Ethyl trichloroacetate

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:

2012-2013

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

other: CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: TOXI-COOP ZRT. H-1103, Budapest, Cserkesz u. 90.
- Age at study initiation: Preliminary test: 11 weeks old (at start of the preliminary test)/ Main test Experiment 1: 10-11 weeks at start of Experiment 1/ Main test Experiment 2: 11 weeks old at start of Experiment 2
- Weight at study initiation: Experiment 1: 17.7-21.6 g; Experiment 2: 18.5 – 21.0 g (The weight variation in animals involved in the study did not exceed ± 20 % of the mean weight.)
- Housing: During acclimatization period: grouped caging in small groups; During the test: grouped caging (4 animals/cage); Type II Polypropylene/polycarbonate; Lignocel® Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co.KG (D-73494 Rosenberg (Germany) Holzmühle 1) (=deep wood sawdust bedding)
- Diet (e.g. ad libitum): Animals received ssniff® SM R/M-Z+H complete diet for rats and mice produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany, ad libitum
- Water (e.g. ad libitum): Tap water from municipal supply, as for human consumption, from a bottle ad libitum
- Acclimation period: Experiment 1: 14 days; Experiment 2: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

IN-LIFE DATES: From: November 14, 2012 To: February 20, 2013

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

Preliminary test: undiluted test item (100 %) and formulations of 50 % or 25 % (w/v) in the vehicle (AOO)
Main test Experiment 1: undiluted test item (100 %) and formulations of 50 %, 25 % or 10% (w/v) in the vehicle (AOO) + vehicle control + untreated control + 25% HCA positive control
Main test Experiment 2: undiluted test item (100 %) and formulations of 95% (w/v) in the vehicle (AOO) + vehicle control + 25% HCA positive control

No. of animals per dose:

Preliminary test: 2 (6 animals used in total)
Main test Experiment 1: 4 (28 animals used in total)
Main test Experiment 2: 4 (16 animals used in total)

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: The test item was adequately miscible with Acetone: Olive oil 4:1 (v/v) mixture (AOO). Since this vehicle is the most preferred one by the relevant guidelines no other vehicles were evaluated. Based on the above AOO was selected to be used as vehicle for the test item in the main test.
- Irritation: No signs of systemic toxicity or local irritation (indicated by an erythema score ≥ 3 and/or an increase of ear thickness ≥ 25 %) were observed at the treatment site (ears) at the tested concentrations.
- Lymph node proliferation response:
In Experiment 1 visually larger lymph nodes than the control was observed in the positive control group only. Visual appearance of the lymph nodes was normal in the negative control groups (both AOO and untreated) and in the test item treated groups.
Significantly increased lymphoproliferation (indicated by an SI ≥ 3) compared to the relevant control (untreated) was noted for Ethyl Trichloroacetate at concentrations of 100 % (when the undiluted test item was used). No significant lymphoproliferation compared to the relevant control (AOO) was observed at concentrations of 50 %, 25 % and 10 % (w/v). The stimulation index values were 6.9, 0.6, 0.6 and 1.5 at concentrations of 100 %, 50 %, 25 % and 10 %, respectively. Significance of the dose-response was evaluated by linear regression using the SI values. No statistically significant dose-response relationship was observed (p = 0.15, r = 0.85).
In Experiment 2 visually larger lymph nodes than the control was observed in the positive control group only. Visual appearance of the lymph nodes was normal in the negative (vehicle) control group (AOO) and in the test item treated groups.
No significantly increased lymphoproliferation (indicated by an SI ≥ 3) compared to the vehicle control was noted for Ethyl Trichloroacetate at the tested concentrations (100 % and 95 %). No dose-related response was observed.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA (Pooled Treatment Group Approach)
The animals were set in order of their body weight. The animals were randomly assigned to control and test groups using a randomization scheme. The randomization was checked by computer software according to the actual body weights verifying the homogeneity and deviations between the groups.
- Criteria used to consider a positive response:
The stimulation index (SI = the DPN of a treated (positive control or test item) group divided by the DPN of the respective negative control group) for each treatment group was also calculated. A stimulation index of 3 or greater is an indication of a positive result.
The test item is considered as a skin sensitizer, if the following criteria are fulfilled:
- That exposure to at least one concentration of the test item 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.

TREATMENT PREPARATION AND ADMINISTRATION:
Each mouse was topically treated with 25 μL of the undiluted test item, appropriate formulations of the test item, the positive control substance (positive control group) or the vehicle (AOO, as negative control group) using a pipette, on the dorsal surface of each ear in both Experiments. After the treatments animals were returned to their cages. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. In Experiment 1 animals in the untreated control group remained untreated.

No animals showed symptoms of systemic toxicity or excessive skin irritation, and no technically failed treatment was observed during the test: all animals treated were processed in both Experiments. Therefore no any treatment group was excluded from the evaluation. The same procedure (as given below) was followed for determination of lymphocyte proliferation in both Experiments.

On Day 6 each mouse was intravenously injected via the tail vein with 250 μL of sterile PBS (1 x PBS, diluted from 10x concentrate with purified water) containing 20 μCi* of 3H-methyl-thymidine using a hypodermic needle with 1 mL sterile syringe. Once injected, the mice were left for 5 hours (± 30 minutes).
* Remark: Braking down of [methyl-3H]-Thymidine in aqueous solution (about 3 % per month) was taken into account when 3HTdR solution was prepared.

Five hours (± 30 minutes) after intravenous injection the mice were sacrificed by cervical dislocation. The draining auricular lymph nodes were excised by making a small incision in the skin between the jaw and the sternum, pulling the skin gently back towards the ears and exposing the lymph nodes. Then the nodes were removed using forceps.
Once removed, the nodes of the mice from each test group were pooled and collected separately in a Petri dish containing a small amount (1-2 mL) of PBS to keep the nodes wet before processing.

A single cell suspension (SCS) of lymph node cells (LNCs), pooled according to groups, was prepared and collected in disposable tubes by gentle mechanical disaggregating of the lymph nodes through a cell strainer using the plunger of a disposable syringe. The cell strainer was washed with PBS (up to 10 mL). LNCs were pelleted with a relative centrifugal force (RCF) of approximately 190 x g for 10 minutes at 4 °C.
After centrifugation, the supernatant was removed, leaving 1-2 mL supernatant above each pellet. The pellets were gently agitated before making up to 10 mL with PBS and resuspending the LNCs. The washing procedure was repeated twice. This procedure was repeated for each group of pooled lymph nodes.

After the final wash, each supernatant was removed leaving a small volume (< 0.5 mL) of supernatant above each pellet. The pellets were gently agitated before suspending the LNCs in 3 mL of 5 % (w/v) trichloracetic acid (TCA, dissolved in purified water) for precipitation of the macromolecules. After incubation with 5 % TCA at 2-8°C overnight (approx. 18 hrs), each precipitate was removed by centrifugation of the samples at approximately 190 x g for 10 minutes at 4°C and decanting the supernatants, than the pellets were re-suspended in 1 mL of 5 % TCA and dispersed using an ultrasonic water bath. Samples were transferred to suitable sized scintillation vials containing 10 mL of scintillation liquid, gently mixed and stored at room temperature protected from light until measurement.
For the measurement of incorporated radioactivity the vials were loaded into a β-scintillation counter and 3HTdR incorporation was measured for up to 10 minutes per sample. The β-counter expresses the 3HTdR incorporation as the amount of radioactive disintegration per minute (DPM). Similarly, background 3HTdR levels were measured in two 1 mL aliquots of 5 % TCA.

Positive control substance(s):

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

Statistics:

Significance of the dose-response was evaluated by linear regression using the SI values.

Positive control results:

The positive control group animals were treated with 25 % (w/v) HCA solution (dissolved in AOO) concurrent to the test item groups in both Experiments. No mortality, cutaneous reactions or signs of toxicity were observed in the positive control group in any Experiment.
Significant lymphoproliferative response (SI ≥ 3) was noted for HCA in both Experiments: the SI values were 11.6 in Experiment 1 and 3.8 in Experiment 2. Although the increase of the lymphoproliferation induced by the positive control was less than expected, it was within the historical control range and met with the validity criteria. Hence the positive control results demonstrated appropriate performance of the test (both Experiments) in accordance with the relevant guidelines and confirmed validity of the assay.

Parameter:

SI

Remarks:

10 %

Value:

1.5

Parameter:

SI

Remarks:

25%

Value:

0.6

Parameter:

SI

Remarks:

50%

Value:

0.6

Parameter:

SI

Remarks:

100%

Value:

6.9

Parameter:

other: disintegrations per minute (DPM)

Value:

0

Test group / Remarks:

10, 25, 50 and 100 w/v

Remarks on result:

other: see Remark

Remarks:

See under 'Any other information on results incl. tables' DPM (disintegration per minute) was measured for each treatment group in both Experiments. The measured DPM values were corrected with the background DPM value. The average of the two measured DPM values of 5 % (w/v) TCA solutions was used as the background DPM value. The results were expressed as DPN (DPM divided by the number of pooled lymph nodes). The stimulation index (SI = the DPN of a treated (positive control or test item) group divided by the DPN of the respective negative control group) for each treatment group was also calculated. A stimulation index of 3 or greater is an indication of a positive result. EC3 value (dose calculated to induce a stimulation index of 3) of the test item was not calculated.

Cellular proliferation data / Observations:

Experiment 1: The stimulation index values were 6.9, 0.6, 0.6 and 1.5 at concentrations of 100 %, 50 %, 25 % and 10 %, respectively. Significance of the dose-response was evaluated by linear regression using the SI values. No statistically significant dose-response relationship was observed (p = 0.15, r = 0.85).

Experiment 2: No significantly increased lymphoproliferation (indicated by an SI ≥ 3) compared to the vehicle control was noted for Ethyl Trichloroacetate at the tested concentrations (100 % and 95 %). No dose-related response was observed.

Table 1a: DPM, DPN and Stimulation Index Values for all Groups in Experiment 1

Test Group Name	Measured DPM/Group	Group* DPM	DPM (DPM/Node)	Stimulation Index Values
Negative (vehicle) control: AOO	6313	6263.5	782.91	1.0
Positive control: 25 % HCA in AOO	72885	72835.5	9104.4	11.6
Ethyl Trichloroacetate 100 % (undiluted)	10536	10486.5	1310.8	6.9
Ethyl Trichloroacetate 50% in AOO	3774	3724.5	465.6	0.6
Ethyl Trichloroacetate 25% in AOO	3578	3528.5	441.1	0.6
Ethyl Trichloroacetate 10% in AOO	9267	9217.5	1152.2	1.5
Untreated control	1566	1516.5	189.6	1.0

 

HCA = α-Hexylcinnamaldehyde

AOO = Acetone: Olive oil 4:1 (v/v) mixture

*Group DPM = measured DPM_group- average DPM_background

Average DPM_background= 49.5

 

Table 1b: DPM, DPN and Stimulation Index Values for all Groups in Experiment 2

Test Group Name	Measured DPM/Group	Group* DPM	DPM (DPM/Node)	Stimulation Index Values
Negative (vehicle) control: AOO	9944	9902.0	1237.8	1.0
Positive control: 25 % HCA in AOO	38072	3803.0	4753.8	3.8
Ethyl Trichloroacetate 100 % (undiluted)	10509	10467.0	1308.4	1.1
Ethyl Trichloroacetate 95% in AOO	13446	13404.0	1675.5	1.4

 

HCA = α-Hexylcinnamaldehyde

AOO = Acetone: Olive oil 4:1 (v/v) mixture

*Group DPM = measured DPM_group- average DPM_background

Average DPM_background= 49.5

 

 

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of the present Local Lymph Node Assay, Ethyl Trichloroacetate tested at concentration of 100 % as the undiluted test item and at 95 %, 50 %, 25 % and 10 % (w/v) formulations in an appropriate vehicle (AOO) was shown to have no sensitization potential.

Executive summary:

The aim of this study was to evaluate the skin sensitization potential of Ethyl Trichloroacetate following dermal exposure in the Local Lymph Node Assay. Selection of test item concentrations were based on the results of a formulation evaluation and also result of a preliminary irritation/toxicity test according to the relevant guidelines.

The test item was a liquid hence applicability of the 100 % concentration (the undiluted test item) was evaluated in order to test the highest test concentration possible. Further test concentrations were achieved by formulation of the test item in AOO. Both the undiluted test item and the formulations were adequately applicable on the ears of animals.

The main test consisted of two independent Experiments. Based on results of the preliminary irritation/toxicity test Ethyl Trichloroacetate was examined in the Experiment 1 at concentrations of 100 % as the undiluted test item and as 50 %, 25 % or 10 % (w/v) formulations in AOO according to the relevant guidelines.

In Experiment 1, 28 female CBA/Ca mice were allocated to 7 groups of four animals each:

- four groups received Ethyl Trichloroacetate at four different concentrations of 100 % (the undiluted test item), 50 %, 25 % and 10 % (w/v),

- the positive control group received α-Hexylcinnamaldehyde (HCA) at concentration of 25 % (w/v),

- the group used as negative control for the groups treated with the test item formulations or positive control substance received the vehicle (AOO) only,

- the group used as negative control for the 100 % test item treated group remained untreated.

The result obtained from Experiment 1 was inconclusive hence a confirmatory experiment (Experiment 2) was performed to clarify the results.

In Experiment 2, 16 female CBA/Ca mice were allocated to 4 groups of four animals each:

- two groups received Ethyl Trichloroacetate at two different concentrations of 100 % (the undiluted test item) and 95 % (w/v),

- the positive control group received α-Hexylcinnamaldehyde (HCA) at concentration of 25 % (w/v),

- the group used as negative control for the groups treated with the test item or positive control substance received the vehicle (AOO) only.

In both Experiments each substance was applied on the external surface of each ear (25 μL/ear) of the animals for three consecutive days (Day 1, 2 and 3). There was no treatment on Days 4, 5 and 6. On Day 6 animals were intravenously injected via the tail vein with tritiated methyl thymidine (3HTdR), than sacrificed approximately 5 hours after the injection. Auricular lymph nodes were removed and processed. The cell proliferation in the local lymph nodes was measured by incorporation of 3HTdR and the obtained values were used to calculate stimulation indices (SI).

The positive control item (25 % HCA in AOO) induced the appropriate stimulation over the control in both Experiments (SI = 11.6 in Experiment 1 and SI = 3.8 in Experiment 2), thus confirming the validity of the assay.

No mortality was observed during the study. No significant, treatment related effect on body weights or any other signs of systemic toxicity were observed in any treatment group during the assay (in any Experiment). No visible signs of irritation or any other local effect were observed at the treatment site (ears) in any treatment group in any Experiment.

In Experiment 1 visually larger lymph nodes than the control was observed in the positive control group only. Visual appearance of the lymph nodes was normal in the negative control groups (both AOO and untreated) and in the test item treated groups. In spite of normal visual appearance of the lymph nodes, significantly increased lymphoproliferation (indicated by an SI ≥ 3) compared to the relevant control (untreated) was noted for Ethyl Trichloroacetate at 100 % concentration (when the undiluted test item was used). No significantly increased lymphoproliferation (indicated by an SI ≥ 3) compared to the relevant control (AOO) was noted for Ethyl Trichloroacetate at the other test concentrations.

The stimulation index values were 6.9, 0.6, 0.6 and 1.5 at test item concentrations of 100 %, 50 %, 25 % and 10 %, respectively. Significance of the dose-response was evaluated by linear regression using the SI values. No statistically significant dose-response relationship was observed (p = 0.15, r = 0.85).

Based on the results it was considered that the positive result observed for the test item at concentration of 100 % should be confirmed, hence Experiment 2 was performed.

Results of Experiment 2 did not confirm the positive result observed in Experiment 1 for the test item at concentration of 100 %. In Experiment 2 visually larger lymph nodes than the control was observed in the positive control group only. Visual appearance of the lymph nodes was normal in the negative (vehicle) control group (AOO) and in the test item treated groups. No significantly increased lymphoproliferation (indicated by an SI ≥ 3) compared to the vehicle control was noted for Ethyl Trichloroacetate at the tested concentrations (100 % and 95 %). No dose-related response was observed.

Since the test was valid and no sign of systemic toxicity or irritation was observed, the proliferation values obtained are considered to reflect the real potential of the test item to cause lymphoproliferation in the Local Lymph Node Assay.

Although significantly increased lymphoproliferation was observed at concentration of 100 % (for the undiluted test item) in Experiment 1 it was not confirmed by the Experiment 2. Based on overall results of the Experiments performed it was concluded that the SI = 6.9 observed for the test item at concentration of 100 % could have been a consequence of the relatively low proliferation value observed for the untreated control (compared to the relevant historical control data). Along with the observation, that no significant dose-related response was observed it was considered as evidence that the test item has no sensitization potential. Based on summarized results of the Experiments performed, according to evaluation criteria of the relevant guidelines it was considered that Ethyl Trichloroacetate is not a sensitizer.

In conclusion, under the conditions of the present Local Lymph Node Assay, Ethyl Trichloroacetate tested at concentration of 100 % as the undiluted test item and at 95 %, 50 %, 25 % and 10 % (w/v) formulations in an appropriate vehicle (AOO) was shown to have no sensitization potential.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

A key study for skin sensitisation potential of Ethyl Trichloroacetate following dermal exposure in the Local Lymph Node Assay was performed with liquid test item at different concentrations in acid oliv oil (Pénzes, 2013). The main test consisted of two independent experiments. In Experiment 1, 28 female CBA/Ca mice were allocated to 7 groups of 4 animals each, receiving concentrations of 100, 50, 25 and 10 % (w/v),positive control α-Hexylcinnamaldehyde at 25 % (w/v), vehicle and negative control (untreated). The results from Experiment 1 were inconclusive hence a confirmatory experiment (Experiment 2) was performed to clarify the results. In Experiment 2, 16 female CBA/Ca mice were allocated to 4 groups of 4 animals each, receiving concentrations of 100 and 95% test item, positive control α-Hexylcinnamaldehyde (HCA) at concentration of 25 % (w/v) and negative control with vehicle only.

In both Experiments each substance was applied on the external surface of each ear (25 μL/ear) of the animals for three consecutive days (Day 1, 2 and 3), no treatment on Days 4, 5 and 6. On Day 6 animals were intravenously injected via the tail vein with tritiated methyl thymidine (3HTdR), then sacrificed approximately 5 hours after the injection. Auricular lymph nodes were removed and processed. The cell proliferation in the local lymph nodes was measured by incorporation of 3HTdR and the obtained values were used to calculate stimulation indices (SI). The positive control item (25 % HCA in AOO) induced the appropriate stimulation over the control in both Experiments (SI = 11.6 in Experiment 1 and SI = 3.8 in Experiment 2), thus confirming the validity of the assay.

No mortality, no significant effect on body weights or any other signs of systemic toxicity were observed in any treatment group during the assay; no visible signs of irritation or any other local effect were observed at the treatment site (ears) in any treatment group in any Experiment. In Experiment 1 stimulation index values were 6.9, 0.6, 0.6 and 1.5 at test item concentrations of 100 %, 50 %, 25 % and 10 %, respectively. Significance of the dose-response was evaluated by linear regression using the SI values. No statistically significant dose-response relationship was observed (p = 0.15, r = 0.85). Based on the results it was considered that the positive result observed for the test item at concentration of 100 % should be confirmed, hence Experiment 2 was performed. Results of Experiment 2 did not confirm the positive result observed in Experiment 1 for the test item at concentration of 100 %. In Experiment 2 visually larger lymph nodes than the control was observed in the positive control group only. Visual appearance of the lymph nodes was normal in the negative (vehicle) control group (AOO) and in the test item treated groups. No significantly increased lymphoproliferation (indicated by an SI ≥ 3) compared to the vehicle control was noted for Ethyl Trichloroacetate at the tested concentrations (100 % and 95 %). No dose-related response was observed.

Since the test was valid and no sign of systemic toxicity or irritation was observed, the proliferation values obtained are considered to reflect the real potential of the test item to cause lymphoproliferation in the Local Lymph Node Assay.

In conclusion, under the conditions of the present Local Lymph Node Assay, Ethyl Trichloroacetate tested at concentration of 100 % as the undiluted test item and at 95 %, 50 %, 25 % and 10 % (w/v) formulations in an appropriate vehicle (AOO) was shown to have no sensitisation potential.

Migrated from Short description of key information:
Under the conditions of the present Local Lymph Node Assay, Ethyl Trichloroacetate tested up to a 100% concentration in an appropriate vehicle (acid olive oil) was shown to have no sensitisation potential.

Justification for selection of skin sensitisation endpoint:
Key study

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the negative results of the LLNA study, classification for skin sensitisation is not warranted according to EC Directive (No.93/21/EEC) and CLP (No. 1272/2008 of 16 December 2008).