Registration Dossier

Administrative data

Description of key information

Skin irritation: not irritating , weight of evidence approach using in vivo (eq. OECD 404, Rel.4) and in vitro (OECD 439, GLP, read-across, Rel.1) studies.

Eye irritation: not irritating (US FHSA method, K, Rel. 2).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From February 18 to 24, 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed according to OECD test guideline No. 439 and in compliance with GLP.
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2013
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Remarks:
UK GLP Compliance Program (inspected on March 12 to 14, 2014 / Signed on May 12, 2014)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from multiple donors
Source strain:
not specified
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™ Reconstructed Human Epidermis Model Kit, SkinEthic Laboratories, Lyon, France
- Tissue batch number(s): 14-EKIN-005
- Production date: 18 February 2014
- Shipping date: 18 February 2014
- Delivery date: 18 February 2014
- Date of initiation of testing: 18-24 February 2014
- Expiry date: 24 February 2014

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: ambient temperature
- Temperature of post-treatment incubation: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Not reported. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item.
- Observable damage in the tissue due to washing: none
- Modifications to validated SOP: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/L
- Incubation time: 3 hours
- Spectrophotometer: Anthos 2001 microplate reader
- Wavelength: 562 nm
- Filter: without reference filter
- Filter bandwidth: not applicable
- Linear OD range of spectrophotometer: not reported

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability:
- Barrier function: 2.2 mg/mL ( ≥ 1.5 mg/mL)
- Morphology: well differenciated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum
- Contamination:
Absence of HIV1 and 2 antobodies, hepatitis C antibodies, hepatitis B antigen HBs
Absence of bacteria, fungus and mycoplasma
- Reproducibility: not reported

NUMBER OF REPLICATE TISSUES: triplicate

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE : the test item did not directly reduce MTT

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the viability after the 15-min exposure period followed by the 42h post-exposure period is less or equal to 50%
- The test substance is considered to be non-irritant to skin if the viability after the 15-min exposure period followed by the 42h post-exposure period 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% w/v
Duration of treatment / exposure:
15 minutes at room temperature
Duration of post-treatment incubation (if applicable):
42 h
Number of replicates:
Triplicate
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
63.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: none
- Direct-MTT reduction: the test item was not able to directly reduce MTT
- Colour interference with MTT: no colour interference were observed

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD562 for the negative control treated tissues was 1.013 and the standard deviation value of the percentage viability was 2.4%. The negative control acceptance criterion was therefore satisfied.
- Acceptance criteria met for positive control: The relative mean tissue viability for the positive control treated tissues was 5.3% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 1.1%. The positive control acceptance criterion was therefore satisfied.
- Acceptance criteria met for variability between replicate measurements:
- Range of historical values if different from the ones specified in the test guideline: The standard deviation calculated from individual percentage tissue viabilities of the three identically treated test item tissues was 8.4%. The test item acceptance criterion was therefore satisfied.
For the previous 35 experiments conducted between May 2013 and February 2014 using this test method, the mean OD of the positive control was 0.084 ± 0.036 and the mean percentage viability was 9.6 ± 4.4. In this same period the mean OD of the negative control was 0.898 ±0.106.
The positive control reflected the ability to respond to an irritant test item under the conditions of the test. The negative control in this study was greater than the mean OD of the negative control in the previous 35 experiments conducted with this test method.

Table 7.3.1/1: Mean OD562 Values and Percentage Viabilities for the Negative Control Item, Positive Control Item and Test Item

 

Item

OD562 of tissues

Mean OD562 of triplicate tissues

± SD of OD562

Relative individual tissue viability (%)

Relative mean viability (%)

± SD of Relative mean viability (%)

Negative Control Item

1.034

1.013

0.024

102.1

100*

2.4

1.018

100.5

0.986

97.3

Positive Control Item

0.062

0.054

0.011

6.1

5.3

1.1

0.042

4.1

0.058

5.7

Test Item

0.687

0.646

0.085

67.8

63.7

8.4

0.702

69.3

0.548

54.1

SD=        Standard deviation

*=         The mean viability of the negative control tissues is set at 100%

 

Interpretation of results:
GHS criteria not met
Conclusions:
Under the test conditions, test item was classified as non-irritant according to the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.
Executive summary:

An in vitro skin irritation study was performed according to the OECD Guideline 439 and in compliance with GLP, using the EPISKINTM reconstructed human epidermis model. Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post‑exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre‑labeled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT‑loaded tissues. At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µL samples were transferred to the appropriate wells of a pre‑labeled 96‑well plate. The optical density was measured at 562 nm.

Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues). The relative mean viability of the test item treated tissues was 63.7% after the 15‑minute exposure period and 42 hours post‑exposure incubation period.

 

The relative mean tissue viability for the positive control treated tissues was 5.3% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 1.1%. The positive control acceptance criterion was therefore satisfied. The mean OD562 for the negative control treated tissues was 1.013 and the standard deviation value of the percentage viability was 2.4%. The negative control acceptance criterion was therefore satisfied. The standard deviation calculated from individual percentage tissue viabilities of the three identically treated test item tissues was 8.4%. The test item acceptance criterion was therefore satisfied.

 

Under the test conditions, test item was classified as non-irritant according to the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

This study is considered as acceptable and satisfies the requirement for skin irritation endpoint.

Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From May 6 to June 8, 1984
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Remarks:
Does not meet important criteria of today standard methods: the exposure period was 24 hours instead of 4 hours, occlusive dressing was used instead of semi-occlusive dressing, and the animals were observed for 3 days instead of 14 days although complete reversibility was not achieved. This study was thus not sufficient on its own to conclude on the skin irritation potential.
Qualifier:
equivalent or similar to guideline
Guideline:
other: Section 1500.41 - Federal Hazardous Substance Act Regulations - 16 CFR
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
not specified
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Ace Animals, Inc., Boyertown, PA.
- Age at study initiation: no data
- Weight at study initiation: 2.3-3.0 kg bw.
- Fasting period before study: no.
- Housing: individually, in stainless steel cages with elevated wire mesh flooring.
- Diet (e.g. ad libitum): Wayne 15% Rabbit ration ad libitum.
- Water (e.g. ad libitum): tap water ad libitum.
- Acclimation period: appropriate time.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15.5 - 23
- Humidity (%): 40-45
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: no data
Type of coverage:
occlusive
Preparation of test site:
other: 2 sites: 1 clipped and 1 abraded
Vehicle:
unchanged (no vehicle)
Controls:
not required
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 mL
Duration of treatment / exposure:
24 hours
Observation period:
72 hours
Number of animals:
6
Details on study design:
TEST SITE
- Area of exposure: back
- % coverage: no data
- Type of wrap if used: gauza patches were placed over the treated areas and an impervious material was wrapped snugly around the trunks of animals to hold the patches in place.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): no (wipping only)
- Time after start of exposure: 24 hours

SCORING SYSTEM: Draize scale, observations at 24 and 72 hours.
Irritation parameter:
erythema score
Basis:
animal #1
Time point:
other: 24/72 h
Score:
2
Max. score:
4
Reversibility:
not fully reversible within: 72 hrs
Remarks on result:
probability of moderate irritation
Remarks:
no 48 h scoring
Irritation parameter:
erythema score
Basis:
animal: #2, #4 and #5
Time point:
other: 24/72 h
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 72 hrs
Remarks on result:
probability of weak irritation
Remarks:
no 48 h scoring
Irritation parameter:
erythema score
Basis:
animal: #3 and #6
Time point:
other: 24/72 h
Score:
0.5
Max. score:
4
Reversibility:
fully reversible within: 72 hrs
Remarks on result:
probability of weak irritation
Remarks:
no 48 h scoring
Irritation parameter:
edema score
Basis:
animal: #1, #2 and #5
Time point:
other: 24/72 h
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 72 hrs
Remarks on result:
probability of weak irritation
Remarks:
no 48h scoring
Irritation parameter:
edema score
Basis:
animal #3
Time point:
24 h
Score:
0.5
Max. score:
4
Reversibility:
fully reversible within: 72 hrs
Remarks on result:
probability of weak irritation
Remarks:
no 48h scoring
Irritation parameter:
edema score
Basis:
animal #5
Time point:
24 h
Score:
2
Max. score:
4
Reversibility:
fully reversible within: 72 hrs
Remarks on result:
probability of weak irritation
Remarks:
no 48h scoring
Irritation parameter:
edema score
Basis:
animal #6
Time point:
24 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Remarks:
no 48h scoring
Irritant / corrosive response data:
- Intact skin:
Well defined erythema in 4/6 animals at the 24-hour reading, still visible in 1/6 animal at the 72-hour reading. Very slight erythema in 2/6 animals at the 24-hour reading, fully reversible within 72 hours.
Severe oedema in 1/6, moderate oedema in 3/6, slight oedema in 1/6 at the 24-hour reading. All fully reversible within 72 hours.
- Abraded skin:
Well defined erythema in 4/6 animals at the 24-hour reading, still visible in 1/6 animal at the 72-hour reading. Very slight erythema in 2/6 animals at the 24-hour reading, fully reversible within 72 hours.
Severe oedema in 3/6, moderate oedema in 1/6, slight oedema in 1/6 at the 24-hour reading. All fully reversible within 72 hours.
Other effects:
None

Table 7.3.1/1: Intact skin - Mean irritant/corrosive response data for all animals at each observation time up to removal of animals from the test

Score at time point / Reversibility

Erythema

Max. score 4

Oedema

Max. score 4

24 h

1.67

1.83

72 h

0.33

0.00

Average 24h and 72h

1

0.9

Reversibility*)

n.c. (1 animal)

c.

Average time for reversion**

72 hours

72 hours

 *) Reversibility: c. = completely reversible; n.c. = not completely reversible; n. = not reversible

**): correspond to the last day for which skin irritation signs in the last animal were observed

 

Table 7.3.1/2: Intact skin - Irritant/corrosive response data for individual animals at each observation time up to removal of animals from the test

 

Score at time point / Reversibility

Erythema

Max. score 4

Oedema

Max. score 4

24 h

2 / 2 / 1 / 2 / 2 / 1

2 / 2 / 1 / 2 / 4 / 0

72 h

2 / 0 / 0 / 0 / 0 / 0

0 / 0 / 0 / 0 / 0 / 0

Average 24h and 72h

2 / 1 / 0.5 / 1 / 1 / 0.5

1 / 1 / 0.5 / 1 / 2 / 0

Reversibility*)

n.c. (animal #1)

c.

Average time for reversion**

72 hours

72 hours

 *) Reversibility: c. = completely reversible; n.c. = not completely reversible; n. = not reversible

**): correspond to the last day for which skin irritation signs in the last animal were observed

 

Table 7.3.1/3: Abraded skin - Mean irritant/corrosive response data for all animals at each observation time up to removal of animals from the test

 

Score at time point / Reversibility

Erythema

Max. score 4

Oedema

Max. score 4

24 h

1.67

2.50

72 h

0.33

0.00

Average 24h and 72h

1

1.25

Reversibility*)

n.c. (1 animal)

c.

Average time for reversion**

72 hours

72 hours

 *) Reversibility: c. = completely reversible; n.c. = not completely reversible; n. = not reversible

**): correspond to the last day for which skin irritation signs in the last animal were observed

 

Table 7.3.1/4: Abraded skin - Irritant/corrosive response data for individual animals at each observation time up to removal of animals from the test

Score at time point / Reversibility

Erythema

Max. score 4

Oedema

Max. score 4

24 h

2 / 2 / 1 / 2 / 2 / 1

4 / 2 / 1 / 4 / 4 / 0

72 h

2 / 0 / 0 / 0 / 0 / 0

0 / 0 / 0 / 0 / 0 / 0

Average 24h and 72h

2 / 1 / 0.5 / 1 / 1 / 0.5

2 / 1 / 0.5 / 2 / 2 / 0

Reversibility*)

n.c. (animal #1)

c.

Average time for reversion**

72 hours

72 hours

 *) Reversibility: c. = completely reversible; n.c. = not completely reversible; n. = not reversible

**): correspond to the last day for which skin irritation signs in the last animal were observed

Interpretation of results:
study cannot be used for classification
Conclusions:
Under the test conditions, the test material induced a slight irritation being reversible in 5 out of 6 animals within the end of the observation period (i.e. 72 hours). This study was not sufficient on its own to conclude on the skin irritation potential.
Executive summary:

In a dermal irritation study performed similarly to the OECD test guideline No. 404, 0.5 mL of undiluted test material was dermally applied on two skin sites (one clipped one abraded) of the back of 6 New Zealand White rabbits.Test sites were covered with an occlusive dressing for 24 hours. Animals were then observed for 72 hours for oedema and erythema. 

Skin irritation was assessed and scored according to the Draize scale 24 and 72 hours after the application.

The mean scores calculated over all the animals tested within 2 scoring times (24 and 72 hours) were 1.0 for erythema and 0.9 for oedema.

Under the test conditions, the test material induced a slight irritation being reversible in 5 out of 6 animals within the end of the observation period (i.e. 72 hours). This study was not sufficient on its own to conclude on the skin irritation potential.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
Cf. IUCLID section 13.2

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source and target substances have similar Physico-Chemical, and Toxicological properties because of their structural similarity.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The target and source substances are both multi-constituents, as reaction mass of stereoisomers of the same substance.
The target and source substances are structurally related, in that both are a reaction mass of stereoisomers of 1-(2,2,6-Trimethylcyclohexyl)hexan-3-ol. They differ by the number of constituents. The two trans 1R,6S diastereoisomers, with both 3R and 3S hydroxyl group, of the source substance are constituents of the target substance, which contains also the trans 1S,6R pair (i.e. 2 pairs of enantiomers).

3. ANALOGUE APPROACH JUSTIFICATION
Based on structural similarity (constituents of the source and the target substance are from the same pool of substance, i.e stereoisomers of 1-(2,2,6-Trimethylcyclohexyl)hexan-3-ol) it is considered appropriate and scientifically justified to read-across the data from the source to the target substance.
The study design (OECD 439, GLP) is adequate and reliable for the purpose of the prediction based on read-across. The test material used represents the source substance as described in the hypothesis in terms of purity and impurities. The results of the studies are adequate for the purpose of classification and labelling.
Therefore, based on the considerations above, it can be concluded that the result of the in vitro skin irritation test conducted with the source substance is highly likely to predict the properties of the target substance and is considered as adequate to fulfil the information requirement of Annex VII, 8.1.

4. DATA MATRIX
Cf. IUCLID section 13.2
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across: supporting information
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
63.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: none
- Direct-MTT reduction: the test item was not able to directly reduce MTT
- Colour interference with MTT: no colour interference were observed

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD562 for the negative control treated tissues was 1.013 and the standard deviation value of the percentage viability was 2.4%. The negative control acceptance criterion was therefore satisfied.
- Acceptance criteria met for positive control: The relative mean tissue viability for the positive control treated tissues was 5.3% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 1.1%. The positive control acceptance criterion was therefore satisfied.
- Acceptance criteria met for variability between replicate measurements:
- Range of historical values if different from the ones specified in the test guideline: The standard deviation calculated from individual percentage tissue viabilities of the three identically treated test item tissues was 8.4%. The test item acceptance criterion was therefore satisfied.
For the previous 35 experiments conducted between May 2013 and February 2014 using this test method, the mean OD of the positive control was 0.084 ± 0.036 and the mean percentage viability was 9.6 ± 4.4. In this same period the mean OD of the negative control was 0.898 ±0.106.
The positive control reflected the ability to respond to an irritant test item under the conditions of the test. The negative control in this study was greater than the mean OD of the negative control in the previous 35 experiments conducted with this test method.

Table 7.3.1/1: Mean OD562 Values and Percentage Viabilities for the Negative Control Item, Positive Control Item and Test Item

 

Item

OD562 of tissues

Mean OD562 of triplicate tissues

± SD of OD562

Relative individual tissue viability (%)

Relative mean viability (%)

± SD of Relative mean viability (%)

Negative Control Item

1.034

1.013

0.024

102.1

100*

2.4

1.018

100.5

0.986

97.3

Positive Control Item

0.062

0.054

0.011

6.1

5.3

1.1

0.042

4.1

0.058

5.7

Test Item

0.687

0.646

0.085

67.8

63.7

8.4

0.702

69.3

0.548

54.1

SD=        Standard deviation

*=         The mean viability of the negative control tissues is set at 100%

 

Interpretation of results:
GHS criteria not met
Conclusions:
Under the test conditions, the source substance item was classified as non-irritant according to the Regulation (EC) No. 1272/2008 (CLP) and to the GHS. The same conclusion applies to the target substance.
Executive summary:

An in vitro skin irritation study was performed according to the OECD Guideline 439 and in compliance with GLP, using the EPISKINTM reconstructed human epidermis model. Triplicate tissues were treated with the source substance for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post‑exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre‑labeled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT‑loaded tissues. At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µL samples were transferred to the appropriate wells of a pre‑labeled 96‑well plate. The optical density was measured at 562 nm.

Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues). The relative mean viability of the test item treated tissues was 63.7% after the 15‑minute exposure period and 42 hours post‑exposure incubation period.

 

The relative mean tissue viability for the positive control treated tissues was 5.3% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 1.1%. The positive control acceptance criterion was therefore satisfied. The mean OD562 for the negative control treated tissues was 1.013 and the standard deviation value of the percentage viability was 2.4%. The negative control acceptance criterion was therefore satisfied. The standard deviation calculated from individual percentage tissue viabilities of the three identically treated test item tissues was 8.4%. The test item acceptance criterion was therefore satisfied.

 

Under the test conditions, the source substance was classified as non-irritant according to the Regulation (EC) No. 1272/2008 (CLP) and to the GHS. The same conclusion applies to the target substance. Read-across justification is attached to Iuclid section 13.

This study is considered as acceptable and satisfies the requirement for skin irritation endpoint.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From August 6 to 13, 1984
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Study comparable to OECD test guideline No. 405 with deviations not affecting the integrity of the result. Even if full reversibility was not achieved for conjunctival reactions within the observation period of 7 days, only minimal responses (score = 1) persisted at the end of the observation period. Complete reversibility would have probably occurred if the observation period was extended to 21 days, as required by the current OECD TG 405.
Qualifier:
according to guideline
Guideline:
other: Section 1500.42 - Federal Hazardous Substances Act Regulations - 16 CFR
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Ace Animals, Inc., Boyertown, USA
- Housing: Animals were housed individually in stainless steel cages with elevated wire mesh flooring.
- Diet: Wayne 15 % Rabbit Ration, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: Animals were acclimated to the laboratory for an appropriate time prior to dosing.

ENVIRONMENTAL CONDITIONS
- Temperature: 60-75 °F
- Humidity: 40-45 %
- Photoperiod: 12 h light/12 h dark

IN-LIFE DATES: From: August 6, 1984 To: August 13, 1984
Vehicle:
unchanged (no vehicle)
Controls:
other: untreated eye served as control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL
Duration of treatment / exposure:
- Test material was not washed from the eyes of animals.
Observation period (in vivo):
7 days
Number of animals or in vitro replicates:
6 animals
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): No

SCORING SYSTEM: According to the 'Draize technique'

TOOL USED TO ASSESS SCORE: No data
Irritation parameter:
cornea opacity score
Basis:
mean
Remarks:
(6 animals)
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
mean
Remarks:
(6 animals)
Time point:
24/48/72 h
Score:
0
Max. score:
2
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Basis:
mean
Remarks:
(6 animals)
Time point:
24/48/72 h
Score:
1
Max. score:
3
Reversibility:
not fully reversible within: 7 days
Remarks on result:
probability of weak irritation
Irritation parameter:
chemosis score
Basis:
mean
Remarks:
(6 animals)
Time point:
24/48/72 h
Score:
1
Max. score:
4
Reversibility:
not fully reversible within: 7 days
Remarks on result:
probability of weak irritation
Irritant / corrosive response data:
- Animals showed conjunctival reactions (redness, chemosis and discharge) which were not fully reversible within 7 days.
Other effects:
None

Table 7.3.2/1: Mean eye irritation response data of 6 animals at each observation time

 

Score at time point / Reversibility

Cornea

Iris

(/2)

Conjunctivae

Opacity

(/4)

Area

(/4)

Redness

(/3)

Chemosis

(/4)

Discharge

(/3)

24 h

0

0

0

1

1

1

48 h

0

0

0

1

1.17

0.5

72 h

0

0

0

1

0.83

0.17

Mean

0

0

0

1

1

0.56

Reversibility

-

-

-

Not completely

reversible

Not Completely reversible

Not Completely reversible

Table 7.3.2/2: Eye irritation response data for each animal at each observation time

Score at time point

Cornea

Iris

(/2)

Conjunctivae

Opacity

(/4)

Area

(/4)

Redness

(/3)

Chemosis

(/4)

Discharge

(/3)

24 h

0 / 0 / 0 / 0 / 0 / 0

0 / 0 / 0 / 0 / 0 / 0

0 / 0 / 0 / 0 / 0 / 0

1 / 1 / 1 / 1 /1 / 1

1 / 2 / 1 / 1 / 1 / 0

0 / 1 / 1 / 2 / 1 / 1

48 h

0 / 0 / 0 / 0 / 0 / 0

0 / 0 / 0 / 0 / 0 / 0

0 / 0 / 0 / 0 / 0 / 0

1 / 1 / 1 / 1 / 1 / 1

1 / 2 / 1 / 1 / 1 / 1

0 / 1 / 1 / 1 / 0 / 0

72 h

0 / 0 / 0 / 0 / 0 / 0

0 / 0 / 0 / 0 / 0 / 0

0 / 0 / 0 / 0 / 0 / 0

1 / 1 / 1 / 1 / 1 / 1

1 / 2 / 1 / 1 / 0 / 0

0 / 0 / 0 / 0 / 1 / 0

Day 4

0 / 0 / 0 / 0 / 0 / 0

0 / 0 / 0 / 0 / 0 / 0

0 / 0 / 0 / 0 / 0 / 0

1 / 1 / 1 / 1 / 1 / 1

1 / 2 / 0 / 0 / 0 / 0

0 / 0 / 0 / 1 / 0 / 0

Day 7

0 / 0 / 0 / 0 / 0 / 0

0 / 0 / 0 / 0 / 0 / 0

0 / 0 / 0 / 0 / 0 / 0

1 / 1 / 1 / 1 / 0 / 0

1 / 1 / 0 / 0 / 0 / 0

0 / 1 / 0 / 0 / 0 / 0

Average 24, 48 and 72 h

0 / 0 / 0 / 0 / 0 / 0

0 / 0 / 0 / 0 / 0 / 0

0 / 0 / 0 / 0 / 0 / 0

1 / 1 / 1 / 1 / 1 / 1

1 / 2 / 1 / 1 / 0.67 / 0.33

0 / 0.67 / 0.67 / 1 / 0.67 / 0.33

Reversibility

 -

-

-

Not completely

reversible

Not Completely reversible

Not Completely reversible

Interpretation of results:
GHS criteria not met
Conclusions:
The calculated mean score for each animal within 3 scoring times (24, 48 and 72 h) were 1 for redness, 1 for chemosis and 0.0 for iris lesions and 0.0 for corneal opacity. Animals showed conjunctival reactions (redness, chemosis and discharge) which were not fully reversible within 7 days.
Even if full reversibility was not achieved for conjunctival reactions within the observation period of 7 days, only minimal responses (score = 1) persisted at the end of the observation period on 4 out of 6 animals. Complete reversibility would have probably occurred if the observation period was extended to 21 days, as required by the current OECD TG 405;   Under the test conditions, the test material is not classified as irritating to the eyes according to the Regulation EC No. 1272/2008 (CLP) and to the GHS.

Executive summary:

In an eye irritation study performed according to US-FHSA method, 0.1 mL of undiluted test material was instilled into the conjunctival sac of one eye of 6 New Zealand White rabbits while the remained untreated eye served as control. The eyes were not rinsed after the instillation of test material. Animals were observed at 24, 48 and 72 h after instillation of test material into eyes and then on Days 4 and 7. The reactions in the conjunctivae (redness, chemosis and discharge), the iris and the cornea (opacity and area involved) were scored according to the Draize scale.  

The calculated mean score for each animal within 3 scoring times (24, 48 and 72 h) were 1 for redness, 1 for chemosis and 0.0 for iris lesions and 0.0 for corneal opacity. Animals showed conjunctival reactions (redness, chemosis and discharge) which were not fully reversible within 7 days.

Even if full reversibility was not achieved for conjunctival reactions within the observation period of 7 days, only minimal responses (score = 1) persisted at the end of the observation period on 4 out of 6 animals. Complete reversibility would have probably occurred if the observation period was extended to 21 days, as required by the current OECD TG 405;   Under the test conditions, the test material is not classified as irritating to the eyes according to the Regulation EC No. 1272/2008 (CLP) and to the GHS.

This study is considered as acceptable and satisfies the requirement for acute eye irritation endpoint.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation / corrosion:

A study was available on the target substance but was not sufficiently robust to be the key study (Biosearch, 1984, Rel.4). Indeed, deviations from OECD Test Guideline No. 404 were noted: the exposure period was 24 hours instead of 4 hours, occlusive dressing was used instead of semi-occlusive dressing, and the animals were observed for 3 days instead of 14 days although complete reversibility was not achieved. This study was thus not sufficient on its own to conclude on the skin irritation potential. Therefore a weight of evidence approach using the studies on the targer and on the source substances was followed. The source substance is considered adequate for read-across purposes as the data relates to a mixture composed of the same isomers that the source substance, but at different ratios (see Iuclid section 13 for additional justification).

A reliable study was identified on the source substance (Harlan, 2014, rel. 2). The skin irritation potential of the test material was evaluated using the EPISKINTM reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. This test was designed to be compatible with the Method B.46 of Commission Regulation (EC) No. 440/2008/EC and was performed in compliance with GLP. The quality criteria required for acceptance of results in the test were satisfied.The relative mean viability of the test item treated tissues was 63.7± 8.4 %, after the 15‑minute exposure period. With a tissue viability > 50%, the test material was not considered to be irritant to skin.

Taken together within a weight-of-evidence approach, data suggest that the substance should not be classified for skin irritation.

Eye irritation:

A key study was identified on the target substance (Biosearch, 1984, Rel.2). In this eye irritation study performed according to US-FHSA method, 0.1 mL of undiluted test material was instilled into the conjunctival sac of one eye of 6 New Zealand White rabbits while the remained untreated eye served as control. The eyes were not rinsed after the instillation of test material. Animals were observed at 24, 48 and 72 h after instillation of test material into eyes and then on Days 4 and 7. The reactions in the conjunctivae (redness, chemosis and discharge), the iris and the cornea (opacity and area involved) were scored according to the Draize scale.

The calculated mean score for each animal within 3 scoring times (24, 48 and 72 h) were 1 for redness, 1 for chemosis and 0.0 for iris lesions and 0.0 for corneal opacity.

Even if full reversibility was not achieved for conjunctival reactions within the observation period of 7 days, only minimal responses (score = 1) persisted at the end of the observation period on 4 out of 6 animals. Complete reversibility would have probably occurred if the observation period was extended to 21 days, as required by the current OECD TG 405.

Justification for classification or non-classification

Harmonised classification:

The substance has no harmonised classification according to the Regulation (EC) No. 1272/2008 (CLP).

 

Self-classification:

Based on the available information no additional self-classification is proposed regarding both skin and eye irritation according to the CLP and to the GHS.

No data was available regarding respiratory irritation, however the substance not being classified for skin and eye irritation, no classification is expected for respiratory irritation.