Reaction mass of 2,4-dimethyl-3a,4,5,6,7,7a-hexahydro-1H-4,7-methanoinden-5-ol and 3,4-dimethyl-3a,4,5,6,7,7a-hexahydro-1H-4,7-methanoinden-5-ol

Endpoint summary

Description of key information

Skin corrosion: Skin corrosion is not expected because the substance does not contain acidic or base groups that indicate corrosion. There are also no other reactive groups identified that could result in corrosion. Furthermore, in the acute dermal toxicity study in male and female rats (IFF, 1980), after single application of 5000 mg/kg bw of the undiluted test substance, no indication of corrosion was observed at the end of the 14 day observation period (see Acute toxicity Endpoint summary).

Skin irritation (OECD TG 439): Irritating

Eye irritation (similar to OECD TG 405): Irreversible damage

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November 23, 2016 - January 13, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

July 28, 2015

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

other: Normal Human-Derived Epidermal Keratinocytes

Cell source:

other: SkinEthic Laboratories (69007 Lyon, France)

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkin
- Tissue batch number(s): 17-EKIN-002
- Delivery date: January 10, 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: not specified
- Temperature of post-treatment incubation: 37 ± 1.5 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: rinsed with PBS until all test material was removed
- Observable damage in the tissue due to washing: not specified

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3h
- Spectrophotometer: Versamax® Molecular Devices, 85737 Ismaning, Germany, version 4.7.1
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Historical neg control OD= 0.61-1.52, Current negative control OD = 1.053

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the viability after 15 minutes exposure is less than or equal to 50%
- The test substance is considered to be non-irritant to skin if the viability after 15 minutes exposure is greater than 50%

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 µL
- Concentration (if solution): undiluted

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL
- Concentration (if solution): undiluted

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL
- Concentration (if solution): 5% SLS solution in deionised water

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Irritation / corrosion parameter:

other: Mean relative tissue viability (%)

Value:

6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: not specified
- Direct-MTT reduction: none
- Colour interference: none

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, after treatment with the negative control the mean absorbance value of the triplicate tissues was 1.053 which was well within the required acceptability criterion of mean OD >= 0.6 till ≤ 1.5 thus showing the quality of the tissues.
- Acceptance criteria met for positive control: yes, treatment with the positive control induced a decrease in the relative absorbance as compared to the negative control to 8.1% (<40%) thus ensuring the validity of the test system.
- Acceptance criteria met for variability between replicate measurements: yes, the rel. standard deviations between tissues of the same treatment group were below 16% (<18%), thus ensuring the validity of the study.
- Range of historical values if different from the ones specified in the test guideline:

Data of 232 studies performed from October 2007 until May 2014

Positive Control
Mean Viability: 19.30%
Rel. Standard Deviation: 10.00%
Range of Viabilities: 1.70% - 35.40%

Negative Control
Mean OD: 1.01
Rel. Standard Deviation: 0.22
Range of ODs: 0.61 – 1.52

Data of 12 studies performed from July 2015 to January 2017

Positive Control
Mean Viability: 15.83%
Rel. Standard Deviation: 11.88%
Range of Viabilities: 4.25% - 32.7%

Negative Control
Mean OD: 0.80
Rel. Standard Deviation: 0.22
Range of ODs: 0.61 – 1.09

The test for colour interference and direct MTT reduction was found to be negative, as 1) the colour of the test item/water mixture did not change during the incubation period compared with the colour of the pure test item and 2) the colour of a test solution containing test substance and MTT did not turn blue/purple after incubation.

Interpretation of results:

other: Skin Irritant Category 2

Remarks:

based on EU CLP (EC 1272/2008 and its updates)

Conclusions:

Under the conditions of this test, the relative mean tissue viability for the test item was determined to be 6%. This value is below the threshold for irritancy of ≤50%. Therefore, the test substance is considered to be a skin irritant.

Executive summary:

This in vitro skin irritation study to assess the irritation potential of Dimeth Cyclormol by means of the Human Skin Model Test, was performed in line with OECD439 and GLP principles. The test item did not directly reduce MTT, and did not cause colour interference. Triplicate tissues of the human skin model EpiSkin™ were treated with the test item, the negative or the positive control for 15 minutes. After washing, and further incubation for approximately 42 hours, tissue viability was determined by measuring MTT reduction. The viability and OD values of respectively the positive and negative control tissues were considered valid. After treatment with the test item the mean relative viability decreased to 6.0%. The value is below the threshold for irritancy of ≤ 50%. Therefore, the test item is considered a skin irritant.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

February 18, 1980 - March 3, 1980

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

non-GLP

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

GLP compliance:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Weight at study initiation: Body weights were 2.0 to 3.5 kg
- Housing: Individually, according to the 'Guide for the care and use of laboratory anminals' (DHEW Publication no. (NIH) 78-23, 1978).
- Diet: daily 100 grams Purina Rabbit Chow supplemented by whole oats
- Water: ad libitum

ENVIRONMENTAL CONDITIONS: standard laboratory conditions

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

Amount applied (volume or weight with unit): 0.1 mL into the right eye of each animal.

Duration of treatment / exposure:

Single instillation on day 1

Observation period (in vivo):

Eyes were examined before testing and at 24, 48 and 72 hours and at 4 and 7 days; any with persistent changes were examined again on day 10 and if needed also on day 15.

Number of animals or in vitro replicates:

6 rabbits

Details on study design:

STUDY DESIGN
0.1 mL the test material was applied into the right eye of each of 6 animals.

TREATMENT
In accordance with OECD 405 (1981). The test substance is placed in the conjunctival sac of one eye of each animal after gently pulling the lower lid away from the eyeball. The lids are then gently held together for about one second. The other eye serves as control.

REMOVAL OF TEST SUBSTANCE
-Washing: No

SCORING SYSTEM
The primary occular irritation potential of the testing material was assessed with procedure of Draize, H.J. "Appraisal of the Safety of chemicals in foods, drugs and cosmetics", Assoc. of food and drug officials of the U.S., Austin, Texas (1959).

OBSERVATIONS
- Irritation: The eyes of each animal were examined approximately 24, 48 and 72 hours and 4 and 7 days after instillation of the test substance. The irritation scores and a description of all other (local) effects were recorded. Additionally at 24 hours and 7 days, the eyes were examined with fluorescein stain under an UV light. Any persistent changes on day 7 were re-evaluated on day 10 using a hand held opthalmoscope and the fluorescein method. The irritation was assessed according to Draize as laid down in OECD 405 (1981).

Irritation parameter:

cornea opacity score

Basis:

other: mean of 6 animals

Time point:

24/48/72 h

Score:

1.5

Max. score:

4

Reversibility:

other: fully reversible 1 on day 7, 3 on day 10, 1 on day 15, 1 judged irreversible at day 10

Remarks on result:

other: mean score per animal: 1.33/2/1/1/2/1.66

Irritation parameter:

iris score

Basis:

other: mean of 6 animals

Time point:

24/48/72 h

Score:

0.39

Max. score:

2

Reversibility:

fully reversible within: 7 days

Remarks on result:

other: mean score per animal: 0/0.33/0/0.66/0.66/0.66

Irritation parameter:

conjunctivae score

Basis:

other: mean of 6 animals

Time point:

24/48/72 h

Score:

1.61

Max. score:

3

Reversibility:

fully reversible within: 7 days

Remarks on result:

other: mean score per animal: 1.33/2/1.33/1/2/2

Irritation parameter:

chemosis score

Basis:

other: mean of 6 animals

Time point:

24/48/72 h

Score:

0.89

Max. score:

4

Reversibility:

fully reversible within: 7 days

Remarks on result:

other: mean score per animal: 0.66/0/0/1/2/1.66

Irritant / corrosive response data:

All six animals developed opacity of the cornea within 24 hours after being dosed. Three rabbits had iritis at 48 hours and four at 72 hours. By Day 7 iritis had subsided in all rabbits and opacity had cleared in one eye. One rabbit had developed pannus. By Day 10 two rabbits still had opacity. In one there were ghost vessels and slight scarring adjudged irreversible. The second animal was retained until Day 15 at which time the opacity had cleared and the Draize score was 0.

Ocular Irritant response data (for the undiluted test substance)

	Time after administration
	24 hours	48 hours	72 hours	Day 4	Day 7	Day 10	15 days
Animal number	1/2/3/4/5/6
Corneal Opacity	2/2/1/1/2/1	1/2/1/1/2/2	1/2/1/1/2/2	1/2/1/2/2/2	0/1/1/1/2/1	-/0/0/0/1/1	-/-/-/-/-/0
Cornea - Area	3/4/4/4/4/4	3/4/3/4/4/4	3/3/3/4/4/4	3/3/3/4/4/4	0/1/1/1/4/1	-/0/0/0/4/1	-/-/-/-/-/0
Iris	0/0/0/0/0/0	0/0/0/1/1/1	0/1/0/1/1/1	0/1/0/1/1/1	0/0/0/0/0/0	-/0/0/0/0/0	-/-/-/-/-/0
Conjunctivae
- Redness	2/2/2/1/2/2	1/2/1/1/2/2	1/2/1/1/2/2	1/2/1/1/2/2	0/0/0/0/0/0	-/0/0/0/0/0	-/-/-/-/-/0
- Chemosis	1/0/0/0/2/2	1/0/0/1/2/2	0/0/0/2/2/1	0/0/0/2/2/1	0/0/0/0/0/0	-/0/0/0/0/0	-/-/-/-/-/0
- Discharge	2/1/1/3/3/3	1/1/0/2/3/2	0/0/0/2/2/3	0/2/0/1/1/2	0/0/0/0/0/0	-/0/0/0/0/0	-/-/-/-/-/0
Control	0/0/0/0/0/0	0/0/0/0/0/0	0/0/0/0/0/0	0/0/0/0/0/0	0/0/0/0/0/0	-/0/0/0/0/0	-/-/-/-/-/0
Weighted score	40/46/26/28/54/34	21/46/17/33/59/57	17/39/17/35/57/57	17/43/17/53/55/55	0/5/5/5/40/5	-/0/0/0/20/5	-/-/-/-/-/0
Total	228	233	222	240	60	25	0
Primary ocular irritation score	38.00	38.83	37.00	40.00	10.00	4.17	0

Mean scores per animal over three timepoints (24-72 hours):

Corneal Opacity: 1.33/2/1/1/2/1.66: this means that 6/6 have a score >= 1 (CLP limit for classification as irritant, if at least in 2 of 3 animals)

Iritis: 0/0.33/0/0.66/0.66/0.66: this means that 0/6 have a score >= 1 (CLP limit for classification as irritant, if at least in 2 of 3 animals)

Redness: 1.33/2/1.33/1/2/2: this means that 3/6 animals have scores of >= 2 (CLP limit for classification as irritant, if at least in 2 of 3 animals)

Chemosis: 0.66/0/0/1/2/1.66: this means that 1/6 animals have a score of >= 2 (CLP limit for classification as irritant, if at least in 2 of 3 animals)

Interpretation of results:

other: Serious Eye Damage, Category 1

Remarks:

in accordance with EU CLP (EC 1272/2008 and its updates)

Conclusions:

Under the conditions of this study, the mean eye irritation scores observed in the animals following grading at 24, 48 and 72 hours indicate an eye irritation potential. However, as in one animal irreversible effects were observed, Dimeth Cyclormol is considered to cause serious eye damage.

Executive summary:

Dimeth Cyclormol was tested in an eye irritation test in rabbits similar to OECD 405 but non-GLP and therefore scored Klimish 2. The right eye of six rabbits was treated with 0.1 ml of the undiluted test article.

Cornea opacity: Mean grade of 24, 48 and 72 hours is 1.5 (6/6 eyes scored >= 1; less than 4/6 eyes scored 3) indicating eye irritation. Opacity occurred within 24 hours after being dosed and one rabbit developed pannus. By day 10 two rabbits still had opacity. In one animal ghost vessels and slight scarring were adjudged irreversible and scoring was terminated. The opacity seen in the other animal had cleared by day 15.

Iris: Mean score during during 24 to 72 hours is 0.39 (less than 4/6 eyes scored 1). By day 7 iritis had subsided in all rabbits.

Conjuctivae: Mean score during 24 to 72 hours is 1.61 (less than 4/6 eyes scored 2).

Conjunctival chemosis: Mean score during 24 to 72 hours is 0.89 (less than 4/6 eyes scored 2).

In conclusion: Based solely on the scores the substance is an eye irritant. In view of the one animal showing irreversible effects, scar formation, the final conclusion is that the substance causes serious eye damage.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro eye irritation study does not need to be conducted because adequate data from an in vivo eye irritation study are available

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Additional information

Skin corrosion

Skin corrosion is not expected because the substance does not contain acidic or base groups that indicate corrosion. There are also no other reactive groups identified that could result in corrosion. Furthermore, in the acute dermal toxicity study in male and female rats (IFF, 1980), after single application of 5000 mg/kg bw of the undiluted test substance, no indication of corrosion was observed at the end of the 14 day observation period (see Acute toxicity Endpoint summary).

Skin irritation

This in vitro skin irritation study to assess the irritation potential of Dimeth Cyclormol by means of the Human Skin Model Test, was performed in line with OECD439 and GLP principles. The test item did not directly reduce MTT, and did not cause colour interference. Triplicate tissues of the human skin model EpiSkin™ were treated with the test item, the negative or the positive control for 15 minutes. After washing, and further incubation for approximately 42 hours, tissue viability was determined by measuring MTT reduction. The viability and OD values of respectively the positive and negative control tissues were considered valid. After treatment with the test item the mean relative viability decreased to 6.0%. The value is below the threshold for irritancy of ≤ 50%. Therefore, the test item is considered a skin irritant.

Eye irritation/damage

Dimeth Cyclormol was tested in an eye irritation test in rabbits similar to OECD 405 but non-GLP and therefore scored Klimish 2. The right eye of six rabbits was treated with 0.1 ml of the undiluted test article.

Cornea opacity: Mean grade of 24, 48 and 72 hours is 1.5 (6/6 eyes scored >= 1; less than 4/6 eyes scored 3) indicating eye irritation. Opacity occurred within 24 hours after being dosed and one rabbit developed pannus. By day 10 two rabbits still had opacity. In one animal ghost vessels and slight scarring were adjudged irreversible and scoring was terminated. The opacity seen in the other animal had cleared by day 15.

Iris: Mean score during during 24 to 72 hours is 0.39 (less than 4/6 eyes scored 1). By day 7 iritis had subsided in all rabbits.

Conjuctivae: Mean score during 24 to 72 hours is 1.61 (less than 4/6 eyes scored 2).

Conjunctival chemosis: Mean score during 24 to 72 hours is 0.89 (less than 4/6 eyes scored 2).

In conclusion: Based solely on the scores the substance is an eye irritant. In view of the one animal showing irreversible effects, scar formation, the final conclusion is that the substance causes serious eye damage.

Justification for classification or non-classification

Based on the available data, Dimeth Cyclormol needs to be classified for skin irritation (Skin Irritant, Category 2 / H315: Causes skin irritation) and for serious eye damage (Serious Eye Damage, Category 1 / H318: Causes serious eye damage) in accordance with the criteria outlined in EU CLP (1272/2008/EC and its updates).