[No public or meaningful name is available]

Endpoint summary

Description of key information

Skin irritation: 

Target: Reaction mass of (4RS,4aRS,8RS,8aRS)-4-ethyl-8,8a-dimethyloctahydro-1(2H)-naphthalenone and (±)-(4RS,4aSR,8SR,8aSR)-4-ethyl-8,8a-dimethyloctahydro-1(2H)-naphthalenone and (±)-(4RS,4aSR,8RS,8aSR)-4-ethyl-8,8a-dimethyloctahydro-1(2H)-naphthalenone: Skin irritant.

Read-across: source data: Reaction mass of (4RS,4aRS,8RS,8aRS)-4-ethyl-8-methyloctahydronaphthalen-1(2H)-one and (4RS,4aSR,8SR,8aRS)-4-ethyl-8-methyloctahydronaphthalen-1(2H)-one and (4RS,4aSR,8SR,8aSR)-4-ethyl-8-methyloctahydronaphthalen-1(2H)-one: Skin irritant, OECD TG 404, 2014.

Read-across justification document available in Section 13.2. 

Eye irritation, in vitro: not eye irritating, OECD TG 438, 2021

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23-09-2013 To: 31-12-2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP. All relevant validity criteria were met with acceptable devations

Remarks:

To facilitate scoring, treated skin areas were re-clipped at least 3 hours before the observations ; this may inadvertently enhance scoring observations due to potential abrasion of the skin. The OECD TG 404 guideline specifies pre-exposure clipping only. Additionally, there was some indications that the test item was not fully removed after 4 hours exposure. This would lead to increased potential for classification due to enhancement of response observations.

Justification for type of information:

Information as to the availability of the in vivo study is provided in 'attached justification'.

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

yes

Remarks:

Deviation: to facilitate scoring, treated skin areas were re-clipped before the observations. Additionally, some indications that the test item was not fully removed post 4-hour exposure. This would lead to increased potential for classification.

Qualifier:

according to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

Deviations:

yes

Remarks:

see above

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2500 (Acute Dermal Irritation)

Deviations:

yes

Remarks:

see above

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries (JMAFF), 12 Nousan, Notification No 8147, November 2000

Deviations:

yes

Remarks:

see above

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: March 2013 ; signature: May 2013

Species:

rabbit

Strain:

New Zealand White

Remarks:

Hsdlf: NZW

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Recognised Supplier
- Age at study initiation: 12 to 24 weeks
- Weight at study initiation: 2574 to 2753 g
- Housing: Individually housed in cages with perforated floors, with shelters and cage enrichment including wooden sticks
- Diet (ad libitum): Global Diet 2030 (Recognised Supplier); provided ad libitum (approximately 100 g per day)
- Water (ad libitum): mains drinking water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0 ± 3.0
- Humidity (%): 40 to 70
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12 h light / 12 h dark

IN-LIFE DATES: From: 23-09-2013 To: 31-12-2013

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.5 mL.
- Concentration (if solution): Not applicable.

VEHICLE
- Amount applied: Not applicable.

Duration of treatment / exposure:

4 hours

Observation period:

72 hours (initial observation); additional observations are made daily up to Days 7 and 14 to assess the reversibility of skin reactions (as appropriate).

Number of animals:

3 males

Details on study design:

TEST SITE
- Area of exposure: Dorsal (2 cm x 3 cm metalline patch secured with adhesive tape)
- % coverage: Not reported
- Type of wrap if used: metalline patch secured with adhesive tape and elastic bandage

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Yes
- Time after start of exposure: Four hours after the application, the dressing was removed and the skin cleaned of residual test item using tap water and watery ethanol (50%v/v). Note: that a sticky residue was reported at the 1 hour observation, indicating incomplete removal.

OBSERVATION TIME POINTS
(indicate if minutes, hours or days): 1 hour, 4 hours, 24 hours, 48 hours and 72 hours. Additional observations daily up to 7 or 14 days, as appropriate

SCORING SYSTEM: Draize Scale:
- Method of calculation:
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) *.......................................................................................... 4
*. Where signs of necrosis or corrosion (injuries in depth) prevent erythema scoring, the
maximum grade for erythema (= 4) is given.
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 millimeter) ........................................................... 3
Severe oedema (raised more than 1 millimeter and extending beyond the area of exposure) 4

Irritation parameter:

erythema score

Basis:

mean

Remarks:

n = 3

Time point:

24/48/72 h

Score:

2.47

Max. score:

4

Reversibility:

not fully reversible within: 14 days

Remarks on result:

positive indication of irritation

Irritation parameter:

edema score

Basis:

mean

Remarks:

n = 3

Time point:

24/48/72 h

Score:

3.43

Max. score:

4

Reversibility:

not fully reversible within: 14 days

Remarks on result:

positive indication of irritation

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

not fully reversible within: 14 days

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

4

Max. score:

4

Reversibility:

not fully reversible within: 14 days

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

2.7

Max. score:

4

Reversibility:

not fully reversible within: 14 days

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

3

Max. score:

4

Reversibility:

not fully reversible within: 14 days

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

2.7

Max. score:

4

Reversibility:

not fully reversible within: 14 days

Irritation parameter:

edema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

3.3

Max. score:

4

Reversibility:

not fully reversible within: 14 days

Irritant / corrosive response data:

Moderate to severe erythema and moderate or severe oedema in the treated skin areas of the three rabbits, which remained present until termination (after 14 days). Reduced flexibility of the skin was noted for one animal at 72 hours and 7 Days after exposure. Scaliness and/or bald skin were noted for all animals 7 days after exposure, and persisted until termination.

Other effects:

All males showed expected gain in body weight during the study.

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of this study, the test item is considered to be irritating to the skin.

Executive summary:

The study was performed to OECD TG 404, EU Method B.4, US EPA OPPTS 870.2500 and Japan (JMAFF) 12 Nousan, Notification No 8147 guidelines to assess the primary skin irritancy potential of the test item in accordance with GLP in New Zealand White rabbits. The test item was applied sequentially. Three rabbits were exposed for 4-hour by semi-occluded application of the test item to the intact clipped skin with 0.5 mL test item introduced under a 2 cm x 3 cm semi-occlusive patch. The patch was secured in position with adhesive tape and elasticated bandage. After exposure to the test item the patches were removed and individual dose sites were scored at approximately 1, 24, 48, and 72 hours. Treated sites were reclipped at least 3 hours before observations.Sticky remnants of the test item were present on the skin on Day 1.Moderate to severe erythema and moderate or severe oedema in the treated skin areaswhich remained present until termination (after 14 days). Reduced flexibility of the skin was noted for one animal at 72 hours and 7 Days after exposure. Scaliness and/or bald skin were noted for all males 7 days after exposure, and persisted until termination. Under the conditions of the study, the test item is considered to be a skin irritant.

Applicant assessment indicates: to facilitate scoring, treated skin areas were re-clipped at least 3 hours before the observations ; this may inadvertently enhance scoring observations due to potential abrasion of the skin. The OECD TG 404 guideline specifies pre-exposure clipping only. Additionally, there was some indications that the test item was not fully removed after 4 hours exposure. Whilst this may not of invalidated the study conclusions, this would lead to highly conservative conclusion due to increased potential for classification due to enhancement of response observations.

Under the conditions of the study, the test item is considered to be irritating to skin, category 2. 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06-11-2020 to 23-02-2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Also conducted according to GLP.

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: ROSS 308

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Recognised supplier (documented in the full study report)
- Number of animals: Not reported.
- Characteristics of donor animals (e.g. age, sex, weight): ca. 7 weeks old (typically) and 2.5 kg.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Freshly isolated heads from ca. 7 weeks old donors were collected from the abattoir and transported at ambient temperature. After collection, the heads were inspected for appropriate quality and wrapped with tissue paper moistened with saline, then placed in a plastic box which was closed (4-5 heads per box). The heads were received at the test facility and processed within 2 hours of collection by the test facility. After removing the head from the plastic box, it was put on soft paper. The eyelids were carefully cut away with scissors, avoiding damaging the cornea. One small drop of 2% (w/v) fluorescein solution was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL physiological saline. Then the fluorescein-treated cornea was examined with a hand-held slit lamp or slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged (i.e. fluorescein retention and corneal opacity scores ≤ 0.5). If the cornea was in good condition, the eyeball was carefully removed from the orbit. The eye was placed onto damp paper and the nictitating membrane was cut away with other connective tissue. The prepared eyes were kept on the wet papers in a closed box so that the appropriate humidity was maintained. Eyes were then clamped in the superfusion apparatus and then re-examined to ensure good condition. Then the eyes were acclimatised for 45 to 60 minutes at (32±1.5°C).
- Time interval prior to initiating testing: <24 hours. Eyes were prepared for testing immediately on same day arrival within 2 hours of colection.
- indication of any existing defects or lesions in ocular tissue samples: None.
- Indication of any antibiotics used: None reported.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.03 ml (or 30 µL of the test item)
- Concentration (if solution): undiluted

VEHICLE
- Amount(s) applied (volume or weight with unit): Not applicable.
- Concentration (if solution): Not applicable.
- Lot/batch no. (if required): Not applicable.
- Purity: Not applicable.

Duration of treatment / exposure:

The test item remained for an exposure period of 10 seconds and was then rinsed from eye using 20 µL of physiological saline solution.
Controls (both negative and positive controls) were similarly applied to the cornea in the negative and positive control groups respectively.

Observation period (in vivo):

Treated eyes were evaluated prior to treatment and at 30, 75, 120, 180 and 240 minutes (±5 minutes) after the eyes had been rinsed with the physiological saline.

Number of animals or in vitro replicates:

Triplicate (n=3)

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES :
Freshly isolated heads from ca. 7 weeks old donors were collected from the abattoir and transported at ambient temperature. After collection, the heads were inspected for appropriate quality and wrapped with tissue paper moistened with saline, then placed in a plastic box which was closed (4-5 heads per box). The heads were received at the test facility and processed within 2 hours of collection by the test facility. After removing the head from the plastic box, it was put on soft paper. The eyelids were carefully cut away with scissors, avoiding damaging the cornea. One small drop of 2% (w/v) fluorescein solution was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL physiological saline. Then the fluorescein-treated cornea was examined with a hand-held slit lamp or slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged (i.e. fluorescein retention and corneal opacity scores ≤ 0.5). If the cornea was in good condition, the eyeball was carefully removed from the orbit. The eye was placed onto damp paper and the nictitating membrane was cut away with other connective tissue. The prepared eyes were kept on the wet papers in a closed box so that the appropriate humidity was maintained. Eyes were then clamped in the superfusion apparatus and then re-examined to ensure good condition. Then the eyes were acclimatised for 45 to 60 minutes at (32±1.5°C).

EQUILIBRATION AND BASELINE RECORDINGS:
At the end of the acclimatization period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a baseline (t=0) for each individual eye. The cornea thickness of the eyes should not change by more than 5% within the -45 min and the zero time. No significant corneal thickness changes (1.8% was observed) was observed in one eye and no changes were noted in the other eyes in the experiment. All eyes were considered to be suitable for the assay.

NUMBER OF REPLICATES :
Triplicate (3) for test item, positive and negative controls.

NEGATIVE CONTROL USED : Yes.

SOLVENT CONTROL USED (if applicable) : Not applicable.

POSITIVE CONTROL USED : Benzalkonium chloride (5%).

APPLICATION DOSE AND EXPOSURE TIME : Application dose: 30 µL of the test item. Exposure time: 10 seconds.

OBSERVATION PERIOD : prior to treatment and at 30, 75, 120, 180 and 240 minutes (+/- 5 minutes) after decontaminated with physiological saline solution.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: The test item and control treated eyes were rinsed additional gentle rinsing with 3x20 mL saline at ambient temperature (taking care not to damage the cornea but attempting to remove all residual test material if possible) after treatment.
- Indicate any deviation from test procedure in the Guideline : Not applicable.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Microscope.
- Damage to epithelium based on fluorescein retention: Microscope.
- Swelling: measured with optical pachymeter on a slit-lamp microscope; slit-width setting: Measured with optical pachymeter on a slit-lamp microscope.
- Macroscopic morphological damage to the surface: Yes.
- Others (e.g, histopathology): Not applicable.

SCORING SYSTEM:
- Mean corneal swelling (%) : See Tables.
- Mean maximum opacity score : See Tables.
- Mean fluorescein retention score at 30 minutes post-treatment : See Tables.

DECISION CRITERIA: In accordance with Guideline OECD TG 438.
The study is considered valid, if:
• the number of eyes meets the requirement of the relevant OECD guideline,
• the negative control item results No Category,
• the positive control item results Category 1 classification.
The results from all eyes used met the quality control standards. The negative control and positive control results were within the historical data range. This study was considered to be valid.

Irritation parameter:

cornea opacity score

Run / experiment:

mean (n=3)

Value:

0.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class I

Irritation parameter:

fluorescein retention score

Run / experiment:

mean (n=3)

Value:

0.33

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class I

Irritation parameter:

percent corneal swelling

Run / experiment:

mean (n=3)

Value:

1.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: value %; ICE Class I

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: None reported.

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The laboratory previsouly demonstrated technical proficiency of the validated method with proficiency test items (information in the public domain). Additionally, concurrent positive control and negative controls were within the current historic control range (HCD) (documented in the full study report), each meeting the validity criteria respectively.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes.
- Acceptance criteria met for positive control: Yes.
- Range of historical values if different from the ones specified in the test guideline:Not applicable. Applicant assessment of the data indicates that the positive controls and negative controls were within the current historic control range (HCD) (documented in the full study report), each meeting the validity criteria respectively.

Table 1.- Ocular reactions

Test Item
Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	1.1%	I
Mean maximum corneal swelling at up to 240 min	1.1%	I
Mean maximum corneal opacity change	0.50	I
Mean fluorescein retention change	0.33	I
Other Observations	None
Overall ICE Class	3xI
Positive Control Item
Observation	Value		ICE Class
Mean maximum corneal swelling at up to 75 min	14.2%		III
Mean maximum corneal swelling at up to 240 min	34.7%		IV
Mean maximum corneal opacity change	4.00		IV
Mean fluorescein retention change	2.83		IV
Other Observations	Slight loosening of epithelium was observed in two eyes (2/3) at 180 minutes after the post-treatment rinse.
Overall ICE Class	3xIV
Negative Control Item
Observation	Value		ICE Class
Maximum corneal swelling at up to 75 min	-1.6%		I
Maximum corneal swelling at up to 240 min	-1.6%		I
Maximum corneal opacity change	0.00		I
Fluorescein retention change	0.00		I
Other Observations	None
Overall ICE Class	3xI

- Corneal Opacity Scores

Maximum score was 0.5 in the test item group. Maximum score was 0.0 in the negative control. In the positive control group the Maximum score was 4.0. No morphological effects were noted in the test item or negative control item treated eyes. In the positive control group, slight loosening of epithelium was observed in two eyes (2/3) at 180 minutes after the post-treatment rinse.

- Fluorescein Retention Scores

Maximum score was 0.5 in the test item group. In the negative control maximum score was 0.0 and in the positive control group the Maximum score was 4.00.

Table 2.- Individual scoring - test item

End Point	Eye Number	Time (after decontamination)
		0 minutes	30 minutes	75 minutes	120 minutes	180 minutes	240 minutes
Corneal opacity	11	0.0	0.0	0.5	0.5	0.5	0.5
	12	0.0	0.5	0.5	0.5	0.5	0.5
	13	0.0	0.5	0.5	0.5	0.5	0.5
Mean		0.00	0.33	0.50	0.50	0.50	0.50
ICE Class		I
Fluorescein Retention	11	0.0	0.5
	12	0.0	0.5
	13	0.5	0.5
Mean difference (30 - min)			0.33
ICE Class		I
Corneal Thickness	11	0.60	0.62	0.61	0.60	0.59	0.59
	12	0.56	0.56	0.56	0.56	0.56	0.56
	13	0.58	0.58	0.58	0.58	0.57	0.57
Mean		0.58	0.59	0.58	0.58	0.57	0.57
Mean Corneal Swelling (%)			1.10%	0.60%	0.00%	-1.10%	-1.10%
ICE Class		I
ICE Classes Combined:		3 x I
Classification:		Category 1

The test was considered acceptable since the concurrent negative or vehicle/solvent items and the concurrent positive controls were identified as GHS Non Classified and GHS Category 1, respectively.

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of this study, the test item was considered to be non-irritant.

Executive summary:

An in vitro eye irritation study of the test item was performed in isolated chicken's eyes. The study was conducted and irritation effects of the test item were evaluated according to the OECD No. 438 guideline (25 June 2018).

After the zero reference measurements, the eyes were held in a horizontal position and the test item was applied onto the centre of the cornea such that the entire surface of the cornea was covered in all cases. After 10 seconds exposure time, the surface of the eyes was rinsed with physiological saline solution. Three eyes were treated with 30 µL test item. The three positive control eyes were treated in a similar way with 30 µL of 5% (w/v) Benzalkonium chloride solution. The negative control eye was treated with 30 µL of physiological saline (0.9% (w/v) NaCl solution). Corneal thickness, corneal opacity and fluorescein retention were measured and any morphological effects (e.g. pitting or loosening of the peithelium) were evaluated.

The results from all eyes used in the study met the quality control standards. The negative control and positive control results were within the historical control data range in experiment. Thus, the study was considered to be valid.

No significant cornea thickness change (mean = 1.1 %) was observed during the four-hour observation period on the test item treated eyes. No significant fluorescein retention change (mean = 0.33) was observed on the test item treated eyes during the observation period. No morphological effect was observed.

 

Based on this in vitro eye irritation assay in isolated chicken eyes, the test item was non-irritant, UN GHS classification: No Category.

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 substance meets the classification criteria under Regulation (EC) No 1272/2008 for skin irritation: category 2: H15: causes skin irritation.

The substance does not meet classification criteria under Regulation (EC) No 1272/2008 for eye irritation.