4-(1,1-dimethylethyl)cyclohexyl acrylate

Endpoint summary

Description of key information

Sensitizing (LLNA, BASF 2016)

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08.04. - 24.05.2016

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

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Vehicle:

methyl ethyl ketone

Concentration:

0.1, 0.5, 2 and 5%

No. of animals per dose:

5 (female)

Details on study design:

Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 0.1, 0.5, 2, and 5% in MEK. The application volume, 25 µL/ear/day, was spread over the entire dorsal surface (8 mm) of each ear once daily for three consecutive days. A further group of mice (control animals) was treated with an equivalent volume of the relevant vehicle alone (control animals).
Five days after the first topical application (day 6) 250 µL of phosphate-buffered saline containing 19.8 µCi of 3H-methyl thymidine (equivalent to 79.1 µCi/mL 3HTdR) were injected into each test and control mouse via the tail vein.
Approximately five hours after treatment with 3HTdR all mice were euthanised by using CO2, which was, after harvesting of the lymph nodes, followed by cervical dislocation to ensure death.
After excision, the lymph nodes were pooled per animal and weighed immediately using an analytical balance.

Positive control substance(s):

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

Statistics:

Where appropriate, the EC3 value was calculated according to the equation
EC3 = (a-c) [(3-d)/(b-d)] + c
where EC3 is the estimated concentration of the test item required to produce a 3-fold increase in draining lymph node cell proliferative activity; (a, b) and (c, d) are respectively the co-ordinates of the two pair of data lying immediately above and below the S.I. value of 3 on the local lymph node assay dose response plot.
All calculations conducted on the DPM values, the ear weights, the lymph node weights and the lymph node cell count were performed with validated program R Script STABW-mitStat.Rnw).
Within the program a statistical analysis conducted on the DPM values, the ear weights, the lymph node weights and the lymph node cell count to assess whether the difference was statistically significant between the test item groups and negative control group. Statistical significance was set at the five per cent level (p < 0.05). Additionally, the Dean-Dixon-Test and Grubb’s Test were used for identification of possible outliers. No outliers were detected.

Key result

Parameter:

SI

Value:

1.2

Test group / Remarks:

0.1% test substance

Key result

Parameter:

SI

Value:

6.7

Test group / Remarks:

2% test substance

Key result

Parameter:

SI

Value:

23

Test group / Remarks:

5% test substance

Parameter:

EC3

Value:

1

The measured lymph node weights and –cell counts of all animals treated were recorded after sacrifice. A statistically significant and biologically relevant increase in lymph node weights and –cell counts was observed in the two highest test item groups in comparison to the vehicle control group. For BALB/c mice, a cut-off value for the lymph node cell count index of 1.55 was reported for a positive response. The indices determined for the lymph node cell count of the two highest test item groups exceeded this threshold.

The measured ear weight of all animals treated was recorded on test day 6 (after necropsy). A biologically relevant or statistically significant increase in ear weights was not observed. Furthermore, the cut-off value (1.1) of the ear weight index for a positive response regarding ear skin irritation reported for BALB/c mice was not exceeded in any of the treated groups.

Test item concentration	Group Calculation
	Mean DPM per animal (2 lymph nodes)^a)	SD	S.I.
Vehicle Control Group (MEK)	1040.1	784.8	1.0
0.1% 2-Propenoic acid, 4-(1,1-dimethylethyl)cyclohexyl ester	1250.5	617.6	1.2
0.5% 2-Propenoic acid, 4-(1,1-dimethylethyl)cyclohexyl ester	1042.1	341.0	1.0
2% 2-Propenoic acid, 4-(1,1-dimethylethyl)cyclohexyl ester	6990.5	1039.8	6.7^S
5% 2-Propenoic acid, 4-(1,1-dimethylethyl)cyclohexyl ester	24055.1	10231.8	23.1^S

^a)      Mean DPM/animal was determined by dividing the sum of the measured values from lymph nodes of all animals within a group by the number of animals in that group (5 animals)

^S      statistically significant (p < 0.05)

Calculation of the EC3 value:

	Test item concentration %	S.I.
Test Group 3	0.5 (a)	1.0 (b)
Test Group 4	2 (c)	6.7 (d)
EC3 = (a-c) [(3-d)/(b-d)] + c = 1% (w/w)

a,b,c,d = Co-ordinates of the two pairs of data lying immediately above and below the S.I. value of 3 on the LLNA dose response plot.

Interpretation of results:

Category 1A (indication of significant skin sensitising potential) based on GHS criteria

Reference 2

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

17.11.2014 - 31.03.2015

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

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Vehicle:

methyl ethyl ketone

Concentration:

5, 10 and 25%

No. of animals per dose:

5 (female)

Details on study design:

Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 5, 10, and 25% in MEK. The application volume, 25 µL/ear/day, was spread over the entire dorsal surface ( 8 mm) of each ear once daily for three consecutive days. A further group of mice (control animals) was treated with an equivalent volume of the relevant vehicle alone (control animals).
Five days after the first topical application (day 6) 250 µL of phosphate-buffered saline containing 20.2 µCi of 3H-methyl thymidine (equivalent to 80.9 µCi/mL 3HTdR) were injected into each test and control mouse via the tail vein.
Approximately five hours after treatment with 3HTdR all mice were euthanised by using CO2, which was, after harvesting of the lymph nodes, followed by cervical dislocation to ensure death.
After excision, the lymph nodes were pooled per animal and weighed immediately using an analytical balance.
Experiment 2:

Positive control substance(s):

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

Statistics:

A statistical analysis was conducted on the DPM values, the ear weights, the lymph node weights and the lymph node cell count to assess whether the difference was statistically significant between the test item groups and negative control group. For all statistical calculations custom made statistical program ´R` Decision Tree was used. Statistical significance was set at the five per cent level (p < 0.05). The Dean-Dixon-Test and the Grubb’s test were used for detection of possible outliers (performed with custom made statistical program ´R` Decision Tree).

Key result

Parameter:

SI

Value:

23.78

Test group / Remarks:

5%

Key result

Parameter:

SI

Value:

23.15

Test group / Remarks:

10 %

Key result

Parameter:

SI

Value:

24.31

Test group / Remarks:

15%

The measured lymph node weights and -cell counts of all animals treated were recorded after sacrifice. A statistically significant and biologically relevant increase in lymph node weights or –cell counts was observed in all test item treated groups in comparison to the vehicle control group. For BALB/c mice, a cut-off value for the lymph node cell count index of 1.55 was reported for a positive response (See Ref. 8). The indices determined for the lymph node cell count of all test item treated groups exceeded this threshold.

The measured ear weight of all animals treated was recorded on study day 6 (after necropsy). A statistically significant increase in ear weights was observed in the high dose group in comparison to the vehicle control group (p<0.05).Furthermore, for BALB/c mice, a cut-off value of 1.1 for the ear weight index was reported for a positive response regarding ear skin irritation (see Ref. 9). The index determined for the high dose group slightly exceeded this threshold (1.14).

Test item concentration	Group Calculation
	Mean DPM per animal (2 lymph nodes)^a)	SD	S.I.
Vehicle Control Group (MEK)	637.9	230.6	1.00
5% Laromer TBCH	15168.9	5493.3	23.78^S
10% Laromer TBCH	14769.9	2340.5	23.15^S
25% Laromer TBCH	15505.5	2918.3	24.31^S

^a)      Mean DPM/animal was determined by dividing the sum of the measured values from lymph nodes of all animals within a group by the number of animals in that group (5 animals)

^S      statistically significant[TK1] 

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Two experiments with different concentrations have been performed with the registered substance. In the first LLNA very high SI (stimulation index) values above 20 were observed at all tested concentrations (5, 10, 25%). It was impossible to calculate an EC3 value, since there was no dose response relationship. Consequently, a second study was performed to check, if very low concentrations (<0.1%) could still cause sensitization. In that case, a specific concentration limit for the registered substance would have been needed. The second LLNA confirmed the SI value at 5%, but also provided a steep dose response with an EC3 not far below 2%, justifying the use of the generic concentration limits for cat. 1A sensitizers.

In detail, the first LLNA utilized concentration of 5, 10, 25% (w/w) of the substance dissolved in MEK (BASF 2016). The highest concentration tested was the highest concentration that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation, as determined by three pre-experiments. The animals showed neither signs of systemic toxicity nor mortality during the course of the study. On day 2 and day 3, all animals treated with the test item showed an erythema of the ear skin (score 1). Additionally, the animals treated with 25% test item concentration showed an erythema of the ear skin (score 1) on day 4.A statistically significant increase in ear weights was observed in the high dose group in comparison to the vehicle control group (p<0.05).Furthermore, for BALB/c mice, a cut-off value of 1.1 for the ear weight index was reported for a positive response regarding ear skin irritation. The index determined for the high dose group slightly exceeded this threshold (index of 1.14) and thus, indicated a slight irritant property of the test item. Stimulation Indices (S.I.) of 23.78, 23.15, and 24.31 were determined with the test item at concentrations of 5, 10, and 25%, respectively. A statistically significant and biologically relevant increase in DPM value and also in lymph node weight and -cell count was observed in all dose groups in comparison to the vehicle control group. Furthermore, the cut-off value of 1.55 for a positive response regarding the lymph node cell count index reported for BALB/c mice (see Ref. 8) was exceeded in all dose groups (indices of 3.71, 3.47, and 3.71, respectively).Thus, the test item was found to be a skin sensitizer.

In the second LLNA lower concentrations of 0.1, 0.5, 2, and 5% (w/w) in MEK were used to derive a threshold for sensitization (BASF 2016). The animals did not show any signs of systemic toxicity or local skin irritation during the course of the study and no mortality was observed. A statistically significant or biologically relevant increase in ear weights was not observed in any treated group in comparison to the vehicle control group. There were no signs of skin irritation. The Stimulation Indices (S.I.) were 1.2, 1.0, 6.7, and 23.1 for 0.1, 0.5, 2, and 5%, respectively. A clear dose response was observed, and the SI value in the 5% dose group confirmed the results of the first study. A statistically significant and biologically relevant increase in DPM value and also in lymph node weight and -cell count was observed in the two highest dose groups (2 and 5%) in comparison to the vehicle control group. Furthermore, the cut-off value of 1.55 for a positive response regarding the lymph node cell count index reported for BALB/c mice was exceeded in the two highest dose groups (2 and 5%, indices of 2.87 and 4.64, respectively). The test item was found to be a skin sensitizer, and an EC3 value of 1 % was derived.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data the test item is classified and labelled as skin sensitiser cat 1A (H317) according to Regulation (EC) No 1272/2008 (CLP), as amended for the eighteenth time in Regulation (EU) 2022/692.