[No public or meaningful name is available]

Acute Toxicity: dermal

Administrative data

Endpoint:

acute toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the study does not need to be conducted because the physicochemical and toxicological properties suggest no potential for a significant rate of absorption through the skin

the study does not need to be conducted because the substance does not meet the criteria for classification as acute toxicity or STOT SE by the oral route and no systemic effects have been observed in in vivo studies with dermal exposure (e.g. skin irritation, skin sensitisation)

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

February, from the 04th to the 20th, 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Version / remarks:

17th December 2001

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Species and strain: Crl:(WI)BR rats.
- Source: TOXI COOP ZRT. Budapest, Hungary.
- Females: nulliparous and non pregnant animals.
- Age at study initiation: young adult rat, 9 weeks old in first and second step.
- Weight at study initiation: 180 - 186 g first step; 180 - 184 g second step.
- Fasting period before study: the day before treatment the animals were fasted. The food but not water was withheld overnight.
- Housing: 5/II (E building). 3 animals/cage; cages type II polypropylene/polycarbonate.
- Diet: animals received ssniff® SM R/M-Z+H complete diet for rats and mic, ad libitum.
- Water: tap water from municipal supply, as for human consumption from bottle, ad libitum.
- Acclimation period: 12 days in first step and 13 days in second step.
- Animal health: only healthy animals were used for the study. Health status was certified by the study director.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C.
- Humidity: 30 - 70 %
- Air changes: 10-15 air exchanges/hour by central air-condition system.
- Photoperiod: artificial light, from 6 a.m. to 6 p.m.

Route of administration:

oral: gavage

Vehicle:

other: Helianthi annui ol. raffinat.

Details on oral exposure:

FORMULATION
- Treatment volume: 10 ml/kg bw.
- Concentration: 200 mg/ml.
- Preparation: formulations were prepared just before the administration and stirred continuously during the treatment.

DOSE SELECTION
Starting dose was selected on the basis of the available information about the test item.
The acute toxic class method was carried out involving a stepwise procedure with the use of 2000 mg/kg bw as the starting dose in three female rats. No animal died in the first step at 2000 mg/kg bw dose level, so treatment with 2000 mg/kg bw was repeated on further three female rats.

Doses:

2000 mg/kg bw

No. of animals per sex per dose:

3 animals/group

Details on study design:

- Duration of observation period following administration: fourteen-day observation period.
- Frequency of observations for mortality: animals were observed individually after dosing at least once during the first 30 minutes, then 1 h, 2 h, 3 h, 4 h after the treatment and twice each day for 14 days thereafter.
- Frequency of observations for mortality: animals were weighed before the application and the food was given back 3 hours after the treatment. The body weights were recorded on day 0 (just before the treatment), on day 7 and on day 15 with a precision of 1 g.
- General state, external appearance, behavior and clinical symptoms: animals were observed individually after dosing at least once during the first 30 minutes,
then 1 h, 2 h, 3 h, 4 h, after the treatment and once each day for 14 days thereafter. Individual observations were performed on the skin and fur, eyes and mucous membranes and also respiratory, circulatory, autonomic and central nervous system, somatomotor activity and behaviour pattern. Particular attention was directed to observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.
- Necropsy of survivors performed: necropsy on Day 15. At the end of the observation period rats were sacrificed under isofluran anaesthesia. After examination of the external appearance, the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed, and any abnormality was recorded with details of its location, colour, shape and size.

Sex:

female

Dose descriptor:

LD50

Effect level:

> 2 000 mL/kg bw

Based on:

test mat.

Mortality:

No death occurred at 2000 mg/kg bw single oral dose. All female rats in step 1 and step 2 survived until the end of the 14-day observation period.

Clinical signs:

other: In group 1 treated with 2000 mg/kg bw dose clinical sign of reaction comprised of brownish-red coloured faeces (6 cases of 57 observations). This symptom (score +4; +3) was detected in all animals on Day 1 and on Day 2. In group 2 treated with 2000 mg/kg

Gross pathology:

All animals survived until the scheduled necropsy on Day 15.
Slight hydrometra was observed in female No.: 8 of the group 1 and in female No.: 11 of the group 2. It is physiological finding and connected to the cycle of the animal.
No pathological changes were found related to the effect of the test item during the macroscopic examination of animals.

Interpretation of results:

other: not classified, according to the CLP Regulation (EC) No 1272/2008

Conclusions:

LD50 (female, rat) > 2000 mg/kg bw

Executive summary:

An acute toxic class method was carried out according to the OECD guideline 423. The experiment involved a stepwise procedure with the use of 2000 mg/kg bw as the starting dose in three female rats. No animal died in the first step at 2000 mg/kg bw dose level, therefore treatment with 2000 mg/kg bw was repeated on further three female rats. No animal died in the second step, too, so the test was finished. Animals were weighed, observed for lethality and toxic symptoms for 14 days after the treatment. Gross pathological examination was carried out 15th day after the treatment.

Lethality, Clinical symptoms and Body weight:

No death occurred during the study. All rats dosed at 2000 mg/kg bw test item survived until the end of the 14-day observation period. In the first step, brownish-red coloured faeces was observed in animals between Day 1 and Day 2. In the second step, brownish-red coloured faeces was observed in animals between Day 1 and Day 2. The observed clinical sign as brownish-red coloured faeces was not related to the systemic toxic effect of the test item, but this alteration was connected with the physical property of the test item. The body weight development was undisturbed in all animals.

Slight hydrometra was observed in one female of the group 1 and in one female of the group 2; this physiological finding is connected to the cycle of the animal. No pathological changes were found related to the effect of the test item during the macroscopic examination of animals.

Conclusion

LD50 (female, rat) > 2000 mg/kg bw

Cross-reference 2

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

February from the 12th to the 20th, 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted 22th July 2010

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: TOXI-COOP ZRT, Budapest.
- Females: female, nulliparous, non-pregnant.
- Age at study initiation: young adult mice. 10-11 weeks old at start of the preliminary test; 10-12 weeks old at start of the main test.
- Weight at study initiation: 18.2-22.5 g. The weight variation in animals involved in the study did not exceed ± 20 % of the mean weight.
- Housing: 4 animals/cage. Cage type II, polypropylene / polycarbonate. Mice were group-housed to allow social interaction, and with deep wood sawdust bedding, to allow digging and other normal rodent activities.
- Diet: animals received ssniff® SM R/M-Z+H complete diet for rats and mice, ad libitum.
- Water: tap water from municipal supply, as for human consumption, from a bottle, ad libitum.
- Acclimation period: 7 days.
- Animal health: only healthy animals were used.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30 – 70 %
- Photoperiod: 12 hours daily, from 6.00 a.m. to 6.00 p.m.

Vehicle:

dimethylformamide

Concentration:

10 %, 5 %, 2.5 % and 1 % (w/v) as formulations in N,N-Dimethylformamide (DMF)

No. of animals per dose:

28 animals, 4 animals/treatment group

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: the test item was adequately soluble in DMF with a maximum concentration of 10 % (w/v). The other vehicles (i.e. Acetone: Olive oil 4:1 (v/v) mixture and purified water) were not suitable at the same concentration (10 %, w/v) and above. Based on this DMF was used for the test item formulations in the main test.
- N. of animals per dose: 6 animals (2 animals/treatment groups).
- Concentration: the test item was formulated in the selected vehicle (DMF) and tested at concentrations of 10 %, 5 % and 2.5 % (w/v).
- Frequence of application: once daily for 3 consecutive days.
- Irritation: no signs of significant irritation (indicated by an increased ear thickness of ≥ 25 % on any day of measurement) were observed in the treatment groups. Although erythema could not be reliably observed due to intensive orange colour of the test item an erythema score < 3 is considered.
- Systemic toxicity: no mortality or any signs of systemic toxicity were observed during the preliminary test. No significant, treatment related effect on body weights was observed.
- Erythema scores:
No erythema 0
Very slight erythema (barely perceptible) 1
Well-defined erythema 2
Moderate to severe erythema 3
Severe erythema (beet redness) to eschar formation preventing grading of erythema 4

MAIN STUDY

TEST PROCEDURES
Treatment
Each mouse was topically treated with 25 μl of the appropriate formulations of the test item, the positive control substance (positive control group) or the vehicles (DMF or AOO as negative control groups) using a pipette, on the dorsal surface of each ear. 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.

Proliferation Assay
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. Therefore no any treatment group was excluded from the evaluation.

Injection of 3HTdR
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 mlsterile syringe. Once injected, the mice were left for 5 hours (± 30 minutes).

Removal and Preparation of Draining Auricular Lymph Nodes
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.

Preparation of Single Cell Suspension of Lymph Node Cells
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 re-suspending the LNCs. The washing procedure was repeated twice. This procedure was repeated for each group of pooled lymph nodes.

Determination of Incorporated 3HTdR
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) trichloroacetic 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.

OBSERVATIONS
Clinical Observations
During the main test (from Day 1 to Day 6) all animals were observed at least once a day for any clinical signs, including systemic toxicity and local irritation. Irritation was monitored by erythema scoring during the whole test. Individual records were maintained.

Measurement of Body Weight
Individual body weights were recorded on Day 1 (beginning of the test) and on Day 6 (prior to 3HTdR injection) with a precision of ± 0.1 g.

Evaluation of the Results
DPM (disintegration per minute) was measured for each treatment group. 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. Based on the results EC3 value (dose calculated to induce a stimulation index of 3) of the test item was not calculated. Dose-response relationship was evaluated by linear regression. All calculations were made by Microsoft Excel Software.

Interpretation of the Results
The test item is considered as a skin sensitizer, if: 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 (SI ≥ 3). However, the strength of the dose-response, the statistical significance and the consistency of the solvent/vehicle and positive control responses may also be used when determining whether a borderline result is declared positive.

ASSAY ACCEPTANCE CRITERIA
- Lymph nodes from all 4 animals in each dose groups were pooled for a valid experiment.
- Skin sensitizing effect was observed at the applied concentration of the positive control (the stimulation index was greater than 3).

Positive control substance(s):

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

Positive control results:

No mortality, cutaneous reactions or signs of toxicity were observed in the positive control group.
Significant lymphoproliferative response (SI ≥ 3) was noted for HCA (SI = 7.0). The results of the positive control item demonstrated appropriate performance of the test in accordance with the relevant guidelines and confirmed validity of the assay.

Parameter:

SI

Value:

1.1

Test group / Remarks:

at the concentration of 10 %

Parameter:

SI

Value:

0.6

Test group / Remarks:

at the concentration of 5 %

Parameter:

SI

Value:

0.5

Test group / Remarks:

at the concentration of 2.5 %

Parameter:

SI

Value:

0.9

Test group / Remarks:

at the concentration of 1 %

Cellular proliferation data / Observations:

Body Weight Measurement: no significant, treatment related effect on body weights was observed during the test.

Clinical Observations and Signs of Irritation: 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 test. No signs of significant irritation or any other significant local effect were observed at the treatment site (ears) in any treatment group. Although erythema could not be reliably observed due to intensive orange colour of the test item an erythema score < 3 is considered in all dose groups.

Proliferation Assay: visually larger lymph nodes than the vehicle control observed in the positive control group only. Visual appearance of the lymph nodes was normal in the negative control groups (both DMF and AOO) and in the test item treated groups.
No significantly increased lymphoproliferation (indicated by an SI ≥ 3) compared to the relevant control (DMF) was noted for test item at the tested concentrations. The stimulation index values were 1.1, 0.6, 0.5 and 0.9 at concentrations of 10 %, 5 %, 2.5 % and 1 %, respectively. No dose-related response was observed (r = 0.56, p = 0.44, evaluated by linear regression using SI values).

ASSAY ACCEPTANCE CRITERIA
The assay acceptance criteria was fulfilled, hence the test was valid.

DPM, DPN and Stimulation Index Values for all Groups in the Main Test

Test Group	Measured DPM/group	Group* DPM	DPN (DPM/node)	Stimulation Index
Vehicle control for the positive control: AOO	8621	8599.5	1074.9	1.0
Positive control: 25 % HCA in AOO	60045	60023.5	7502.9	7.0
Test item 10 % in DMF	6112	6090.5	761.3	1.1
Test item 5 % in DMF	3621	3599.5	449.9	0.6
Test item 2.5 % in DMF	2714	2692.5	336.6	0.5
Test item 1 % in DMF	5171	5149.5	643.7	0.9
Vehicle control for the test item: DMF	5720	5698.5	712.3	1.0

HCA = α-Hexylcinnamaldehyde; AOO = Acetone: Olive oil 4:1 (v/v) mixture; DMF= N,N-Dimethylformamide; *Group DPM = measured DPMgroup- average DPMbackground Average DPMbackground = 21.5

Interpretation of results:

other: not classified, according to the CLP Regulation (EC) No 1272/2008

Conclusions:

not sensitising

Executive summary:

The maximum achievable test item concentration was 10 % (w/v) in N,N-Dimethylformamide (DMF). Based on the preliminary test results the test item was examined in the LLNA as 10 %, 5%, 2.5 % or 1 % (w/v) formulations in DMF according to the OECD guidelines 429. In the main test 28 female CBA/Ca mice were allocated. No mortality, no significant treatment related effect on body weights, any other signs of systemic toxicity or any sign of significant irritation or any other local effect were observed in any treatment group during the test. Visually larger lymph nodes than the control were observed in the positive control group only. Visual appearance of the lymph nodes was normal in the test item treated groups and in the negative control groups (both DMF and AOO). No significantly increased lymphoproliferation (indicated by an SI > 3) compared to the vehicle control was noted for test item at the tested concentrations. The stimulation index values were 1.1, 0.6, 0.5 and 0.9 at concentrations of 10 %, 5 %, 2.5 % and 1 % (w/v), respectively. No dose-related response was observed (p = 0.44, r = 0.56).

The positive control item (25 % HCA in AOO) induced the appropriate (SI > 3) stimulation over the control (SI value was 7.0), thus confirming the validity of the assay.

Conclusion

According to evaluation criteria of the relevant guidelines the lack of a significantly increased lymphoproliferation (indicated by an SI ≥ 3) up to the maximum achievable concentration (based on solubility) of 10 % (w/v) and the lack of a dose-related response are considered evidence that test item has no skin sensitization potential.