3,5-di(undecan-2-yl)heteropolycycle

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:

guideline study

Qualifier:

according to guideline

Guideline:

other: ECVAM Skin Irritation Validation Study

Principles of method if other than guideline:

This study was conducted using a reconstruced human epidermis model and was in accordance with the following references:
- ECVAM international validation study on in vitro tests for acute skin irritation: Report on the validity of the EPISKIN and EpiDerm assays and on the Integrity Funktion Test (Altern Lab Anim. 2007 Dec; 35 (6): 559-601.
- European Commission: Institute for Health and Consumer Protection and European Centre for the Validation of Alternative Methods (ECVAM): Statement on the validity of in vitro tests for skin irritation (2007)

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Remarks:

EpiSkin Standard Model

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:

15 µL

Duration of treatment / exposure:

15 min

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

3

Irritation / corrosion parameter:

other: Optical density (OD)

Run / experiment:

Time point: 15 min

Value:

0.85

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

0.82

Positive controls validity:

valid

Remarks:

0.13

Remarks on result:

other:

Remarks:

Max. score: 1.6

Irritation / corrosion parameter:

other: Rel. Absorbance

Run / experiment:

Time point: 15 min.

Value:

104

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100.0

Positive controls validity:

valid

Remarks:

16.2

Other effects / acceptance of results:

no other effects

Dose group	Treatment 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	0.8909	0.8028	0.7786	0.8241	0.0591	100.0	7.2
Positive Control	15 min	0.1243	0.1320	0.1432	0.1331	0.0095	16.2	1.2
Sa 190	15 min	0.8821	0.7955	0.8939	0.8571	0.0537	104.0	6.5

* Mean of two replicate wells after blank correction /

** relative absorbance [rounded values]:100 (absorbance_{test item)}/ (absorbance_{negative control})

Interpretation of results:

GHS criteria not met

Conclusions:

Classification: not irritating

Executive summary:

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item Sa 190 is not irritant to skin.

This in vitro study was performed to assess the irritation potential of Sa 190 by means of the Human Skin Model Test.

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

15 μL of the melted test item were applied to each tissue and spread to match the tissue size.

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

The test item as well as the positive and negative controls were washed off the skin tissues after 15 minutes treatment. After further incubation for 42 hours the tissues were treated with the MTT solution for 3 hours following 72 hours 10 minutes extraction of the colorant from the cells. The amount of extracted colorant was determined photometrically at 570 nm.

After treatment with the negative control the absorbance values were well above the required acceptability criterion of mean OD ≥ 0.6 for the 15 minutes treatment interval thus showing the quality of the tissues.

Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control for the 15 minutes treatment interval 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 (see chapter 12).

After treatment with the test item Sa 190 the mean relative absorbance value did not decrease (104.0%) 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.

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item Sa 190 is not irritant to skin.

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:

2017-12-19 to 2018-01-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

28 July 2015

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

22 July 2015

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 irritation of chemicals

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:

30 +/- 2 min.

Duration of post- treatment incubation (in vitro):

- post soak incubation: 12 +/- 2 min.
- post treatment incubation: 120 +/- 15min
- MTT incubation: 3h

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 [%]

Run / experiment:

Mean tissues 1 and 2

Value:

97.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100

Positive controls validity:

valid

Remarks:

20.5

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

TEST ACCEPTANCE CRITERIA

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 blue/purple. Since the 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 = 0.99%

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

NSMTT1 [%] = [meanODKT1 - ODKU)/ODNK] * 100 = -0.34%

NSMTT2 [%] = [meanODKT2 - ODKU)/ODNK] * 100 = 2.32%

NSMTT1 - NSMTT2 =± 2.66%

NSMTT was ≤ 60% (0.99%) 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 = 97.0%

 

The mixture of 50 µL test item per 1 mL A. dest showed colouring as compared to the solvent. Since the mean relative tissue viability of the test item treated tissues (TM) was above the 60% threshold value coloured tissue controls were performed for quantitative correction of results.

NSC_living [%] = [OD_TVT/OD_NK]*100 = 0.47%

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

NSC1 [%] = [OD_TVT1/OD_NK] * 100 = 0.39%

NSC2 [%] = [OD_TVT2/OD_NK] * 100 = 0.54%

NSC1 – NSC2 = ±0.15%

NSC_livingwas ≤ 60% (0.47%) relative to the negative control of living epidermis and could therefore be used for determination of theNSC-corrected mean relative tissue viability (NSCCV) according to the following formula:

NSCCV [%] = viability_TM[%] – NSC_living[%] = 96.4%

 

Since the test item showed non-specific MTT-reduction and non-specific colouring of living tissues, a third control for non-specific colour in killed tissues (NSC_killed) was performed to avoid a possible double-correction for colour interference.

The non-specificcolour of additional killed tissues (NSC_killed) was calculated according to the following formula:

NSC_killed [%] = [OD_TKT/OD_NK]*100 = 1.45%

The true tissue viability was then calculated as the percent tissue viability obtained with living tissues minus NSMTT minus NSC_livingplus NSC_killed.

True Tissue Viability = [%] mean tissue viability – NSMTT –NSC_living+ NSC_killed= 97.9%

Result of the Test Item Sa 190

Name	Negative Control		Positive Control		Test item
Tissue	1	2	1	2	1	2
OD570values	1.798	1.775	0.400	0.405	1.763	1.755
	1.847	1.837	0.401	0.424	1.817	1.777
OD570values (blank-corrected)	1.754	1.731	0.356	0.361	1.719	1.711
	1.803	1.794	0.357	0.380	1.774	1.733
mean of the duplicates	1.778	1.762	0.356	0.371	1.746	1.722
mean OD	1.770*		0.364		1.734
TODTT- NSMTT	-		-		1.717
TODTTNSMTT und NSCliving	-		-		1.707
mean sd OD	0.011		0.010		0.017
tissue viability [%]	100.5	99.5	20.1	20.9	98.6	97.3
relative tissue viability difference [%]^***	0.9		0.8		1.4
mean tissue viability [%]	100.0		20.5**		98.0
mean tissue viability [%] - NSMTT corrected	-		-		97.0
mean tissue viability [%] - NSMTT and NSClivingcorrected	-		-		96.4
True Tissue Viability	-		-		97.9

 

^*              Corrected mean OD₅₇₀ of 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	0.074	0.075	0.067	0.115	1.798	1.775
	0.075	0.076	0.071	0.117	1.847	1.837
OD570(Blank Corrected)	0.030	0.031	0.023	0.071	1.754	1.731
	0.031	0.032	0.027	0.073	1.803	1.794
mean OD570 (mean of 2 aliquots)	0.031	0.031	0.025	0.072	1.778	1.762
total mean OD570 (mean of the replicate tissues)	0.031		0.049		1.770
SD OD570(of the replicate tissues)	0.000		0.033		0.011
NSMTT [%]	0.99				-
Relative Tissue Viability [%]	-				100.5	99.5
Mean Relative Tissue Viability [%]	-				100.0
SD Tissue Viability [%]	-				0.6
CV [% Viabilities]	-				0.6

Result of the NSC_living Control

NSCliving	TVT		Negative Control
Tissue	1	2	1	2
absolute OD570 -values	0.050	0.053	2.098	2.048
	0.051	0.055	2.111	2.021
absolute OD570 - Blank corrected values	0.007	0.010	2.055	2.005
	0.008	0.012	2.068	1.978
mean OD570 (mean of 2 aliquots)	0.008	0.011	2.062	1.991
total mean OD570 (mean of replicate tissues)	0.010		2.027
SD OD570 (of the 2 replicate tissues)	0.002		0.050
NSCliving[%]	0.47		-
Relative Tissue Viability [%]	-		101.7	98.3
Mean Relative Tissue Viability [%]	-		100.0
SD Tissue Viability [%]***	-		2.5
CV [% Viabilities]	-		2.5

 

Result of the NSC_killed Control 

NSCkilled	TKT		Negative Control
Tissue	1	2	1	2
Absolute OD570 -values	0.065	0.078	2.098	2.048
	0.064	0.082	2.111	2.021
Absolute OD570 - Blank corrected values	0.022	0.035	2.055	2.005
	0.021	0.039	2.068	1.978
Mean OD570 (mean of 3 aliquots)	0.022	0.037	2.062	1.991
Total Mean OD570 (Mean of Replicate Tissues)	0.029		2.027
SD OD570 (of the 2 replicate tissues)	0.011		0.050
NSCkilled[%]	1.45		-
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 190 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% (97.9%; NSMTT-,NSC_living and NSC_killed-corrected).

The controls confirmed the validity of the study. The mean absolute OD₅₇₀of the two negative control tissues was > 0.8 and < 2.5 (1.814). 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% (1.4%).

Reference 2

Endpoint:

eye 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: Test procedure according to validation study and GLP

Qualifier:

according to guideline

Guideline:

other: HET-CAM Test

Principles of method if other than guideline:

This study was conducted according to the procedures indicated by the folowing internationally accepted guideline and recomendation:
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, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

For each replicate about 300 µl of the test item were applied directly to the chorioallantoic membrane in order to cover at least 50 % of the membrane. 300 µl of each of the positive controls ( 1% solution in deion. water) and the negative control (physiological sodium chloride solution, 0.9% w/w) were applied to three eggs each.

Duration of treatment / exposure:

300 s

Observation period (in vivo):

300 s

Number of animals or in vitro replicates:

- test item: 6
- controls: 3 each

Details on study design:

CAM Preparation
On day 9, the day of the performance of the experiment, the eggs were candled once again in order to determine the position of the egg's oxygen bubble. The position was marked on the eggshell. Along the marking the eggshell was sawed with an electric saw. Afterwards, the shell above the oxygen bubble was removed. The egg skin beneath was covered with 0.9% (w/v) NaCl in deion. water (saline, produced in-house) in order to separate the egg skin from the chorioallantoic membrane beneath the skin. After 3 minutes incubation at 37.5 ± 0.5 °C the egg skin was removed from the membrane using forceps.

Treatment
In this HET-CAM assay the test item was tested pure. Six eggs were treated with the test item.
For each replicate about 300 μL of the test item were applied directly to the membrane in order to cover at least 50% of the membrane. 300 μL of each of the positive controls and the negative control were applied to three eggs each. During the observation period of 300 seconds any lesions in close proximity to the covered membrane were monitored and recorded.

Observations
The membranes of the eggs were observed for 300 seconds. Lesions of the underlying blood vessels were monitored and noted. In particular three endpoints were observed and the time point at which an effect occurs was recorded. The three endpoints were:
• haemorrhage
• coagulation
• lysis of the blood vesse

Irritation parameter:

in vitro irritation score

Run / experiment:

HET-CAM 300 s

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

0

Positive controls validity:

valid

Remarks:

9.92 (DDS); 19.12 (NaOH)

Other effects / acceptance of results:

no other effects

Mean irritancy index of 6 eggs: Test Item = 0.00

Mean irritancy index of 3 eggs:

Positive control (1% Sodium dodecyl sulphate) = 9.92

Positive control (0.1 N Sodium hydroxide solution) = 19.12

Negative control (Physiological sodium chloride solution (0.9%)) = 0.00

Interpretation of results:

GHS criteria not met

Remarks:

Criteria used for interpretation of results: INVITTOX

Conclusions:

Classification: not irritating

Executive summary:

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item Sa 190 does not possess any irritating potential.

This in vitro study was performed to assess the irritating potential of SAT 080004 by detection of damages in blood vessels under the chorioallantoic membrane of chicken eggs incubated for 9 days (Hen's Egg Test - Chorioallantoic Membrane Test, HET-CAM.

The test item was tested pure.

The observation time was 5 minutes at room temperature.

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

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

1 % solution of sodium dodecyl sulphate (SDS) and 0.1 N sodium hydroxid (NaOH) were used as positive controls.

The positive controls induced severe irritation on the blood vessels (mean irritancy indices of 9.92 for SDS and 19.12 for NaOH).

The mean irritancy indices of the controls are well comparable with the historical control values and are within the acceptance criteria. Therefore, it was concluded that the test was valid.

No irritating effects were observed during 5 min incubation with the test substance Sa 190. The calculated mean irritancy index is 0.00.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

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.