3-(p-methoxyphenyl)-2-methylpropionaldehyde

Endpoint summary

Description of key information

Local lymph node assay (OECD TG 429): EC3 = 23.63%, skin sensitiser category 1B; The NOEC is 15%.

HRIPT at 5%: absence of skin sensitisation

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:

25 Aug 2004 to 31 Aug 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The study is conducted in 2004 before REACH required in vitro tests first (2016)

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

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Jackson Laboratories, Bar Harbor, ME 04609
- Age at study initiation: 9 to 11 weeks at start of dosing
- Weight at study initiation: 18 to 26 grams at the outset (Day 1) of the study
- Housing: Animals were group housed (5 per cage) upon receipt in compliance with National Research Council "Guide for the Care and Use of Laboratory Animals". No other species were kept in the same room.
- Diet: Animals had access to Certified Rodent Chow 7012C ad libitum.
- Water: Tap water was available ad libitum, via water bottles.
- Acclimation period: Study animals were acclimated to their housing for six to seven days prior to their first day of dosing.
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.7 to 25.6
- Humidity (%): 39 to 54
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

other: Diethyl phthalate/ethanol in a ratio of 3:1

Concentration:

7.5, 15 and 30 %

No. of animals per dose:

5

Details on study design:

JUSTIFICATION FOR DOSE LEVELS
Doses were selected by the sponsor. The highest dose that will be used in the intended application will not exceed 20%. However, as a conservative measure, and for a higher margin of safety, 30% was chosen as the high dose.

MAIN STUDY
Mice were treated on the dorsal surface of both ears, once per day on Days 1, 2, and 3. Approximately 24 ± 2 hours between applications of test article was maintained. On Day 6 the mice were injected i.v. with 20 pCi of 3H-thymidine in 250 µL of sterile saline. Five hours later the mice were euthanized by CO2 asphyxiation. Ear thickness measurements of each mouse were recorded and the draining auricular lymph nodes were removed. At removal, the number of nodes collected per animal were recorded, and the nodes were examined for size/appearance and the data recorded. Any unexpected observations were noted in study records. 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 a vial containing scintillation fluid. An additional 1 mL of TCA was used to rinse the tube, and was also transferred to the scintillation fluid. Incorporation of 3H-thymidine was measured in a ß-scintillation counter.

IN-LIFE OBSERVATIONS AND MEASUREMENTS
- Mortality/ morbidity: Daily on days 1 to 6.
- Clinical observations: Prior to dose administration and a minimum of once post-dose on Days 1 to 3. Clinical observations were also performed once daily on Days 4-6. Particular attention was given to the application sites.
- Body weight: Animals were weighed at the time of randomization/selection, and on Days 1 and 6

Positive control substance(s):

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

Statistics:

- The mean DPM for each group will be evaluated using SYSTAT version 9.01, developed by SPSS, Inc. Increases in 3H-thymidine incorporation relative to the vehicle-treated control will be derived for each group and recorded as stimulation indices (Sl).
- Individual DPM values will be analyzed by log transformation (base 10) of the data. The evaluation of the equality of means for the DPM, body weight and ear thickness data will be made by a one-way analysis of variance using the F distribution to assess statistical significance. If statistically significant differences between the means are found, a Dunnett's test will be used to determine the degree of significance from the control means.
- If the data indicates that the test article is positive, the EC3 will be calculated by the formula:
EC3 = c+[(3-d)/(b-d)](a-c)
where the data points lying immediately above and below the Sl value of 3 have the co-ordinates (a,b) and (c,d) respectively.

Positive control results:

The positive control, 35% (v/v) HCA, resulted in a stimulation index (Sl) of 5.97. A 3-fold or greater increase in proliferative activity relative to the concurrent vehicle treated control is considered a positive response. In addition, the response with the positive control in this study was also statistically significant (p<0.001 ) when compared to the vehicle control group.

Key result

Parameter:

other: NOEC

Value:

15

Remarks on result:

other: 15%

Key result

Parameter:

EC3

Value:

23.63

Parameter:

SI

Value:

1.51

Remarks on result:

other: 7.5%

Parameter:

SI

Value:

2.01

Remarks on result:

other: 15%

Parameter:

SI

Value:

3.73

Remarks on result:

other: 30%

Cellular proliferation data / Observations:

CLINICAL OBSERVATIONS AND MORTALITY:
There was no mortality and all animals appeared normal throughout the study. The application sites on the mice from the groups treated with the positive control appeared wet on Days 2-3. Wet appearance to the application sites was also observed in three animals treated with the test article at a concentration of 15% on Day 3. There were no other findings.

BODY WEIGHTS
Mean body weights at Day 1 and Day 6 and mean changes in body weights were evaluated. There were no statistically significant differences observed between any of the treatment groups. Therefore, the test article did not appear to cause any overt toxicity.

OTHER OBSERVATIONS
- At termination, the lymph nodes from the mice treated with the positive control were enlarged but appeared normal. The lymph nodes from the mice in the vehicle and all test article treated animals were normal in size and appearance.
- At termination both ears of each mouse were also measured. Statistically significant decrease (p < 0.01) in the ear measurements were observed in the group treated with 30% test article when compared to the vehicle group. However, this in not biologically significant.

Interpretation of results:

other: skin sensitiser Category 1B

Remarks:

in accordance with EU CLP (1272/2008 and its updates)

Conclusions:

The SI values calculated for the substance concentrations 7.5, 15 and 30% were 1.51, 2.01, and 3.73, respectively. These results show that the test substance could elicit a SI ≥ 3. The calculated EC3 was 23.63% and therefore the test substance was considered to be a skin sensitiser 1B under the conditions of the test. The NOEC is 15%.

Executive summary:

The skin sensitisation potential of the test substance has been tested using the Local Lymph Node Assay according to OECD TG 429 and GLP principles. The application of the test substance at concentrations of 7.5, 15 and 30% in a vehicle of Diethyl phthalate/ethanol in a ratio of 3:1 for three consecutive days did result in SI values of 1.51, 2.01, and 3.73, respectively. The calculated EC3 values was 23.63% and the NOEC is 15%. No mortality or toxicological relevant clinical observations and no effects on body weight were reported. Based on the results, the substance has to be classified as a skin sensitizer (category 1B).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

LLNA according to OECD429

The skin sensitisation potential of the test substance has been tested using the Local Lymph Node Assay according to OECD TG 429 and GLP principles. The application of the test substance at concentrations of 7.5, 15 and 30% in a vehicle of Diethyl phthalate/ethanol in a ratio of 3:1 for three consecutive days did result in SI values of 1.51, 2.01, and 3.73, respectively. The calculated EC3 values was 23.63% and the NOEC is 15%. No mortality or toxicological relevant clinical observations and no effects on body weight were reported. Based on the results, the substance has to be classified as a skin sensitizer (category 1B).

Human Repeated Insult Patch Test

In addition human sensitisation data is available as well. In a recent study the basic schedule was kept in the induction phase every Monday, Wednesday and Friday till 9 applications had been made in approximately 3 weeks. Test concentration is 5%. During the induction patches were placed at the left side of the back. Technicians removed the patches 24 hours after application, except for Saturdays on which the patients removed the patches themselves. After removal 24 hours without treatment were conducted, except for the 48 hours treatment free period after the Friday application. After two weeks a challenge patch was applied to the virgin right side of the back and removed after 24 hours. Reactions to the challenge were assessed after 24, 48, 72 and 96 hours. None of the 104 subjects tested was sensitized by the sample. It was therefore concluded that the test substance is not sensitizing. 

Other information

Several older studies from the RIFM database are available as well: when applied at 0.5% none of 38 subjects had any sensitization reaction (1965). In another HRIPT, when applied at 2.5%, 5/53 subjects exhibited significant irritation (1980). The 5 subjects were challenged and no responses were observed. When applied at 1% (1979) or 10%, 0/50 subjects showed any responses.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The substance is not a respiratory sensitiser because there are no human data that indicate respiratory sensitisation. In addition the potential for respiratory sensitization of the substance is assessed using the integrated evaluation strategy for respiratory sensitization data in the ECHA guidance (R7A, Fig. 7.3-2).

1)           The substance is not a strong skin sensitizer;

2)           The substance does not belong to the di-isocyanates;

3)           The substance has not structural alerts or is structurally related to chemicals causing respiratory sensitization as presented in Table R.7.3-1 in the ECHA guidance of 2008 or those provided in the following document: http://ec.europa.eu/health/scientific_committees/docs/annex6_respiratory.pdf

Using this ITS in the ECHA guidance it can be concluded that the substance is not a respiratory sensitiser.

Justification for classification or non-classification

Based on the available data, the substance needs to be classified and labelled for skin sensitisation category 1B (H317) according to EU CLP (EC No. 1272/2008 and its updates) but not for respiratory sensitisation.