1-[2-(2-chloroethoxy)phenylsulfonyl]-3-(4-methoxy-6-methyl-1,3,5-triazin-2-yl)urea;triasulfuron (ISO)

Endpoint summary

Description of key information

- Skin sensitisation: non sensitising, female, mice, OECD TG 429, Török 2012

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:

05 Sep 2012 to 11 Sep 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2010

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

GLP compliance:

yes (incl. QA statement)

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
- Sex: Females
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 11 weeks old
- Weight at study initiation: 19.1 – 23.1 g
- Housing: Group caging / mice were provided with glass tunnel-tubes
- Diet: Autoclavable complete diet for rats and mice – breeding and maintenance. Ad libitum
- Water: tap water. Ad libitum
- Acclimatisation period: 19 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 15-20
- Photoperiod: 12 hours daily, from 6.00 a.m. to 6.00 p.m.

- IN-LIFE DATES: From: 05 September 2012 To: 11 September 2012

Vehicle:

dimethyl sulphoxide

Concentration:

25, 10 and 5 % (w/v)

No. of animals per dose:

5

Details on study design:

PRE-SCREEN TEST:
A preliminary irritation/ toxicity test was performed on mice using two doses of the test substance, 25 and 10 % (w/v), respectively. The 25 % (w/v) formulation was considered to be a suitable maximum dose level for the study. The application of the material and the local effects on the animals were considered acceptable for a valid LLNA.

MAIN STUDY:
Animal Assignment and Treatment:
Groups of five female mice were treated with: 25, 10 and 5 % (w/v) of the test substance. The negative control group received DMSO and the positive control group received 25 % a-Hexylcinnamaldehyde in DMSO concurrent to the test substance groups.
The test solutions were applied on the dorsal surface of the ears (25 μL/ear) for 3 consecutive days (days 1, 2 and 3). There was no treatment on days 4, 5 and 6. On day 6, the animals were killed and cell proliferation in the local lymph nodes was measured by incorporation of tritiated methyl thymidine. The values obtained were used to calculate stimulation indices (SI).

Observations:
Clinical observations: During the study (days 1-6), all animals were observed at least once daily for any clinical signs, including local irritation and systemic toxicity.
Body weight: Individual body weights were recorded on day 1 and 6.

Terminal Procedures:
On day 6, animals were intravenously injected with 250 μL of sterile phosphate buffered saline (PBS) containing approximately 20 μCi of 3HTdR. Five hours after intravenous injection, the mice were humanely killed and the draining auricular lymph nodes were excised, and placed in separate Petri dishes containing PBS to keep the nodes wet before processing. The nodes of each animal were processed individually.

Preparation of Lymph Node Cells:
A single cell suspension (SCS) of lymph node cells (LNCs) was prepared by gentle mechanical disaggregating of the lymph nodes and collected in disposable tubes . LNCs was pelleted by centrifugation with a relative centrifugal force (RCF) of 190 x g (approximately) for 10 minutes at 4 °C. After centrifugation, supernatants were discarded. Pellets were gently resuspended and 10 mL of PBS was added to the tubes. The washing step was repeated twice. This procedure was repeated for each group of lymph nodes of each individual animal.
The suspensions were centrifuged and the supernatants were removed leaving a small volume (<0.5 mL) of supernatant above each pellet. Each pellet was gently agitated before suspending the LNCs in 3 mL of 5 % trichloroacetic acid (TCA) for precipitation of macromolecules. After incubation with 5 % TCA at 2-8 °C overnight precipitate was recovered by centrifugation at 190 x g for 10 minutes at 4 °C), and supernatants were removed and pellets were resuspended in 1 mL of 5 % TCA solution and dispersed using an ultrasonic water bath. Each precipitate was transferred to a suitable sized scintillation vial with 10 mL of scintillation liquid and thoroughly mixed. The vials were loaded into a β-scintillation counter and 3HTdR incorporation was measured for up to 10 minutes per sample.
The β-counter expressed the 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM). Background radiation levels were measured in two 1 mL aliquots of 5 % TCA.

Interpretation of Results:
The test substance is regarded as a sensitiser if both of the following criteria are fulfilled:
- That exposure to at least one concentration resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index.
- The data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

Acceptability of the Test:
The Local Lymph Node Assay is considered valid if it meets the following criteria:
- the DPN value of the negative (vehicle) control group falls within the range of historical laboratory control data,
- the positive control substance produces a significant lymphoproliferative response increases (SI > 3),
- each treated and control group includes at least 4 animals,
- the test substance does not cause serious systemic or local toxicity.

Positive control substance(s):

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

Statistics:

Disintegrations per minute (DPM) were measured for each animal of nodes (correcting for background radioactivity). The results were expressed as disintegrations per node (DPN) by dividing the DPM by the number of lymph nodes.
Stimulation index (SI =mean DPN of treated group divided by mean DPN of the appropriate control group) for each treatment group was calculated. A stimulation index of 3 or greater is the criteria for defining a positive result.

Key result

Parameter:

EC3

Remarks on result:

other: No EC3 could be established based on absence of skin sensitisation.

Parameter:

SI

Value:

0.8

Test group / Remarks:

5 %

Remarks on result:

other: (5% applied dose)

Parameter:

SI

Value:

1.9

Test group / Remarks:

10 %

Remarks on result:

other: (10% applied dose)

Parameter:

SI

Value:

1.3

Test group / Remarks:

25 %

Remarks on result:

other: (25% applied dose)

Cellular proliferation data / Observations:

No mortality, systemic toxicity or local irritation was observed during the study and there were no treatment related effects on body weight in any treated groups.

Since there were no confounding effects of irritation at any dose level and no systemic toxicity at the applied concentrations, the proliferation values obtained are considered to reflect the real potential of the test item to cause lympho-proliferation in the Local Lymph Node Assay.

Table 1: Skin sensitisation potential of the test substance.

Concentration of test substance (%w/v)	Animal Number	Disintegrations per minute (dpm)	No. of Nodes	dpm per lymph node	Group DPN	Test : control ratio SI
Negative Control (DMSO)	203	563.5	2	281.8	135.4	N/A
	241	179.5	2	89.8
	252	168.5	2	84.3
	250	198.5	2	99.3
	197	243.5	2	121.8
Test substance 25 % (w/v) in DMSO	242	98.5	2	49.3	181.0	1.3
	253	298.5	2	149.3
	229	148.5	2	74.3
	243	1017.5	2	508.8
	213	246.5	2	123.3
Test substance 10 % (w/v) in DMSO	251	256.5	2	128.3	253.9	1.9
	232	259.5	2	129.8
	228	351.5	2	175.8
	245	373.5	2	186.8
	234	1297.5	2	648.8
Test substance 5 % (w/v) in DMSO	214	119.5	2	59.8	103.8	0.8
	222	312.5	2	156.3
	249	124.5	2	62.3
	218	192.5	2	96.3
	248	288.5	2	144.3

N/A not applicable

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of the present assay, the test substance in a suitable vehicle was shown to have no skin sensitisation potential (non-sensitiser) in the local lymph node assay. No EC3 value was derived.

Executive summary:

The study was performed according to OECD TG 429 under GLP. In this study, the test substance solutions (25, 10 and 5 % test substance formulated in DMSO) were applied to 5 female CBA/J Rj mice per group on the dorsal surface of ears of experimental animals (25 μL/ear) for 3 consecutive days (days 1, 2 and 3). The negative control group received the vehicle (DMSO). There was no treatment on days 4, 5 and 6. On day 6, 5 hours prior to termination, animals were intravenously injected via the tail vein with tritiated methyl thymidine (3HTdR). Cell proliferation in the local lymph nodes was measured by incorporation of 3HTdR and the values obtained were used to calculate stimulation indices (SI).

No mortality, systemic toxicity or local irritation was observed during the study. No treatment related effects were observed on animal body weights in any treated groups. The observed SI values were 1.3, 1.9 and 0.8 at concentrations of 25, 10 and 5 % (w/v), respectively. In a positive control study, (α-hexylcinnamaldehyde) was shown to have the capacity to cause skin sensitisation when applied as 25 % w/v preparations in DMSO, confirming the validity of the protocol used for this study.

Under the conditions of the present assay, the test substance in a suitable vehicle was shown to have no skin sensitisation potential (non-sensitiser) in the local lymph node assay. No EC3 value was derived.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

All available data were assessed and the studies representing the worst-case effects were included as key studies. Other studies are included as supporting information. The in vitro data requirements were waived based on the availability of adequate in vivo data. Dosing with the test material in mice did not result in any signs of skin sensitisation. The key studies are considered to be worst-case and were selected for the CSA.

 

Skin sensitisation

A Local Lymph Node Assays in mice is available. The key study was performed according to OECD TG 429 under GLP (Török 2012). In this study, the test substance solutions (25, 10 and 5 % test substance formulated in DMSO) were applied to 5 female CBA/J Rj mice per group on the dorsal surface of ears of experimental animals (25 μL/ear) for 3 consecutive days (days 1, 2 and 3). The negative control group received the vehicle (DMSO). There was no treatment on days 4, 5 and 6. On day 6, 5 hours prior to termination, animals were intravenously injected via the tail vein with tritiated methyl thymidine (3HTdR). Cell proliferation in the local lymph nodes was measured by incorporation of 3HTdR and the values obtained were used to calculate stimulation indices (SI).

No mortality, systemic toxicity or local irritation was observed during the study. No treatment related effects were observed on animal body weights in any treated groups. The observed SI values were 1.3, 1.9 and 0.8 at concentrations of 25, 10 and 5 % (w/v), respectively. In a positive control study, (α-hexylcinnamaldehyde) was shown to have the capacity to cause skin sensitisation when applied as 25 % w/v preparations in DMSO, confirming the validity of the protocol used for this study.

Under the conditions of the present assay, the test substance in a suitable vehicle was shown to have no skin sensitisation potential (non-sensitiser) in the local lymph node assay. No EC3 value was derived.

 

The other skin sensitisation study was performed according to OECD TG 406 under GLP (Maurer 1983). Under the experimental conditions employed, no differences between the test group and the vehicle-treated controls were seen, after either intradermal or epidermal challenge appli­cation of the test substance. The test substance was found to be devoid of skin-sensitising (contact allergenic) potency in male and female albino guinea pigs.

Justification for classification or non-classification

Based on the available information the test material cannot be classified as skin sensitiser in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008.