N-isopropylhydroxylamine

Endpoint summary

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:

5-25 April 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to test guidelines and in accordance with GLP

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

other: CBA/J

Sex:

female

Details on test animals and environmental conditions:

Sex: Female, nulliparous and non-pregnant.
Species/Strain: Mouse, CBA/J
Age/Body weight: Preliminary Animals: Young adult (11 weeks)
Test and Control Animals: Young adult (12 weeks)/20.5-24.1 grams at experimental start.
Source: Received from Harlan, Indianapolis, IN on March 15, 2011 (Preliminary Irritation, Test and Control Groups).

Housing: The animals were individually housed in plastic solid bottom cages with bedding during the dosing and resting phase of the study. After final weighing until sacrifice, animals were housed in their respective dose groups in plastic cages with bedding. Bedding in the plastic, solid bottom cages was changed at least once per week. All caging conformed to the size recommendations in the most recent Guide for the Care and Use of Laboratory Animals (Natl. Res. Council, 2011).
Animal Room Temperature and Relative Humidity Ranges: 19-24ºC and 42-63%, respectively.
Animal Room Air Changes/Hour: 12. Airflow measurements are evaluated regularly and the records are kept on file at Eurofins PSL.
Photoperiod: 12-hour light/dark cycle
Acclimation Period: 29 or 36 days
Food: Purina Rodent Chow #5001 ad libitum
Water: Filtered tap water was supplied ad libitum by an automatic water dispensing system.
Contaminants: There were no known contaminants reasonably expected to be found in the food or water at levels which would have interfered with the results of this study. Analyses of the food and water are conducted regularly and the records are kept on file at Eurofins PSL.

Vehicle:

propylene glycol

Concentration:

Preliminary Toxicity Testing
Four test substance concentrations and the vehicle control were used. Test substance concentrations of 15%, 10%, 5% and 2.5% were tested to determine the highest achievable level that avoids overt systemic toxicity and excessive local irritation. The top dose of 15% was selected based on maximum solubility/compatibility of the test material with the vehicle while maintaining a pipettable solution that could be applied to the mouse ear (documented in the raw data)

Definitive Study - 0, 1, 5 and 15%

No. of animals per dose:

Preliminary Study - 2 mice/group

Definitive Study - 5 mice/group

Details on study design:

Preliminary Toxicity Testing
Four test substance concentrations and the vehicle control were used. Test substance concentrations of 15%, 10%, 5% and 2.5% were tested to determine the highest achievable level that avoids overt systemic toxicity and excessive local irritation. The top dose of 15% was selected based on maximum solubility/compatibility of the test material with the vehicle while maintaining a pipettable solution that could be applied to the mouse ear (documented in the raw data). Each group consisted of two mice. The ears of each mouse were scored for erythema and edema prior to dosing on Days 1, 2, 3, and prior to termination on Day 6.

Twenty-five μL of the appropriate dilution of the test substance concentration or the vehicle alone was applied to the dorsum of both ears of each mouse for three consecutive days (Days 1, 2 and 3). Application was done using an appropriate size micropipette to accurately deliver 25 μL. The dose was gently spread as evenly as possible over the dorsal surface of the ear using the tip of the pipette. No treatment was made on Days 4 and 5. On Day 6, each site was evaluated for local irritation (erythema & edema).

Animals were observed daily for signs of toxicity. The Study Director and Sponsor used this data in conjunction with any pre-existing data to select the maximum concentration to be tested. Test substance concentrations of 1%, 5% and 15% w/w mixtures in propylene glycol were selected for testing.

Selection of Animals/Dose Levels
Prior to dosing, the animals were weighed and the ears were checked for any abnormalities or clinical signs of diseases or injury. Twenty-five healthy naive female mice without pre-existing ear irritation were selected and distributed (5 mice per group).

Sample Preparation
Dilutions of the test substance were prepared as w/w mixtures in propylene glycol. Concentrations of 1%, 5% and 15% were selected for the main test based on results of the preliminary screening test. A single concentration of a 25% w/w mixture of HCA in propylene glycol was prepared as a positive control. All dosage preparations were freshly prepared on the day of administration.

Test Substance Application
Beginning on Day 1, a volume of 25 μL of the vehicle, the appropriate test substance concentration or the positive control substance was applied to the dorsum of both ears of each mouse once per day for three consecutive days (Days 1, 2, and 3) using a micropipette. During application, the material was gently spread as evenly as possible over the dorsal surface of the ear using the tip of the pipette.

Dermal Scoring
Prior to each application (Days 1, 2 and 3), the ears were evaluated for erythema and edema according to the modified Draize scoring system3. All ears were also evaluated on Day 6.

3H-methyl Thymidine Injections
On Day 6 of the study (three days after the final topical application) 250 μL of sterile phosphate buffered saline (PBS, Lot #: 060M8222) containing 20 μCi of 3H-methyl thymidine (Lot #201103) was injected intravenously via the tail vein of each mouse.

Lymph node assessment
Approximately five hours after the injection, the draining auricular lymph nodes from all animals were excised. The lymph nodes were pooled for each individual mouse. A single cell suspension of lymph node cells (LNC) was prepared in PBS by gently massaging the lymph nodes between the frosted ends of two microscope slides over a collection vessel. The slides were then rinsed briefly with PBS into the vessel. The contents of the vessel were transferred to a centrifuge tube and washed with an excess of PBS and centrifuged for approximately 10 minutes at 1750 rpm, with a g-force of 283.15g. This process was carried out twice. In both cases, the supernatant was decanted and discarded following each centrifugation. After the second wash, approximately 5 mL of 5% trichloroacetic acid (TCA, Lot #: 101712) was then added to the sediment and the tube was vortexed briefly. The DNA was then precipitated in the TCA at approximately 4°C overnight (approximately 18 hours).

Following the overnight precipitation of the DNA, the tubes were centrifuged again for approximately 10 minutes and the supernatant was discarded. The resulting precipitate was re-suspended using 1 mL of TCA and transferred to 10 mL of scintillation fluid. Incorporation of 3H-methyl thymidine was measured by Β-scintillation counting and expressed as disintegrations per minute, minus background dpm.

Clinical Observations
All test and preliminary mice were observed for signs of mortality, gross toxicity, and/or behavioral changes daily. All test mice were euthanized via overdose of inhaled Isoflurane anesthetic on Day 6.

Body Weights and Body Weight Gain
All main test mice were weighed on Day 1 (prior to the first application) and prior to sacrifice on test Day 6. Body weight gain (or loss) was then calculated for each group.

EVALUATION
The mean and standard deviation of the dpm values were calculated for each dose group. A stimulation index (SI) was derived for each experimental group by dividing the mean dpm of each experimental group by the mean dpm of the vehicle control group. Any test material that produces a SI ≥ 3 in the LLNA is normally considered “positive” for dermal sensitization potential (Kimber et al., 1994). While a SI ≥ 3 was originally developed empirically, a robust statistical evaluation indicated that it is an acceptable practical value for hazard identification (Basketter et al., 1999a). Furthermore, by determining EC3 values (estimated concentration resulting in a 3-fold SI), one can compare relative sensitization potency of chemicals and/or formulations (Basketter et al., 1999b). While a test material that produces a SI of ≥ 3 in the LLNA should be considered “positive” for contact sensitization (Kimber et al., 1994), recent opinions have suggested circumstances in which the LLNA result and sensitization potential should be further considered in the context of additional scientific judgment (Ryan et al., 2000; Basketter et al., 1998; Basketter et al., 2006). Based on the EC3 values derived from the LLNA, it has been proposed that contact allergens can be categorized as weak (≥10%-≤100%), moderate (≥1%-<10%), strong (≥0.1%-<1%), or extreme (<0.1%) (ECETOC, 2003).

If applicable, the EC3 value will be calculated to determine the concentration of test substance that induces a stimulation index of 3.0. It is determined by linear interpolation between two points of the stimulation indices axis, one above (a,b) and one below (c,d) the stimulation index of 3.0. If all dose levels induce a stimulation index greater than 3.0 or less than 3.0, the EC3 will not be calculated.

{EC3=c+[(3-d)/(b-d)] x (a-c)}

The final interpretation of the biological significance of the responses will be based on both statistical outcome and scientific judgment.

References
Basketter, D. A., Gerberick, G. F. and Kimber I. (1998). Strategies for identifying false positive responses in predictive skin sensitization tests. Food and Chemical Toxicology 36, 327-333.

Basketter, D. A., Lea, L. J., Cooper, K., Stocks, J., Dickens, A., Pate, I., Dearman R., J., and Kimber, I. (1999a). Threshold for classification as a skin sensitizer in the local lymph node assay: a statistical evaluation. Food and Chemical Toxicology 37(12), 1167-1174.

Basketter, D. A., Lea, L. J., Dickens, A., Briggs, D., Pate, I., Dearman, R. J., and Kimber, I. (1999b). A comparison of statistical approaches to the deviation of EC3 values from local lymph node assay dose responses. Journal of Applied Toxicology 19, 261-266.

Basketter, D. A., McFadden, J., Evans, P., Andersen, K. E., and Jowsey, I. (2006). Identification and classification of skin sensitizers: identifying false positives and false negatives. Contact Dermatitis 55, 268-273.

Boverhof, D. R., Wiescinski, C. M., Botham, P., Lees, D., Debruyne, E., Repetto-Larsay, M., Ladics, G., Hoban, D., Gamer, A., Remmele, M., Wang-Fan, W., Ullmann, L. G., Mehta, J., Billington, R. and Woolhiser, M. R. (2008). Interlaboratory validation of 25% Pluronic L92 surfactant as a suitable, aqueous vehicle for testing pesticide formulations using the murine local lymph node assay. Toxicol. Sci. 105(1), 79-85.

ECETOC (European Centre for Ecotoxicology and Toxicology of Chemicals). Contact Sensitisation: Classification According to Potency. Technical Report No. 87. Brussels, April 2003 (ISSN-0773-8072-87).

Kimber, I., Dearman, R. J., Scholes, E. W., and Basketter, D. A. (1994). The local lymph node assay: developments and applications. Toxicology 93, 13-31.

Ryan, C. A., Gerberick, G. F., Cruse, L. W., Basketter, D. A., Lea, L., Blaikie, L., Dearman, R. J., Warbrick, E. V., and Kimber, I. (2000). Activity of human contact allergens in the murine local lymph node assay. Contact Dermatitis 43, 95-102.

Positive control substance(s):

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

Statistics:

Statistical analysis was performed on mean body weights, body weight gains and DPM values. Significance was judged at p <0.05. The treated groups and vehicle control group were compared using a One-Way Analysis of Variance (ANOVA), followed by comparison of the treated groups to control by Dunnett’s t-test for multiple comparisons. Where variances are considered significantly different by Bartlett’s test, groups were compared using a non-parametric method (Kruskal-Wallis non parametric analysis of variance followed by Dunn’s test) (INSTAT Biostatistics, Graph Pad Software, San Diego, CA). Outlier analysis was conducted using Grubbs test (1969).

Positive control results:

Proper conduct of the LLNA was confirmed via a positive response with 25% HCA, a moderate contact sensitizer, which elicited a proliferative response with a Stimulation Index (SI) value of 4.58 relative to vehicle controls.

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

During the preliminary screening study, mice received three daily doses of 15%, 10%, 5% and 2.5% of Isopropylhydroxylamine (IPHA). The top dose of 15% was selected based on maximum solubility/compatibility of the test material with the vehicle while maintaining a pipettable solution that could be applied to the mouse ear (documented in the raw data). Very slight erythema was noted in mice dosed with the vehicle control and at concentrations of 15%, 10%, 5% and 2.5%. Based on the results of the screening assay, 1%, 5% and 15% were evaluated in the main study. All mice appeared active and healthy, and there were no consistent treatment-related effects on body weight over the course of the study. Between Days 2 and 6, very slight erythema was noted in the vehicle control and treated groups dosed with 1%, 5% and 15% test substance. Treatment of mice with 1%, 5% and 15% of Isopropylhydroxylamine (IPHA) resulted in stimulation index values of 1.02, 1.15 and 1.12, respectively, relative to vehicle control mice. As a stimulation index (SI) of greater than 3.0 was not observed, Isopropylhydroxylamine (IPHA) was considered negative for dermal sensitization potential.

During the preliminary screening study, mice received three daily doses of 15%, 10%, 5% and 2.5% of Isopropylhydroxylamine (IPHA). The top dose of 15% was selected based on maximum solubility/compatibility of the test material with the vehicle while maintaining a pipettable solution that could be applied to the mouse ear (documented in the raw data). Very slight erythema was noted in mice dosed with the vehicle control and at concentrations of 15%, 10%, 5% and 2.5%.

Based on the results of the screening assay, 1%, 5% and 15% were evaluated in the main study. All mice appeared active and healthy, and there were no consistent treatment-related effects on body weight over the course of the study. Between Days 2 and 6, very slight erythema was noted in the vehicle control and treated groups dosed with 1%, 5% and 15% test substance.

Treatment of mice with 1%, 5% and 15% of Isopropylhydroxylamine (IPHA) resulted in stimulation index values of 1.02, 1.15 and 1.12, respectively, relative to vehicle control mice. As a stimulation index (SI) of greater than 3.0 was not observed, Isopropylhydroxylamine (IPHA) was considered negative for dermal sensitization potential.

A table summarizing the results of the LLNA is found below:

	Mean DPM	Stimulation Index^1
Group 1-Vehicle Control	492.50	-
Group 2- 1% Test substance	499.93	1.02
Group 3- 5% Test substance	565.50	1.15
Group 4- 15% Test substance	551.55	1.12
Group 5- Positive Control	2254.84	4.58

¹ The stimulation index is derived by dividing the dpm of each experimental group by the dpm of the vehicle control group. A stimulation index of greater than or equal to 3.0 generally indicates a positive response.

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: expert judgment

Conclusions:

Based on the results of this study, Isopropylhydroxylamine (IPHA) is considered to lack dermal sensitization potential in the LLNA at concentrations of less than or equal to 15%.
Proper conduct of the LLNA was confirmed via a positive response with 25% HCA, a moderate contact sensitizer.

Executive summary:

A dermal sensitization test was conducted with mice to determine the potential for Isopropylhydroxylamine (IPHA) to produce sensitization after repeated topical applications.

Three concentrations of the test substance in propylene glycol or the vehicle alone were topically applied to twenty healthy test mice (5 mice/group) for three consecutive days. Three days after the last application, the mice were given a 20 μCi IV injection of 3H-methyl thymidine. Five hours later, the draining (auricular) lymph nodes were harvested and prepared for analysis in a scintillation counter. The results are presented in disintegrations per minute per mouse (dpm/mouse). Each animal’s ears were also evaluated for erythema and edema prior to each application and again on Day 6, prior to the IV injection. A positive control group (five animals) was maintained under the same environmental conditions and treated with a 25% w/w mixture of alpha-Hexylcinnamaldehyde Technical (HCA) in propylene glycol in the same manner as the test animals.

A table summarizing the results of the LLNA is found below:

	Mean DPM	Stimulation Index^1
Group 1-Vehicle Control	492.50	-
Group 2- 1% Test substance	499.93	1.02
Group 3- 5% Test substance	565.50	1.15
Group 4- 15% Test substance	551.55	1.12
Group 5- Positive Control	2254.84	4.58

Based on the results of this study, the test substance is considered to lack dermal sensitization potential in the LLNA at concentrations of less than or equal to 15%. Proper conduct of the LLNA was confirmed via a positive response with 25% HCA, a moderate contact sensitizer.

¹ The stimulation index is derived by dividing the dpm of each experimental group by the dpm of the vehicle control group. A stimulation index of greater than or equal to 3.0 generally indicates a positive response.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

A dermal sensitization test (LLNA) was conducted with mice to determine the potential for Isopropylhydroxylamine (IPHA) to produce sensitization after repeated topical applications. Based on the results of this study, the test substance is considered to lack dermal sensitization potential in the LLNA at concentrations of less than or equal to 15%. Proper conduct of the LLNA was confirmed via a positive response with 25% HCA, a moderate contact sensitizer.

Migrated from Short description of key information:
One study OECD 429 guideline study with IPHA using the LLNA in CBA/J mice

Justification for selection of skin sensitisation endpoint:
The study was conducted according to test guidelines and in accordance with GLP

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

IPHA was not found to be a sensitizer up to 15% and therefore is not classifiable under GHS.