
Value:
2.191
Test group / Remarks:
Positive control
Remarks on result:
other: meets the criterion for a positive response
Parameter:
ECt
Remarks on result:
not determinable
Cellular proliferation data / Observations:
The negative control, and test item at 25%, 50%, and 100% all resulted in negative responses (SI <1.6). The positive control resulted in a positive response (SI >1.6). The positive response was also statistically significant (p<0.05) versus the negative control. EC1.6 value could not be calculated as all SI values for the test material were <1.6.

No deaths occurred during the study period and no signs of toxicity were observed. No local reactions were observed in the control or test animals; all five positive control females exhibited very slight erythema (barely perceptible) on both ears on Day 3, but this was not perceptible on Day 6. Bodyweights were unaffected by treatment. The test mterial at 25%, 50%, and 100%, and the negative control overall resulted in no change in ear thickness over the 6-day period. The positive control overall resulted in a mean increase of 0.03 mm in ear thickness over the 6-day period. No gross findings were observed on the lymph nodes of the animals.

Interpretation of results:
GHS criteria not met
Conclusions:
Based on the results of this study, [D-Glucose, reaction products with nitric acid and sodium nitrite (1:1), sodium salts] was found to be negative for sensitizing potential.
Executive summary:

The skin sensitisation potential of [D-Glucose, reaction products with nitric acid and sodium nitrite (1:1), sodium salts] was investigated in a 2016 local lymph node assay (LLNA) performed to GLP and OECD 442B. 25 μL of the test material at concentrations of 0, 25, 50 and 100% was applied to the dorsum of both ears of groups of five female CBA mice on three consecutive days (Days 1-3).  All mice received an intraperitoneal injection of 5 mg BrdU on Day 5 and were sacrificed on Day 6.  The draining auricular lymph nodes from each mouse were grossly assessed, excised and combined. For each animal, a single-cell suspension of lymph node cells was prepared by gentle disaggregation. BrdU incorporation was measured by ELISA. The results for each treated group were expressed as the mean Stimulation Index (SI), relative to the control group. Mean SI values of 1.332, 1.266 and 1.147 were calculated for mice treated with concentrations of 25%, 50% and 100% test material, respectively. Values are below the threshold of 1.6 and therefore do not indicate any skin sensitisation potential. A mean SI value for the positive control group (hexyl cinnamic aldehyde) of 2.191 is above 1.6, thereby demonstrating the sensitivity of the assay. No evidence of skin sensitisation potential for [D-Glucose, reaction products with nitric acid and sodium nitrite (1:1), sodium salts] was therefore seen under the conditions of this study.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

The skin sensitisation potential of [D-Glucose, reaction products with nitric acid and sodium nitrite (1:1), sodium salts] was investigated in a 2016 local lymph node assay (LLNA) performed to GLP and OECD 442B. 25 μL of the test material at concentrations of 0, 25, 50 and 100% was applied to the dorsum of both ears of groups of five female CBA mice on three consecutive days (Days 1-3).  All mice received an intraperitoneal injection of 5 mg BrdU on Day 5 and were sacrificed on Day 6.  The draining auricular lymph nodes from each mouse were grossly assessed, excised and combined. For each animal, a single-cell suspension of lymph node cells was prepared by gentle disaggregation. BrdU incorporation was measured by ELISA. The results for each treated group were expressed as the mean Stimulation Index (SI), relative to the control group. Mean SI values of 1.332, 1.266 and 1.147 were calculated for mice treated with concentrations of 25%, 50% and 100% test material, respectively. Values are below the threshold of 1.6 and therefore do not indicate any skin sensitisation potential. A mean SI value for the positive control group (hexyl cinnamic aldehyde) of 2.191 is above 1.6, thereby demonstrating the sensitivity of the assay. No evidence of skin sensitisation potential for [D-Glucose, reaction products with nitric acid and sodium nitrite (1:1), sodium salts] was therefore seen under the conditions of this study.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

No evidence of skin sensitisation potential was seen in a LLNA performed with the submission substance. Classification for skin sensitisation is therefore not required according to the CLP Regulation.

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