Reaction mass of 1-[rac-(1R,6S)-2,2,6-trimethylcyclohexyl]pentan-3-ol isomer 1 and 1-[rac-(1S,6S)-2,2,6-trimethylcyclohexyl]pentan-3-ol

Endpoint summary

Description of key information

Skin irritation

No skin irritation was observed in the Acute Dermal Toxicity study. Therefore, no new study on skin irritation was conducted.

Eye irritation

No eye irritation was observed in the ICE and RhCE tests. Therefore, the test item is not classified with regard to eye irritation/corrosion.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the study does not need to be conducted because an acute toxicity study by the dermal route does not indicate skin irritation up to the relevant limit dose level (2 000 mg/kg body weight)

Reference 2

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2000-06-05 to 2000-07-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD 402 (1987)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl : CD® (SD) IGS BR

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: males - 201 to 207 g, females - 202 to 222 g
- Housing: in suspended polypropylene cages furnished with woodflake. The animals were housed individually during the 24-hour exposure period and in groups of five, by sex, for the remainder of the study.
- Diet: ad libitum during the study
- Water: ad libitum during the study
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 30 - 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

- Dose volume: 2.21 mL/kg
- Dose level: 2000 mg/kg

Duration of treatment / exposure:

24 hours

Observation period:

14 days

Number of animals:

5 females and 5 males

Details on study design:

TEST SITE
- Area of exposure: the back and flanks of each animal
- % coverage: 10 % of the total body surface area
- Type of wrap: surgical gauze
- On the day before treatment the back and flanks of each animal were clipped free of hair using veterinary clippers.

REMOVAL OF TEST SUBSTANCE
- Washing: wiped with cotton wool moistened with distilled water
- Time after start of exposure: 24 hours

TEST MATERIAL
- Amount(s) applied: 2.21 mL/kg

SCORING SYSTEM:
- Method of calculation: The skin reactions were scored using a 0-4 scale (Draize) as follows: Erythema and Eschar formation: No erythema = 0, very slight erythema (barely perceptible) = 1, well-defined erythema = 2, moderate to severe erythema = 3, severe erythema (beet redness) to slight eschar formation (injuries in depth) = 4; Oedema formation - No oedema = 0, very slight oedema (barely perceptible) = 1, slight oedema (edges of area well-defined by definite raising) = 2, moderate oedema (raised approximately 1 mm) = 3, severe oedema (raised more than 1 mm and extending beyond the area of exposure) = 4.

- Duration of observation period following administration: 14 days
- Frequency of observations: at 30 min, 1, 2 and 4 hours after dosing and subsequesntly once daily for 14 days.
- Frequency of weighings: prior to application on day 0 and on day 7 and 14
- Necropsy of survivors performed: yes

Irritation parameter:

erythema score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

edema score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritant / corrosive response data:

No signs of dermal irritation or systemic toxicities were observed in the test animals after a single application of test material.

Other effects:

No death animals were observed during the study. All animals showed an expected gain in bodyweight during the study. No abnormalities were noted at necropsy.

Interpretation of results:

GHS criteria not met

Conclusions:

The test item showed no skin irritation effects on the tested rats in the Acute Dermal Toxicity study.

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

Remarks:

eye corrosion

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

from 2018-02-09 to 2018-05-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

chicken

Strain:

other: Gallus gallus e.g. Ross 308 Broiler

Details on test animals or tissues and environmental conditions:

- Species: Spring chickens ( Gallus gallus e.g. Ross 308 Broiler)
- Number: Multiple chicken heads (three eyes for the test item, three eyes for the positive control item and two eyes for the negative control item)
- Sex: Male or female
- Age of chicken (at slaughter): Approximately 56 days old
- Weight of chicken (at slaughter): Approximately 3 kg
- Storage, temperature and transport conditions of ocular tissue: Heads were removed immediately after the chickens had been humanely killed at the source, for use on the same day. Following slaughter, the intact chicken heads were placed into individual plastic compartments within a plastic box in order to minimize any damage to the eyes. The base of each compartment was lined with a paper towel moistened with isotonic saline. The heads were transported to the test facility at ambient temperature.
- Time interval prior to initiating testing: The time interval between collection of chicken heads and placing the eyes in the superfusion chamber following enucleation was minimized.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

0.03 mL per eye of the test item

Duration of treatment / exposure:

10 seconds

Duration of post- treatment incubation (in vitro):

observations prior to treatment and at 30, 75, 120, 180 and 240 minutes after decontamination with isotonic saline

Number of animals or in vitro replicates:

3 eyes for the test item
3 eyes for the positive control
2 eyes for the negative control

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
Eyelids were carefully excised. The integrity of the cornea was measured with a drop of 2 % (w/v) sodium fluorescein to the surface of the cornea and then rinsed with isotonic saline after a few seconds. The treated eyes were examined for damage to the cornea. When the fluorescein retention and corneal opacity scores were ≤ 0.5, the eyes were selected for the test. Acceptable eyes were dissected from the skull and pulled from the orbit by holding the nictitating membrane firmly with surgical forceps. Enucleated eyes were transferred to an appropriate clamp keeping the cornea vertical. They were then transferred to chambers within the superfusion apparatus ensuring the corneas received sufficient isotonic saline from the saline drip. The temperature of the chambers was at 32.0 ± 1.5 °C.

EQUILIBRATION AND BASELINE RECORDINGS
Once all eyes were placed in the superfusion apparatus, the eyes were examined again to ensure the eyes had not been damaged by the dissection procedure. Corneal thickness measurements are taken with a depth measuring device at the center of each cornea. After the approval process the eyes were incubated for 45 minutes for equilibrium purposes. Time zero measurements for corneal thickness and opacity were taken to serve as a baseline. The baseline for the fluorescein measurements were taken at dissection.

NUMBER OF REPLICATES
3 eyes for the test item
3 eyes for the positive control
2 eyes for the negative control

NEGATIVE CONTROL USED
0.03 mL of the negative control item was applied ot the cornea of each negative control eye.

POSITIVE CONTROL USED
0.03 mL of the positive control item, Benzalkonium chloride (5% v/v), was applied and after 10 seconds was rinsed with 20 mL isotonic saline.

APPLICATION DOSE AND EXPOSURE TIME
0.03 mL of the test item were applied to the cornea. The tets item was used in its initial state. The test item remained in place for 10 seconds and was then rinsed from the eye using 20 mL of 0.9 % (w/v) sodium chloride solution.

OBSERVATION PERIOD
observations prior to treatment and at 30, 75, 120, 180 and 240 minutes after decontamination with isotonic saline

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: 20 mL of 0.9 % (w/v) sodium chloride solution after 10 seconds exposure

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: calculated with the most densely opacified areas for scoring
- Damage to epithelium based on fluorescein retention: calculated at the 30 minute time interval only
- Swelling: assessed from corneal thickness measurements
- Macroscopic morphological damage to the surface: observations for pitting, sloughing, roughening of the corneal surface, and adhering of test item were made

Irritation parameter:

cornea opacity score

Run / experiment:

240 mins

Value:

1.7

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class: III

Irritation parameter:

fluorescein retention score

Run / experiment:

30 mins

Value:

1.5

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class: II

Irritation parameter:

percent corneal swelling

Run / experiment:

240 mins

Value:

8.96

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class: III

Table 1. Summary of test items results for all endpoints

Mean Corneal Opacity (ICE class)	Mean Fluorescein Retention (ICE class)	Mean Corneal Thickness% Compared to Time Zero (ICE class)					Combination of the 3 Endpoints
		30 mins	75 mins	120 mins	180 mins	240 mins
1.7 (III)	1.5 (II)	8.02 (II)	13.21 (III)	8.49 (II)	8.02 (II)	8.96 (II)
		(III)					1 x II, 2 x III
Classification:		No Prediction Can Be Made

Corneal Opacity Scores

Scattered or diffuse areas; details of the iris are clearly visible was noted in one test item treated eye and Easily discernible translucent area; details of the iris are slightly obscured were noted in two test item treated eyes.

Complete corneal opacity; iris invisible was noted in all positive control treated eyes.

Very faint opacity was noted in the negative control treated eyes.

No morphological effects were noted in the test item or negative control item treated eyes. Sloughing was noted in two of the positive control treated eyes.

Fluorescein Retention Scores

Very minor single cell staining was noted in one test item treated eye. Focal or confluent dense single cell staining was noted in two test item treated eyes. Confluent large areas of the cornea retaining fluorescein were noted in all positive control treated eyes. No fluorescein retention to very minor single cell staining was noted in the negative control treated eyes.

Positive control item

Maximal mean score for corneal opacity:       4.0       ICE Class IV

Mean score of Fluorescein Retention:            3.0       ICE Class IV

Maximal mean corneal swelling compared to time zero:       37.98%       ICE Class IV

Negative Control Item

Maximal mean score for corneal opacity:       0.5       ICE Class I

Mean score of Fluorescein Retention:            0.3       ICE Class I

Maximal mean corneal swelling compared to time zero:       0.00%       ICE Class I

Interpretation of results:

other: not classified in Category 1

Conclusions:

The substance is not to be classified as severely eye damaging (Cat. 1).

Executive summary:

This ex vivo study was performed to assess the eye irritation potential of the test item by means of the Isolated Chicken Eye Test according to OECD 438 and GLP. The study was performed to evaluate the possible corrosivity or severe irritancy potential of the test item as measured by its ability to induce toxicity in an enucleated chicken eye. 0.03 mL of the test item were applied onto the cornea of each of three enucleated eyes. A further three enucleated eyes were treated with positive control item (Benzalkonium chloride). A further two enucleated eyes were treated with Sodium chloride for control purposes. Treatment with the test item showed inconclusive results with regard to eye irritation. In conclusion, the substance is not to be classified as severely eye damaging (Cat. 1).

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

from 2018-03-19 to 2018-07-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

9 October 2017

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: MatTek Corporation Protocol

Version / remarks:

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; 29 June 2015.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

other: EpiOcular tissues

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability
This study was performed to assess the eye irritation potential of the test item. In a prevalidation study performed by Avon Products Inc. and MatTek Corporation, the in vitro eye test using the human cornea model EpiOcular™ and measurement of cell viability by dehydrogenase conversion of MTT into a blue formazan salt have turned out as a sufficiently promising predictor for eye irritancy potential.

- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live
The EpiOcular™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. It consists of highly organized basal cells which progressively flatten out as the apical surface of the tissue is approached, analogous to the normal in vivo corneal epithelium. The EpiOcular™ tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELL, 10 mm Ø).

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 50 µL

Duration of treatment / exposure:

30 min

Duration of post- treatment incubation (in vitro):

120 min

Number of animals or in vitro replicates:

2

Details on study design:

- Details of the test procedure used:
Preparation:
EpiOcular™ tissues were shipped at 2 - 8 °C on medium-supplemented agarose gels in a 24-well plate. On day of receipt of the EpiOcular™ tissues, the equilibration step (15 minutes at room temperature in the 24-well shipping container) started. 1.0 mL of DMEM-medium is aliquoted into the appropriate wells of pre-labeled 6-well plates. According to an expert statement the tissues can be stored over night at 2 - 8 °C after receipt..
Each 24-well shipping container was removed from its plastic bag under sterile conditions and its surface was disinfected by wiping with 70 % isopropanol- or ethanol soaked wipes. The sterile gauze was removed and each tissue was inspected for air bubbles between the agarose gel and insert. Cultures with air bubbles under the inserts greater than 50 % of the insert area were not used. The tissues were carefully removed from the 24-well shipping containers using sterile forceps. The insert was then transferred aseptically into the 6-well plates and pre-incubated at standard culture conditions for one hour in the Assay Medium at 37 °C and the EpiOcular™ tissues were incubated at standard culture conditions overnight (16 to 24 hours).

Experimental performance
After the overnight incubation, the tissues were pre-wetted with 20 μL of Ca2+Mg2+ free-DPBS. The tissues were incubated at standard culture conditions (37 ± 1.5 °C, 5 ± 0.5 % CO2, 95 % RH) for 30 minutes. After the 30 minute Ca2+Mg2+ free-DPBS pre-treatment, the test and control items were tested by applying 50 μL topically on the EpiOcular™ tissues. At the end of the treatment time, the test item was removed by extensively rinsing the tissues with Ca2+Mg2+-free DPBS (brought to room temperature). Three clean beakers containing a minimum of 100 mL each of Ca2+Mg2+ free-DPBS were used per test item. After the washing steps, the remaining liquid was decanted onto the absorbent material followed by the immediate transfer of the tissues to 5 mL of previously warmed Assay Medium (room temperature) in a pre-labelled 12-well plate for 12 minute immersion temperature (post-soak) at room temperature. Subsequently, each insert was removed from the Assay Medium, the medium was decanted off the tissue, and the inserts were blotted on absorbent material, and transferred to the appropriate well of the pre-labelled 6-well plate containing 1 mL of warm Assay Medium. The tissues were incubated for about 120 minutes at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5 % CO2 (post-treatment incubation).

- RhCE tissue construct used, including batch number: EpiOcular™ kits and MTT-100 kits (Lot No. 27039)

- Doses of test chemical and control substances used: 50 µL

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods: 30 min exposure at 37 °C; 12 min post-soak immersion at room temperature; 120 min post-exposure at 37 °C

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals: Since the test item did not dye water or isopropanol and did not directly reduced MTT, additional controls are not required.

- Number of tissue replicates used per test chemical and controls: 2

- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device: 570 nm without reference filter using a microplate reader

- Description of the method used to quantify MTT formazan:
At the end of the post-treatment incubation, each insert was removed from the 6-well plate and gently blotted on absorbent material. The tissues were placed into the 24-well plate containing 0.3 mL of MTT solution. Once all the tissues were placed into the 24-well plate, the plate was incubated for 180 minutes at standard culture conditions. Each insert was removed from the 24-well plate after 180 minutes, the bottom of the insert was blotted on absorbent material, and then transferred to a pre-labelled 24-well plate containing 2.0 mL of isopropanol in each designated well so that isopropanol was flowing into the insert on the tissue surface. The plates were sealed with a standard plate sealer, and were stored overnight at 2 - 8 °C in the dark. To extract the MTT, the plates were placed on an orbital plate shaker and shaken for 2 to 3 hours at room temperature. At the end of the extraction period, the tissue was pierced and the liquid within each insert was decanted into the well from which it was taken. The extract solution was mixed and two 200 μL aliquots were transferred to the appropriate wells of a pre-labelled 96-well plate. The OD was determined at 570 nm.

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model
If the test item-treated tissue viability is > 60% relative to the negative control treated tissue viability, the test item is labeled non-irritant.
If the test item-treated tissue viability is ≤ 60% relative to negative control treated tissue viability, the test item is labeled irritant.

- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria: historical positive and negative control data are provided by the conducting laboratory

- Complete supporting information for the specific RhCE tissue construct used: Certificate of Analysis on test system quality is provided by the supplier

- Positive and negative control means and acceptance ranges based on historical data:
Acceptance range negative control: OD is > 0.8 and < 2.5.
The mean relative viability of the positive control is below 50% of the negative control viability.

- Acceptable variability between tissue replicates for positive and negative controls: < 20%

- Acceptable variability between tissue replicates for the test chemical: < 20%

Irritation parameter:

other: mean viability value in %

Value:

100.18

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- Optical pre-experiment on test item's colour change potential in water or isopropanol: no change was observed.
- Optical evaluation of the MTT-reducing capacity: no blue/purple colour was observed.
- Mean relative absorbance value of the test item: 100.18 %; the test item was not irritant to eye.
- Concerning acceptance criteria:
-- The negative control OD is > 0.8 and < 2.5 (was 2.285 and 2.400).
-- The mean relative viability of the positive control is below 50 % of the negative control viability (was 42.83 %).
-- The difference of viability between the two relating tissues of a single item is < 20 % (values were between 0.82 % and 3.14 %) in the same run (for positive and negative control tissues and tissues of single test items).

Results after treatment for 30 minutes with the test item and the controls

Treatment Group	Tissue No.	OD 570 nm Well 1	OD 570 nm Well 2	Mean OD of 2 Wells	Mean OD of 2 Wells blank corrected	Mean OD of Treatment Group blank corrected	Rel. Viability [%] Tissue 1, 2 *	Absolute Value of the Difference of Rel. Viability Tissue 1,2 [%]	Mean Rel. Viability [%]**
Blank		0.037	0.037	0.037
Negative Control	1	2.397	2.333	2.365	2.328	2.317	100.5	0.95	100.0
	2	2.400	2.285	2.343	2.306		99.5
Positive Control	1	1.057	1.021	1.039	1.002	0.992	43.2	0.82	42.83
	2	1.022	1.017	1.020	0.983		42.4
Test Item	1	2.281	2.362	2.322	2.285	2.321	98.6	3.14	100.18
	2	2.409	2.380	2.394	2.357		101.8

* Relative viability [rounded values]: 100 x (absorbance _{test item / positive control / negative control}) / (mean absorbance _{negative control})

** Mean viability [rounded values]: 100 x (mean absorbance _{test item / positive control / negative control}) / (mean absorbance _{negative control})

Interpretation of results:

GHS criteria not met

Conclusions:

The test item showed no eye irritation potential under the experimental conditions of the study.

Executive summary:

The eye irritation potential of the test item was assessed in an in vitro Human Cornea Model Test according to OECD 492. Each 50 μL of the test item, the negative control (deionised water) or the positive control (methyl acetate) were applied to each of duplicate tissue for 30 minutes. Irritating effects were not observed following incubation with the test item. In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item does not possess an eye irritating potential.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

A study according OECD 402 (1987) and Commission Directive 92/69/EEC Method B.3 was performed to assess the acute dermal toxicity of the test material in the Sprague-Dawley CD strain rat. A group of 10 animals (5 males and 5 females) was given a single 24-hour, semioccluded dermal application to intact skin at a dose level of 2000 mg/kg bodyweight. The animals were observed for 14 days after the day of treatment and were then killed for gross pathological examination. No death animals were observed during the study. No signs of systemic toxicity or dermal irritation were noted. All animals showed an expected gain in bodyweight during the study period. No abnormalities were noted at necropsy of the surviving animals. The acute dermal LD50 of the test material in the Sprague-Dawley CD strain rat was found to be greater than 2000 mg/kg bodyweight. No skin irritaion was observed during the study. Therefore, no new skin irritation study was conducted.

Eye irritation

OECD 438

This ex vivo study was performed to assess the eye irritation potential of the test item by means of the Isolated Chicken Eye Test according to OECD 438 and GLP. The study was performed to evaluate the possible corrosivity or severe irritancy potential of the test item as measured by its ability to induce toxicity in an enucleated chicken eye. 0.03 mL of the test item were applied onto the cornea of each of three enucleated eyes. A further three enucleated eyes were treated with positive control item (Benzalkonium chloride). A further two enucleated eyes were treated withSodium chloridefor control purposes. Treatment with the test item showed inconclusive results with regard to eye irritation. In conclusion, the substance is not to be classified as severely eye damaging (Cat. 1).

OECD 492

The eye irritation potential of the test item was assessed in an in vitro Human Cornea Model Test according to OECD 492. Each 50 μL of the test item, the negative control (deionised water) or the positive control (methyl acetate) were applied to each of duplicate tissue for 30 minutes. Irritating effects were not observed following incubation with the test item. In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item does not possesses an eye irritating potential.

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. As a result the substance is not considered to be classified for skin corrosion/irritation and for eye irritation/corrosion under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.