4-hydroxy-3,5-dimethoxybenzonitrile

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008-12-17 to 2009-02-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

4-hydroxy-3,5-dimethoxybenzonitrile

Cas Number:

72684-95-8

IUPAC Name:

4-hydroxy-3,5-dimethoxybenzonitrile

Test material form:

solid

Specific details on test material used for the study:

Test Material: Syringonitrile
- Composition of test material, percentage of components: syringonitrile 99.8%
- Expiration date of the lot/batch: May 2010
-Physical description: pale yellow powder
-solubility: soluble in water
-stability: test substance was expected to be stable for the duration of the testing.

methyl-3H Thymidine
Source: Amersham Batch #B512 received on Jan. 28, 2009. The following information related to the characterization of the radioisotope was provided on the product specification sheet.
Specific Activity: 25 Ci/mmol; 925 GBq/mmol
MW: 244
Radioactive concentrations: 37 MBq; 1 mCi/mL
Date of analysis: Nov. 12, 2008
Radiochemical Purity: 95.1% (HPLC)
Thymine content: 0.2%
Exp date: Jan. 28, 2010

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Received from Jackson Labs on Dec. 31, 2008
- Age at study initiation: Preliminary irritation group: young adult (8 weeks)
Test and Native control group: young adult
- Weight at study initiation: 20.6-24.9 grams
- Housing: suspended stainless steel caging with mesh floors or plastic perforated bottom caging which conform to the size recommendations in the most recent Guide for the Care and Use of Laboratory Animals DHEW (NIH). Litter paper was placed beneath the cage and was changed at least three times per week.
- Diet (e.g. ad libitum): Purina Rodent Chow ad libitum
- Water (e.g. ad libitum): filtered tap water was supplied ad libitum by an automatic water dispensing system.
- Acclimation period: 27 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-24°C
- Humidity (%): 9-32%
- Photoperiod (hrs dark / hrs light):12-hr light/dark cycle

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

0, 10%, 25%, 50% in DMF

No. of animals per dose:

5 mice per group per concentration for the main study (total of 5 groups)
For the preliminary study, 3 mice per group (total of 3 groups)

Details on study design:

In the preliminary screening test, 3 mice were treated with daily application of 25 μL of various dilutions of Mediator SNP to the dorsal of each ear for three consecutive days for signs of irritation and toxicity. On day 6, each site was evaluated for local reactions (edema and erythema). From these results, the concentrations to be tested in the main assay were selected with the maximum concentration equal to a 50% w/w mixture in DMF.
In the main test, groups of 5 mice each were treated with the test material at concentrations of 0, 10, 25% or 50% in DMF to the dorsal surface of each ear for three consecutive days (Days 1, 2, and 3). On day 6 (three days after the third topical application), all mice were injected via the tail vein with 250 μL of phosphate buffered saline containing 3H-methyl thymidine (for a total of 20 μCi per mouse). Five fours following the administration of 3HTdR, all mice were killed and the auricular lymph nodes were drained. The lymph node cells were collected, washed and suspended in appropriate medium. DNA was then precipitated by the addition of 5 mL of 5% trichloroacetic acid (TCA). Following the overnight precipitation of DNA, the tubes were centrifuged and the supernatant discarded. The precipitate was resuspended in TCA and 3HTdR incorporation was measured by β-scintillation counting. A group of 5 mice receiving 25% HCA (alpha-hexylcinnamaldehyde technical) in DMF served as positive control group.

Positive control substance(s):

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

Statistics:

Statistical analysis was performed when individual lymph nodes were analyzed. Significance was judged at p<0.05. The treated groups and negative 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 were 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).

Results and discussion

Positive control results:

Well defined erythema was noted in mice from the positive control group. A stimulation index (SI) of greater than 3.0 was not observed in any of the test groups. The positive control (HCA) at 25% produced a dermal sensitization response with SI = 11.57.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

2.18

Test group / Remarks:

Group 2: 10%

Results 2

Parameter:

SI

Value:

2.02

Test group / Remarks:

Group 3: 25%

Results 3

Parameter:

SI

Value:

1.83

Test group / Remarks:

Group 4: 50%

Results 4

Parameter:

SI

Value:

11.57

Test group / Remarks:

Group 5: positive control

Cellular proliferation data / Observations:

No signs of systemic toxicity were noted in all groups of mice treated with Mediator SNP. There were no effects on body weights throughout the entire experiment. There was no irritation observed on any ear treated with the vehicle control or Mediator SNP at 0, 10, 25 or 50%. Well defined erythema was noted in mice from the positive control group. A stimulation index (SI) of greater than 3.0 was not observed in any of the test groups. The positive control (HCA) at 25% produced a dermal sensitization response with SI = 11.57.

Any other information on results incl. tables

Mean DPM/Stimulation Index
	Mean DPM	Stimulation Index
Group 1 – Vehicle control	567.06	NA
Group 2 – 10%	1237.16	2.18
Group 3 – 25%	1148.08	2.02
Group 4 – 50%	1039.03	1.83
Group 5 – Positive control	6563.31	11.57

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of this assay, Mediator SNP (syringonitrile) at concentrations equal to or below 50% are not considered to be a dermal sensitizer. The response observed with the positive control validates the test system used in this study.

Executive summary:

The objective of this study was to assess the skin sensitization potential of Mediator SNP (4-hydroxy-3,5- dimethoxybenzonitrile) after repeated topical applications.

In the preliminary screening test, 3 mice were treated with daily application of 25 μL of various dilutions of Mediator SNP to the dorsal of each ear for three consecutive days for signs of irritation and toxicity. On day 6, each site was evaluated for local reactions (edema and erythema). From these results, the concentrations to be tested in the main assay were selected with the maximum concentration equal to a 50% w/w mixture in DMF.

In the main test, groups of 5 mice each were treated with the test material at concentrations of 0, 10, 25% or 50% in DMF to the dorsal surface of each ear for three consecutive days (Days 1, 2, and 3). On day 6 (three days after the third topical application), all mice were injected via the tail vein with 250 μL of phosphate buffered saline containing3H-methyl thymidine (for a total of 20 μCi per mouse). Five hours following the administration of 3HTdR, all mice were killed and the auricular lymph nodes were drained. The lymph node cells were collected, washed and suspended in appropriate medium. DNA was then precipitated by the addition of 5 mL of 5% trichloroacetic acid (TCA). Following the overnight precipitation of DNA, the tubes were centrifuged and the supernatant discarded. The precipitate was resuspended in TCA and 3HTdR incorporation was measured by β-scintillation counting. A group of 5 mice receiving 25% HCA (alpha hexylcinnamaldehyde technical) in DMF served as positive control group.

A stimulation index (SI) is derived from each experimental group. Statistically significant increases in cell proliferation in the treated groups compared to the vehicle control group and/or SI of greater than or equal to 3.0 generally indicate a positive response.

No signs of systemic toxicity were noted in all groups of mice treated with Mediator SNP. There were no effects on body weights throughout the entire experiment. There was no irritation observed on any ear treated with the vehicle control or Mediator SNP at 0, 10, 25 or 50%. Well defined erythema was noted in mice from the positive control group. A stimulation index (SI) of greater than 3.0 was not observed in any of the test groups. The positive control (HCA) at 25% produced a dermal sensitization response with SI = 11.57.

Under the conditions of this assay, Mediator SNP at concentrations equal to or below 50% are not considered to be a dermal sensitizer. The response observed with the positive control validates the test system used in this study.