3,5-bis(2,4-dimethylcyclohex-3-en-1-yl)polyheterocycle

Endpoint summary

Description of key information

Skin Irritation

REACH_not irritating | Human skin model | OECD Draft Guideline | #key study#

Eye Irritation

REACH_not irritating | OECD 492 EpiOcular | #key study#

REACH_not irritating | HET-CAM | #key study#

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study according to: OECD Guideline for the Testing of Chemicals, Draft Proposal for a New Guideline, In Vitro Skin Irritation: Reconstructed Human Epidermis (RhE) Test Method, 11 December 2009, Vers. 4

Qualifier:

according to guideline

Guideline:

other: OECD Guideline for the Testing of Chemicals, Draft Proposal for a New Guideline, In Vitro Skin Irritation: Reconstructed Human Epidermis (RhE) Test Method, 11 December 2009, Vers. 4

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Remarks:

EpiSkin

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

skin obtained from plastic surgery from multiple donors

Source strain:

not specified

Vehicle:

unchanged (no vehicle)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

10 µL

Duration of treatment / exposure:

15 min

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

3

Irritation / corrosion parameter:

other: Mean rel. MTT absorbance

Run / experiment:

15 min treatment

Value:

104.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100.0

Positive controls validity:

valid

Remarks:

9.5

Other effects / acceptance of results:

After treatment with the test item Sa 57 the mean relative absorbance value did not decrease (104.3%) compared to the negative control. Therefore, the test item is not considered to possess an irritant potential.

Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour.

Table 1: Results after treatment with Sa 57

 

Dose group	Treat-ment Interval	Absorbance 570 nm Tissue 1*	Absorbance 570 nm Tissue 2*	Absorbance 570 nm Tissue 3*	Mean Absorbance of 3 Tissues	Standard Deviation of 3 Tissues	Rel. Absorbance [% of Negative Control]**	Standard Deviation [%]
Negative Control	15 min	0.857	0.936	0.896	0.896	0.039	100.0	4.4
Positive Control	15 min	0.061	0.087	0.107	0.085	0.023	9.5	2.6
Sa 57	15 min	0.988	0.902	0.916	0.935	0.046	104.3	5.1

 *       Mean of two replicate wells after blank correction
**      relative absorbance [rounded values]:

After treatment with the test item Sa 57 the mean relative absorbance value did not decreas (104.3%) compared to the negative control. This value is well above the threshold for irritancy of ≤50%. Therefore, the test item is not considered to possess an irritant potential.

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item Sa 57 is not irritant to skin.

Executive summary:

This in vitro study was performed to assess the irritation potential of Sa 57 by means of the Human Skin Model Test.

Three tissues of the human skin modelEpiSkin™ were treated with the test item, the negative or the positive control for 15 minutes.

Approximately 10 µL of the neat test item were applied to each tissue and spread to match the tissue size.

10 µL of either the negative control (deionised water) or the positive control (5% Sodium lauryl sulfate) were applied to each tissue.

After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD 0.6 and ≤ 1.5 for the15 minutes treatment interval thus showing the quality of the tissues and the specific batch of the tissue model.

Treatment with the positive control induced a decrease in the relative absorbance as compared to the negative control to 9.5% thus ensuring the validity of the test system.

The standard deviations between the % variabilities of the test item, the positive and negative controls were below 18% thus ensuring the validity of the study.

The control values are well in the range of our historical data.

After treatment with the test item Sa 57 the mean relative absorbance value did not decrease (104.3%) compared to the negative control. Therefore, the test item is not considered to possess an irritant potential.

According to the current prediction model no classification and labeling of Sa 57 is necessary with respect to skin irritation under EU and UN classification.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study according to: INVITTOX Protocol No. 47: HET-CAM Test.

Qualifier:

according to guideline

Guideline:

other: INVITTOX Protocol No. 47: HET-CAM Test. http://ecvam-sis.jrc.it

GLP compliance:

yes (incl. QA statement)

Species:

other: HET-CAM

Strain:

not specified

Vehicle:

unchanged (no vehicle)

Controls:

yes

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

Approx. 100 mg of the neat test item were applied onto the CAM

Duration of treatment / exposure:

5 min

Details on study design:

Approx. 100 mg of the neat test item were applied onto the CAM in order to cover at least 50% of the CAM. The CAM was exposed to the test item for 5 minutes. During this 5 minutes reactions on the blood vessels in and under the CAM in close proximity to the test item were monitored. The CAMs of three eggs were treated with the test item. In addition to the test item a negative control (0.9% NaCl w/v)), two positive controls (1% SDS solution in deionised water and 0.1 N NaOH), and a reference item (5% (w/v) Texapon) were tested.

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean (5 min. exposure)

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Table 1: Results with Sa 57, as well as negative and positive controls, and the reference item

Test Group	Time until Haemorrhage [s]	Time until Lysis [s]	Time until Coagulation [s]	Irritancy Index	Mean Irritancy Index
Negative Control	301	301	301	0.00	0.00
	301	301	301	0.00
	301	301	301	0.00
Positive Control 0.1 N NaOH	10	45	17	19.34	19.26
	9	41	16	19.48
	11	57	20	18.96
Positive Control 1% SDS	13	30	301	11.12	11.11
	14	28	301	11.15
	14	32	301	11.06
Reference Item 5% AS Texpon	17	35	301	10.94	10.78
	22	47	301	10.58
	20	38	301	10.82
Test Item	301	301	301	0.00	0.00
	301	301	301	0.00
	301	301	301	0.00

Mean irritancy index of 3 eggs: Test Item = 0.00

Mean irritancy index of 3 eggs: Positive Control (Sodium dodecyl sulphate) = 11.11

                                                        Positive Control (Sodium hydroxide solution) = 19.26

                                                        Reference Item (Texapon) = 10.78

Interpretation of results:

GHS criteria not met

Remarks:

other: INVITTOX Protocol No. 47: HET-CAM Test. http://ecvam-sis.jrc.it

Conclusions:

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item Sa 57 does not possess any irritating potential.

Executive summary:

This in vitro study was performed to assess the irritating potential of Sa 57 by detection of damages in blood vessels under the chorioallantoic membrane of 9 day incubated chicken eggs.

Each 300 µL of the test item, the positive and the negative controls, and the reference item were applied to three eggs each such that at least 50 % of the membrane was covered. During the observation period for 300 seconds any lesions in close proximity to the covered membrane were monitored and recorded. The observation time was 5 minutes at room temperature.

Physiological sodium chloride solution (0.9 % (w/v) in deionised water) was used as negative control.

The negative control showed no irritating effect on the blood vessels under the membrane. The irritancy mean index is 0.00.

A 1 % solution of SDS and 0.1 NaOH were used as positive controls.

The positive controls induced a severe irritation on the blood vessels. The calculated mean irritancy indices are 11.11 for 1 % SDS and 19.26 for 0.1 N NaOH. The reference itemTexapon ASV Spezial (5 % AS) was chosen because of its known eye irritating properties and induced a severe irritation on the blood vessels. The calculated mean irritancy index is 10.78.

The mean irritancy indices of the controls are well comparable with the historical control values and are within the acceptance criteria. The result of the reference item demonstrated a correct interpretation. Therefore, it was concluded that the test was valid.

No irritating effects were observed during 5 min incubation with Sa 57. The calculated irritancy mean index is 0.00.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-12-20 to 2018-01-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guidline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline for the Testing of Chemicals No. 492: (OECD Guideline for the Testing of Chemicals No. 492: Reconstructed human Cornea-like Epithelium (RhCE) test method for identifying chemicals not requiring classification for eye irritation.

Version / remarks:

28 July 2018

Qualifier:

according to guideline

Guideline:

other: EURL ECVAM DB-ALM Method Summary No. 164: EpiOcular™ Eye Irritation Test - Summary

Version / remarks:

22 July 2018

Qualifier:

according to guideline

Guideline:

other: EpiOcular™ Eye Irritation Test (OCL-200-EIT) For the prediction of acute ocular irritation of chemicals For use with MatTek Corporation’s Reconstructed Human EpiOcular™ Model, r irritation of chemicals with MatTek

Version / remarks:

29 June 2015

GLP compliance:

yes (incl. QA statement)

Remarks:

(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Germany)

Species:

other: Reconstructed human cornea-like epithelium (RhCE) EpiOcular

Details on test animals or tissues and environmental conditions:

The test was carried out with the EpiOcular™ reconstructed human cornea-line epithelium (RhCE) model (MatTek). The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified, highly differentiated squamous epithelium morphologically similar to that found in a human cornea. The EpiOcular™ RhCE tissue construct consists of at least 3 viable layers of cells and a non-keratinised surface, showing a cornea-like structure analogous to that found in vivo.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

1. Negative Control 50 μL Aqua dest.
2. Positive Control 50 μL methyl acetate
3. Test Item 50 μL (undiluted)

Duration of treatment / exposure:

incubation: 30 +/-2 min.

Duration of post- treatment incubation (in vitro):

post soak: 12 +/- 2 min.
post treatment: 120 +/- 15 min.

Number of animals or in vitro replicates:

The test was performed on a total of 2 tissues per dose group.

Details on study design:

The test was performed on EpiOcular, a reconstituted three-dimensional human corneal epithelium model. Hereby, the test item was applied topically. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a 30 min exposure period and 120 min post-treatment period and compared to those of the concurrent negative controls.

Irritation parameter:

other: Mean relative tissue viability (% negative control) %

Run / experiment:

1

Value:

104.9

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

Value Cut off pass/fail
Mean Absolute OD570 nm NK 1.814 0.8 < NK < 2.5 pass
Mean Relative Viability PC [%] 20.5 < 50% pass
Max. Difference of % Viability [%] 1.4 < 20% pass

The mixture of 50 µl test item per 1 mL MTT medium showed reduction of MTT as compared to the solvent. The mixture turned red. Sincethe mean relative tissue viability of the test item treated tissues (TM) was above the 60% threshold value killed tissue controls were performed for quantitative correction of results.

NSMTT [%] = [(OD_KT- OD_KU)/OD_NK] * 100 = 0.23%

Difference of NSMTT of the two duplicate tissues must be < 20%, otherwise not accepted.

NSMTT1 [%] = [meanODKT1 - ODKU)/ODNK] * 100 = 0.25%

NSMTT2 [%] = [meanODKT2 - ODKU)/ODNK] * 100 = 0.20%

NSMTT1 - NSMTT2 = ± 0.05%

NSMTT was ≤ 60% (0.23%) relative to the negative control of living epidermis and could therefore be used for determination of the killed control corrected viability (KCCV) according to the following formula:

KCCV [%] = viability_TM- NSMTT = 104.9%

The mixture of 50 µL test item per 1 mL A. dest. and per 2 mL isopropanol showed no colouring as compared to the solvent.Therefore, NSC_living equalled 0%.

Result of the Test Item Sa 57

Name	Negative Control		Positive Control		Test item
Tissue	1	2	1	2	1	2
OD570values	1.798	1.775	0.400	0.405	1.914	1.917
	1.847	1.837	0.401	0.424	1.898	1.893
OD570values (blank-corrected)	1.754	1.731	0.356	0.361	1.870	1.873
	1.803	1.794	0.357	0.380	1.854	1.849
mean of the duplicates	1.778	1.762	0.356	0.371	1.862	1.861
mean OD	1.770*		0.364		1.861
TODTT	-		-		1.857
mean sd OD	0.034		0.011		0.012
tissue viability [%]	100.5	99.5	20.1	20.9	105.2	105.1
relative tissue viability difference [%]^***	0.9		0.8		0.1
mean tissue viability [%]	100.0		20.5**		105.1
mean tissue viability [%] - NSMTT corrected	-		-		104.9

^*              Corrected mean OD₅₇₀of the negative control corresponds to 100% absolute tissue viability

^**             Mean relative tissue viability of the positive control is < 50%

^***            Relative tissue viability difference of replicate tissues is < 20%

Result of the NSMTT control

NSMTT	KU		KT		Negative Control
Tissue	1	2	1	2	1	2
absolute OD570 -values	0.076	0.076	0.082	0.081	2.098	2.048
	0.076	0.078	0.081	0.081	2.111	2.021
OD570(Blank Corrected)	0.033	0.032	0.038	0.037	2.054	2.004
	0.033	0.034	0.037	0.037	2.067	1.977
mean OD570 (mean of 2 aliquots)	0.033	0.033	0.038	0.037	2.061	1.990
total mean OD570 (mean of the replicate tissues)	0.033		0.037		2.026
SD OD570(of the replicate tissues)	0.000		0.000		0.050
NSMTT [%]	0.23				-
Relative Tissue Viability [%]	-				101.7	98.3
Mean Relative Tissue Viability [%]	-				100.0
SD Tissue Viability [%]	-				2.5
CV [% Viabilities]	-				2.5

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions the test item showed no irritant effects. The test item is classified as “non-irritant“ in accordance with UN GHS “No Category”

Executive summary:

In the present study the eye irritating potential of Sa 57 was analysed. Since irritant substances are cytotoxic to the corneal epithelium after a short time exposure the cytotoxic effects of the test item on EpiOcular™, a reconstituted three-dimensional human corneal epithelium model, were determined. Hereby, the test item was applied topically. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a 30 min exposure period and 120 min post-treatment period and compared to those of the concurrent negative controls.

The test item showed no irritant effects. The mean relative tissue viability (% negative control) was > 60% (104.9%).The controls confirmed the validity of the study. The mean absolute OD₅₇₀ of the two negative control tissues was > 0.8 and < 2.5 (1.814, 2.098). The mean relative tissue viability (% negative control) of the positive control was < 50% (20.5%). The maximum inter tissue difference of replicate tissues of all dose groups was < 20% (0.9%).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Justification for classification or non-classification

The test substance did not produce skin irritation and eye irritation. The substance does not need to be classified for irritation according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.