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EC number: 220-239-6 | CAS number: 2682-20-4
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Administrative data
Description of key information
Skin sensitization - A local lymph node assay (LLNA) study in CBA/J mice was conducted accoridng to OECD 429 and US EPA OPPTS 870.2600 guidelines and in accordance with the Principles of Good Laboratory Practice (GLP). Groups os mice were exposed to methylisothiazolone at nominal concentrations of 0.1, 0.3, 0.6, 0.9, 1.2 and 1.5% in 4:1 acetone/olive oil and evaluated for skin sensitization reactions.Based on the data from this study, methylisothiazolone at 1.35, 1.57 and 1.8% induced a hypersensitivity response and therefore is considered to be a potential sensitizer.
A skin senitization (Buehler test) study in guinea pigs was conducted according to OECD 406 and US EPA OPP 81 -6 guidelines and in accordance with the Principles of Good Laboratory Practice (GLP). Groups of guinea pigs were exposed dermally (epicutaneous, occlusive) to concentrations of 5000, 7500, 15000 and 30000 ppm, (a.i.). Under the conditions of this study, RH-24,573 produced delayed contact hypersensitivity in guinea pigs. The EC50's for induction and elicitation are estimated to be >5000 ppm a.i. (at a challenge co ncentration of 15,000 ppm a.i.) and >15,000 ppm a.i. (at an induction concentration of 30,000 ppm a.i.) RH-24,573, respectively.
Key value for chemical safety assessment
Skin sensitisation
Link to relevant study records
- Endpoint:
- skin sensitisation: in vivo (non-LLNA)
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 27 January - 4 March 1988
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: GLP/Guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 406 (Skin Sensitisation)
- Deviations:
- not specified
- Qualifier:
- according to guideline
- Guideline:
- EPA OPP 81-6 (Skin Sensitisation)
- Deviations:
- not specified
- GLP compliance:
- yes
- Type of study:
- Buehler test
- Justification for non-LLNA method:
- This study was conducted before the adoption of LLNA guideline
- Species:
- guinea pig
- Strain:
- Hartley
- Sex:
- male/female
- Details on test animals and environmental conditions:
- One hundred fifty-two outbred Hartley guinea pigs [Crl:(HA)BR] were obtained from Hazleton Research Animals, Denver, PA, on January 06, 1988. Upon arrival animals were placed into an animal room, examined for physical abnormalities, assigned unique animal numbers (identified by cage cards), and acclimated to the study room for approximately three weeks. Fifty of the 152 animals that were originally placed in one study room were used in the present study. Animal care quarantine procedures were in effect during the first week of acclimation. The animals were individually housed in stainless steel cages (16" x 9-1/4" x 7") with wire mesh bottoms and fronts. Cages were suspended above absorbent paper liners which were changed daily. The animals were housed in an environmentally controlled room with controls set to maintain a temperature of 75°F with a relative humidity of
40-60%. Temperature and relative humidity were monitored 24 hrs a day and the light cycle was automatically controlled 12 hrs on and 12 hrs off. All guinea pigs had free access to filtered tap water (via automatic watering) and Certified Purina Guinea Pig Chow #5026, throughout the acclimation and test period except during the 6-hr periods of restraint during dosing. One day prior to the first induction dose, the guinea pigs were randomized into groups using computer generated random numbers. Each animal was then identified by a cage card, indicating the unique animal number, sex, test material, group number and the protocol number. At the initiation of the induction phase, the animals were approximately 5 weeks old and their body weights ranged from 365 to 534 grams. The guinea pigs fit comfortably in the restrainers throughout the study. - Route:
- epicutaneous, occlusive
- Vehicle:
- water
- Concentration / amount:
- 5000, 7500, 15000 and 30000 ppm a.i.
- Route:
- epicutaneous, occlusive
- Vehicle:
- water
- Concentration / amount:
- 5000, 7500, 15000 and 30000 ppm a.i.
- No. of animals per dose:
- 4 guinea pigs
- Details on study design:
- Dosinq Procedure
The procedures for dosing are those that are described in the method of Buehler (1). This procedure was followed for the animals of the naive control and induced groups during the induction and challenge phases. The day before application of the test substance, the hair was removed from the back of each guinea pig with electric clippers equipped with a surgical clipping head. Prior to each dosing, the guinea pig was placed in a restrainer, and 0.4 ml of the test substance was pipetted onto a patch*. The patch was then placed on the shaved skin of the guinea pig and the application site was occluded with rubber dental dam (0.01 inches thick). At the end of the 6-hr exposure period, each animal was removed from its restrainer and the patch and dam discarded. The exposure sites were washed with paper towels soaked in lukewarm tap water, the skin dried with paper towels, and the animals returned to their cages. Animals in the naive control group received a "SHAM" treatment during the induction phase. The exposure site was shaved, the animals were restrained, a blank patch was placed on the application site which was occluded for 6 hrs, and the shaved sites were washed in the same manner as the induced group.
Assessment of Primary Skin Irritation
Range-finding skin irritation tests were conducted on four naive guinea pigs to select doses to be used for the induction and challenge phases. Four concentrations of RH-24,573 (i.e., 5000, 7500, 15000 and 30000 ppm a.i.) were dissolved in distilled water and applied to four guinea pigs. The application sites were changed among the guinea pigs for each of the above concentrations to minimize any site-to-site variation in irritation response. The test substance was applied as described below. All application sites were depilated prior to scoring. Primary skin irritation was scored at 24 and 48 hrs after patch removal.
Assessment of Delayed Contact Hypersensitivit(DCH)
The closed patch method of Buehler (1) which has been validated in our laboratory with dinitrochlorobenzene (3) was used. Ten induction doses were applied to the shaved backs of guinea pigs over a 3.5 week period (3 doses/week; Mondays, Wednesdays and Fridays). Each dose (6-hr exposure period) consisted of 0.4 ml of the test material at 1000, 5000, 15000 or 30000 ppm a.i. in distilled water. Guinea pigs in the naive control group received a blank patch. Two weeks after the last induction dose, all guinea pigs in the naive control and RH-24,573 induced groups were challenged with three concentrations of RH-24,573 in distilled water (i.e., 1000, 5000 and 15,000 ppm a.i.). Nineteen to 22 hrs after the challenge application, the backs of the guinea pigs were depilated with Neet lotion hair remover. The depilatory was liberally applied to the clipped application sites and allowed to remain on the animals for approximately 20 minutes. The animals were rinsed with lukewarm running tap water, blotted dry, and returned to their cages. Two to 5 hrs after depilation (24 hrs after removal of the challenge patch) the erythema reactions at the application sites were scored according to the method of Ritz and Buehler (2)
Score Erythema Reaction
0................No reaction
+/-...........Slight patchy erythema
1................Slight confluent or moderate patchy erythema
2................Moderate erythema
3................Severe erythema
The application sites were re-evaluated 24 hrs later (i.e., 48 hrs after the challenge application).
Evaluation of Results
The results were evaluated by comparing the incidence of erythema reactions (at either 24 or 48 hrs) in the control groups to the incidence in the induced group. Grades of + were considered to be representative of insignificant erythema responses. Only those scores of grade 1 or greater were considered positive responses.
References:
1. Buehler, E.V. (1965), Delayed Contact Hypersensitivity in the Guinea Pig, Arch. Dermatol., 91, 171-175.
2. Ritz, H.L., and Buehler, E.V. (1980) Current Concepts in Cutaneous Toxicity, pp. 25-40. Eds. V.A. Drill and P. Lazar. Academic Press, New York.
*The "Hill-top" patches used were composed of a Parke-Davis Readi-Bandage modified by the addition of a centered Webril pad (20 x 20 mrn). - Challenge controls:
- Guinea pigs in the naive control group received a blank patch. Two weeks after the last induction dose, all guinea pigs in the naive control groups were challenged with three concentrations of RH-24,573 in distilled water (i.e., 1000, 5000 and 15,000 ppm a.i.)
- Positive control substance(s):
- yes
- Remarks:
- Dinitrochlorobenzene has been used in the lab to validate the method.
- Positive control results:
- Dinitrochlorobenzene has been used in the lab to validate the method.
- Key result
- Reading:
- 1st reading
- Hours after challenge:
- 48
- Group:
- test chemical
- Dose level:
- 15000ppm
- No. with + reactions:
- 3
- Total no. in group:
- 10
- Clinical observations:
- Erythema
- Remarks on result:
- positive indication of skin sensitisation
- Interpretation of results:
- sensitising
- Remarks:
- Migrated information Criteria used for interpretation of results: expert judgment
- Conclusions:
- Under the conditions of this study, RH-24,573 produced delayed contact hypersensitivity in guinea pigs. The EC50's for induction and elicitation are estimated to be >5000 ppm a.i. (at a challenge concentration of 15,000 ppm a.i.) and >15,000 ppm a.i. (at an induction concentration of 30,000 ppm a.i.) RH-24,573, respectively.
- Executive summary:
The delayed contact hypersensitivity (DCH) potential of RH-24,573 (Lot No. ASU 2379, TD No. 88-001, 99.8% a.i.) was tested in young adult outbred Hartley guinea pigs using the closed patch method of Buehler. Four groups of guinea pigs (5/sex/group) received ten 6-hr induction doses (3 doses/week, for 3.5 weeks) of 0.4 ml of the test material at the following concentrations: 1000, 5000, 15,000 or 30,000 ppm a.i. in distilled water. These animals and a group of 10 naive control guinea pigs (i.e., receiving no induction treatments) were challenged 2 weeks after the last induction dose. All guinea pigs were challenged with RH-24,573 at 1000, 5000 and 15,000 ppm a.i. in distilled water. Erythema reactions were scored at 24 and 48 hrs after the challenge exposure.
No erythema reactions were observed in the noninduced control animals following challenge with 1000, 5000 and 15,000 ppm a.i. RH-24,573.
In groups of guinea pigs induced with RH-24,573 at 1000, 5000, 15,000 and 30,000 ppm a.i., the incidence of erythema responses following challenge with 1000 ppm a.i. RH-24,573 was 0/10, 0/10, 1/10 and 0/10, respectively. In these same induction groups, the incidence of erythema following challenge at 5000 ppm a.i. RH-24,573 was 0/10, 2/10, 1/10 and 2/10, respectively, and at 15,000 ppm a.i. RH-24,573 was 1/10, 6/10, 3/10 and 5/10, respectively.
The concentrations of RH-24,573 required to induce and elicit a response in 50% of the animals (EC50) are estimated to be ≥5000 ppm a.i. for induction at a challenge concentration of 15,000 ppm a.i. and ≥15,000 ppm a.i. for elicitation at an induction concentration of 30,000 ppm a.i.. These induction and elicitation concentrations are approximately 20 to 40 fold greater than those required to induce and elicit a 50% incidence of sensitization with Kathon® 886 biocide.
CONCLUSION
Under the conditions of this study, RH-24,573 produced delayed contact hypersensitivity in guinea pigs. The EC50’s for induction and elicitation are estimated to be >5000 ppm a.i. (at a challenge concentration of 15,000 ppm a.i.) and >15,000 ppm a.i. (at an induction concentration of 30,000 ppm a.i.) RH-24,573, respectively
- Endpoint:
- skin sensitisation: in vivo (LLNA)
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 7 December 2002 - 8 March 2003
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: GLP/Guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
- Deviations:
- not specified
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.2600 (Skin Sensitisation)
- Deviations:
- not specified
- 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:
- Supplier: Jackson Laboratories, Bar Harbor, ME
Sex: Females
Mouse - CBA/J
Age Range at Initiation of Treatment: 9 weeks
Weight Range at Initiation of Treatment: 18-23 grams
Acclimation/Quarantine:
Following arrival at Calvert, mice were assessed as to their general health by a member of the veterinary staff or other authorized personnel. Mice were acclimatedlquarantined for 9 days prior to treatment initiation, during which each mouse was observed at least once daily for any abnormalities or for the development of infectious disease.
Animal Husbandry:
Animals were group housed 5 per cage in compliance with the National Research Council "Guide for the Care and Use of Laboratory Animals". Calvert is a USDA registered and a fully AAALAC accredited facility.
The animal room environment was controlled (targeted conditions: temperature 18 to 26C, relative humidity 30 to 70%, 12 hours artificial light and 12 hours dark).
Temperature and relative humidity were monitored daily. During the acclimation period the temperature was out of range from 21 Feb to 23 Feb 2003 for about 36 hours when the temperature was as low as 17.2C. From 23 Feb to 24 Feb 2003 the temperature was out of range for about 19 hours when the temperature was as low as 16.1°C. The relative humidity was out of range on 23 Feb 2003 for a period of about 3 hours and went as low as 26%. From 25 Feb to 6 Mar 2003 the relative humidity was out of range. During this period the relative humidity was as low as 6%. These deviations from the protocol did not adversely affect the results of the study.
All animals had access to Certified Rodent Diet #7012C (Harlan Teklad) or equivalent ad libitum, unless otherwise specified. The lot number(s) and specifications of each lot used are archived at Calvert.
Water was provided to the animals ad libitum, via water bottles unless otherwise specified. Periodic analyses of the water are performed and the results are archived at Calvert.
There were no known contaminants in the diet or water, which at the levels detected would be expected to interfere with the purpose, conduct or outcome of the study.
Allocation to Treatment Groups:
Only mice considered suitable for use were placed on the study. Prior to treatment initiation, all mice were weighed and assigned to treatment groups using a computer generated randomization method based on body weight. - Vehicle:
- acetone/olive oil (4:1 v/v)
- Concentration:
- Methylisothiazolone was prepared at nominal concentrations of 0.1, 0.3, 0.6, 0.9, 1.2 and 1.5% in 4: 1 acetone/olive oil.
- No. of animals per dose:
- 5 females/dose level
- Details on study design:
- The test article, positive or vehicle control were applied daily (25 ul/ear) on the dorsal surface of both ears for 3 days according to Table 1.
Mice were weighed on Days 1 and 6. Mice were treated on the dorsal surface of both ears, once per day on Days 1, 2, and 3. Any irritation observed after test article application was recorded. On Day 6 the mice were injected i.v. with 20 uCi of 3Hthymidine in 250 ul of saline. Five hours later the mice were euthanized with C02 and the draining auricular lymph nodes removed. A single cell suspension was prepared from the lymph nodes of each mouse. Cells were washed twice with phosphate buffered saline (PBS) and precipitated with 5% trichloroacetic acid (TCA) overnight at 2-8°C. The pellets were recovered by centrifugation and resuspended in 1 ml of TCA and transferred to 10 ml of scintillation fluid. Incorporation of 3H-thymidine was measured in a B-scintillation counter.
OBSERVATIONS
Morbidity/Mortality:
Animals were observed daily.
Gross Signs of Toxicity:
On the Days of dose administration, animals were observed prior to dose and immediately post dose. On Days 4-6, animals were observed once a day for gross signs of toxicity. Any irritation observed after test article application was recorded.
Body Weight:
Animals were weighed on Days 1 and 6.
METHOD OF ANALYSIS
The mean DPM for each group was determined. Increases in 3H-thymidine incorporation relative to vehicle-treated control were derived for each group and recorded as stimulation indices (SI). The criterion for a positive response is that one or more concentrations of a test article elicits a 3-fold or greater increase in isotope incorporation relative to the vehicle control. - Positive control substance(s):
- hexyl cinnamic aldehyde (CAS No 101-86-0)
- Statistics:
- Evaluation of equality of means was made by a one way analysis of variance using the F distribution to assess statistical significance using Systat (version 9.01, SYSTAT, Inc.). If statistically significant differences between the means were found, a Dunnett's test was used to determine the degree of significance from control means.
- Positive control results:
- The positive control, 50% Hexylcinnamaldehyde (HCA) in acetone:olive oil, resulted in a stimulation index greater than 3 (12.19) indicating a positive response. This response compared to the vehicle control was also statistically significant (p < 0.01).
- Parameter:
- SI
- Remarks on result:
- other: The test article was also positive at 1.35, 1.57, and 1.8% (measured concentrations) (Table 2). The stimulation indices at these concentrations were 6.64,4.73 and 6.62, respectively. The lower concentrations of the test article were negative.
- Parameter:
- other: disintegrations per minute (DPM)
- Remarks on result:
- other: See Table 2.
- Interpretation of results:
- sensitising
- Remarks:
- Migrated information Criteria used for interpretation of results: expert judgment
- Conclusions:
- Based on the data from this study, methylisothiazolone at 1.35, 1.57 and 1.8% induced a hypersensitivity response and therefore is considered to be a potential sensitizer.
- Executive summary:
The test article, methylisothiazolone, was tested for its capacity to induce a hypersensitivity response in mice as measured by the proliferation of lymphocytes in the draining lymph nodes.
CBA/J female mice were treated on the dorsal surface of both ears once per day for 3 days with methylisothiazolone at either 0.15, 0.45, 0.76, 1.35, 1.57, or 1.8% (measured concentrations), the vehicle (acetone:olive oil, 4: I), or the positive control, 50% Hexylcinnamaldehyde (HCA). On Day 6, the mice were injected with 20 μCi of 3H-thymidine. Five hours later, the mice were euthanized and the draining auricular lymph nodes were removed. The lymph node cells were precipitated with 5% trichloroacetic acid (TCA) and the pellets counted in a ß-scintillation counter to determine incorporation of the 3H-thymidine.
This study was run twice. In the first trial, an error was found in the preparation of the dose solution. Therefore the study was repeated. Only the results from the second trial are reported here.
The positive control, 50% Hexylcinnamaldehyde (HCA) in acetone:olive oil, resulted in a stimulation index greater than 3 (12.19) indicating a positive response. This response compared to the vehicle control was also statistically significant (p<0.01).
The test article was also positive at 1.35, 1.57, and 1.8%. The stimulation indices at these concentrations were 6.64, 4.73 and 6.62, respectively. The lower concentrations of the test article were negative.
Mean body weights at Days 1 and 6 and mean changes in body weights for each treatment group were compared to the vehicle control group. No statistically significant differences were observed for any of the test article treated groups when compared to the vehicle control group. Therefore, the test article did not cause any overt toxicity.
Based on the data from this study, methylisothiazolone at 1.35, 1.57, and 1.8% (measured concentrations) induced a hypersensitivity response and therefore is considered to be a potential sensitizer.
Referenceopen allclose all
Assessment of Primary Skin Irritation
Minimal to no erythema was observed at 24 or 48 hrs in guinea pigs treated with the test material at the following concentrations; 5000, 7500, 15,000 and 30,000 ppm a.i.. Concentrations of RH-24,573 up to and including 30,000 ppm a.i. could be used for both induction and challenge.
Assessment of Delayed Contact Hypersensitivity (DCH)
No erythema reactions were observed in the noninduced control animals following challenge with 1000, 5000 and 15000 ppm a.i. RH-24,573.
In groups of guinea pigs induced with RH-24,573 at 1000, 5000, 15000 and 30000 ppm a.i., the incidence of erythema responses following challenge with 1000 ppm a.i. RH-24,573 was 0/10, 0/10, 1/10 and 0/10, respectively. In these same induction groups, the incidence of erythema following challenge at 5000 ppm a.i. RH-24,573 was 0/10, 2/10, 1/10 and 2/10, respectively, and at 15000 ppm a.i. RH-24,573 was 1/10, 6/10, 3/10 and 5/10, respectively.
The concentrations of RH-24,573 required to induce and elicit a response in 50% of the animals (EC50) are estimated to be 15,000 ppm a.i. for induction at a challenge concentration of 15,000 ppm a.i. and 15,000 ppm a.i. for elicitation at an induction concentration of 30,000 ppm a.i.. These induction and elicitation concentrations are approximately 20 to 40 fold greater than those required to induce and elicit a 50% incidence of sensitization with Kathon 886 biocide.
Table 1 A Summary of Incidences of Erythema of Grade 1 or Greater in Guinea Pigs During the Challenge Phase*
Induction | Erythema Responses after Challenge with: | ||
Treatment | RH-24,573 at 1000 ppm in distilled water |
RH-24,573 at 5000 ppm in distilled water |
RH-24,573 at 15,000 ppm in distilled water |
None | 0/10 | 0/10 | 0/10 |
RH-24,573 at 1000 ppm a.i. in distilled water | 0/10 | 0/10 | 1/10 |
RH-24,573 at 5000 ppm a.i. in distilled water | 0/10 | 2/10 | 6/10 |
RH-24,573 at 15,000 ppm a.i. in distilled water | 1/10 | 1/10 | 3/10 |
RH-24,573 at 30,000 ppm a.i. in distilled water | 0/10 | 2/10 | 5/10 |
*The maximum number of animals exhibiting erythema of grade 1 or greater at either 24 and/or 48 hrs, over the total number of animals in that group.
All test article treated and vehicle treated mice appeared normal throughout the study. On Day 3 the mice treated with HCA had decreased activity. No irritation was observed in any of the mice treated with the test article or controls. This study was run twice. In the first trial, an error was found in the preparation of the dose solution. Therefore the study was repeated.
Mean body weights at Days 1 and 6 and mean changes in body weights for each treatment group were compared to the vehicle control group. No statistically significant differences were observed for any of the test article treated groups when compared to the vehicle control group. Therefore, the test article did not cause any overt toxicity.
Table 2 Results of Local Lymph Node Assay
Group | Treatment | Measured Dose | DPM (mean + sem) | SI (Test/control Ratio) | Results1 |
1 | Acetone/olive oil (4:1 v/v) | - | 460 + 47 | - | - |
2 | Methylisothiazolone | 0.15% | 955 + 321 | 2.08 | - |
3 | Methylisothiazolone | 0.45% | 1104 + 185 | 2.40 | - |
4 | Metylisothiazolone | 0.76% | 1028 + 323 | 2.23 | - |
5 | Methylisothiazolone | 1.35% | 3055 + 705 | 6.64 | + |
6 | Methylisothiazolone | 1.57% | 2176 + 1193 | 4.73 | + |
7 | Methylisothiazolone | 1.8% | 3045 + 664 | 6.62 | + |
8 | HCA | 50% | 5607 + 2218** | 12.19 | + |
1 Test/control ratio of 3.0 or greater represents a positive result
**Statistically significant difference compared to the vehicle control group (P
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed (sensitising)
Respiratory sensitisation
Endpoint conclusion
- Endpoint conclusion:
- no study available
- Additional information:
No study available
Justification for classification or non-classification
Based on the results of the study, this material would be classified as Skin sensitizer, Category 1, H317
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