Registration Dossier

Administrative data

Description of key information

Skin irritation/corrosion: Test method 16 CFR 1500.41. GLP study. The substance was determined to be a dermal irritant.

Eye irritation: Test method according to an in-house validated in-vitro Enucleated Eye Test. The substance was determined to be a severe eye irritant.

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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Results recorded at 24 and 72 hours only.
Qualifier:
according to guideline
Guideline:
other: FHSA standard 16 CFR 1500.41
GLP compliance:
yes (incl. QA statement)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Millbrook Breeding Labs
- Age at study initiation: Young adult
- Weight at study initiation: 2.1 - 2.7 kg
- Housing: 1/cage in suspended cages
- Diet (e.g. ad libitum): Fresh PMI Rabbit Chow
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 1 w eek

ENVIRONMENTAL CONDITIONS
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle
Type of coverage:
occlusive
Preparation of test site:
shaved
Vehicle:
unchanged (no vehicle)
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight w ith unit): 0.5 ml
Duration of treatment / exposure:
24 hours
Observation period:
72 hours
Number of animals:
1 male/ 2 female
Details on study design:
TEST SITE
- Area of exposure: 2.5 x 2.5 cm
- Type of wrap if used: Plastic

REMOVAL OF TEST SUBSTANCE
- Washing (if done): gentle wiping
SCORING SYSTEM: Draize
Irritation parameter:
erythema score
Remarks:
Sample 1500-1, animal 1
Basis:
mean
Time point:
24/48/72 h
Score:
3
Max. score:
4
Reversibility:
not fully reversible within: 72 hours
Remarks on result:
other:
Remarks:
time point only at 24h and 72 h.
Irritation parameter:
erythema score
Remarks:
Sample 1500-1, animals 2 and 3
Basis:
mean
Time point:
24/48/72 h
Score:
3.5
Max. score:
4
Reversibility:
not fully reversible within: 72 hours
Remarks on result:
other:
Remarks:
time point only at 24h and 72 h.
Irritation parameter:
edema score
Remarks:
Sample 1500-1, animal 1
Basis:
mean
Time point:
24/48/72 h
Score:
2.5
Max. score:
3
Reversibility:
not fully reversible within: 72 hours
Remarks on result:
other:
Remarks:
time point only at 24h and 72 h.
Irritation parameter:
edema score
Remarks:
Sample 1500-1, animals 2 and 3
Basis:
mean
Time point:
24/48/72 h
Score:
4
Max. score:
4
Reversibility:
not fully reversible within: 72 hours
Remarks on result:
other:
Remarks:
time point only at 24h and 72 h.
Irritation parameter:
erythema score
Remarks:
Sample AF6155, animals 1, 2 and 3
Basis:
mean
Time point:
24/48/72 h
Score:
3
Max. score:
4
Reversibility:
not fully reversible within: 72 hours
Remarks on result:
other:
Remarks:
time point only at 24h and 72 h.
Irritation parameter:
edema score
Remarks:
Sample AF6155, animal 1
Basis:
mean
Time point:
24/48/72 h
Score:
2
Max. score:
2
Reversibility:
not fully reversible within: 72 hours
Remarks on result:
other:
Remarks:
time point only at 24h and 72 h.
Irritation parameter:
edema score
Remarks:
Sample AF6155, animal 2
Basis:
mean
Time point:
24/48/72 h
Score:
3.5
Max. score:
4
Reversibility:
not fully reversible within: 72 hours
Remarks on result:
other:
Remarks:
time point only at 24h and 72 h.
Irritation parameter:
edema score
Remarks:
Sample AF6155, animal 3
Basis:
mean
Time point:
24/48/72 h
Score:
3
Max. score:
3
Reversibility:
not fully reversible within: 72 hours
Remarks on result:
other:
Remarks:
time point only at 24h and 72 h.
Irritation parameter:
erythema score
Remarks:
Sample 1600-1, animals 1, 2 and 3
Basis:
mean
Time point:
24/48/72 h
Score:
1.5
Max. score:
2
Reversibility:
not fully reversible within: 72 hours
Remarks on result:
other:
Remarks:
time point only at 24h and 72 h.
Irritation parameter:
edema score
Remarks:
Sample 1600-1, animal 1
Basis:
mean
Time point:
24/48/72 h
Score:
0.5
Max. score:
1
Reversibility:
not fully reversible within: 72 hours
Remarks on result:
other:
Remarks:
time point only at 24h and 72 h.
Irritation parameter:
edema score
Remarks:
Sample 1600-1, animal 2
Basis:
mean
Time point:
24/48/72 h
Score:
1
Max. score:
1
Reversibility:
not fully reversible within: 72 hours
Remarks on result:
other:
Remarks:
time point only at 24h and 72 h.
Irritation parameter:
edema score
Remarks:
Sample 1600-1, animal 3
Basis:
mean
Time point:
24/48/72 h
Score:
1.5
Max. score:
2
Reversibility:
not fully reversible within: 72 hours
Remarks on result:
other:
Remarks:
time point only at 24h and 72 h.
Irritation parameter:
erythema score
Remarks:
Sample 1650-1, animals 1 and 3)
Basis:
mean
Time point:
24/48/72 h
Score:
1.5
Max. score:
2
Reversibility:
not fully reversible within: 72 hours
Remarks on result:
other:
Remarks:
time point only at 24h and 72 h.
Irritation parameter:
erythema score
Remarks:
Sample 1650-1, animal 2
Basis:
mean
Time point:
24/48/72 h
Score:
1
Max. score:
1
Reversibility:
not fully reversible within: 72 hours
Remarks on result:
other:
Remarks:
time point only at 24h and 72 h.
Irritation parameter:
edema score
Remarks:
Sample 1650-1
Basis:
mean
Time point:
other: 24 and 72 hours
Score:
0
Max. score:
0
Remarks on result:
other:
Remarks:
time point only at 24h and 72 h.
Irritation parameter:
edema score
Remarks:
Sample 1650-1)
Basis:
mean
Time point:
24/48/72 h
Score:
1
Max. score:
1
Reversibility:
not fully reversible within: 72 hours
Remarks on result:
other:
Remarks:
time point only at 24h and 72 h.
Irritation parameter:
edema score
Remarks:
Sample 1650-1)
Basis:
mean
Time point:
24/48/72 h
Score:
1.5
Max. score:
2
Reversibility:
not fully reversible within: 72 hours
Remarks on result:
other:
Remarks:
time point only at 24h and 72 h.
Interpretation of results:
irritating
Conclusions:
Samples 1500-1 and AF6155 were irritating, how ever, samples 1600-1 and 1650-1 were not irritating.
Executive summary:

In a primary dermal irritation study, 3 young adult New Zealand white rabbits were dermally exposed to CNSL Distilled for 24 hours. Animals then were observed for 72 hours. Irritation was scored by the method of Draize. In this study, CNSL, Distilled is a dermal irritant.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Remarks:
in vitro
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to OECD guidelines and to GLP.
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
rabbit
Strain:
New Zealand White
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1ml
Duration of treatment / exposure:
10 seconds
Observation period (in vivo):
pre-enucleation, post equilibration and 60, 120, 180, and 240 minutes following treatment.
Number of animals or in vitro replicates:
In vitro - 3 eyes and 2 control eyes.
Details on study design:
It is a legal and ethical duty under the Animals (Scientific Procedure) Act 1986 that in the interest of animal welfare, the unnecessary use of animals is avoided, and that any testing which is likely to produce severe responses in animals is minimised.
Available informaiton indicated that the test material had the potential to produce severe effects in a rabbit eye and to confirm this initial assessment, a Rabbit Enucleated Eye Test was performed.
A study was performed to assess the ocular irritancy potential of the test material in the rabbit following application onto the cornea of the enucleated eye. The assay has undergone inter-laboratory validation and has been shown to reliably detect test materials that are negligible, or moderate to severe ocular irritants.
The New Zealand White strain rabbit used in these laboratories has been shown to produce satisfactory responses using known ocular-irritants and non-ocular irritants during in-house validation. The results of the study are believed to be of value in predicting the ocular irritancy potential of the test material in man.
Irritation parameter:
cornea opacity score
Value:
>= 4
Vehicle controls validity:
not specified
Negative controls validity:
not specified
Positive controls validity:
not specified
Irritation parameter:
fluorescein retention score
Value:
>= 4
Vehicle controls validity:
not specified
Negative controls validity:
not specified
Positive controls validity:
not specified
Irritation parameter:
percent corneal swelling
Value:
>= 25
Vehicle controls validity:
not specified
Negative controls validity:
not specified
Positive controls validity:
not specified
Other effects / acceptance of results:
The data for all endpoints w as assessed and an estimate of the test material ocular irritancy potential w as made based on the follow ing cut-off values:
REET Parameter REET cut-off value
Maximum Corneal Opacity > = 4
Maximum fluorescein uptake > = 4
Mean corneal swelling > = 25%
Corneal epithelium observations Any pitting, mottling or sloughing

Moderate loss of transparency was noted in all test eyes during the study.

Corneal swelling of the test eyes was considerably greater than that observed in the control eyes over the same period.

Pitting and sloughing of the corneal epithelium was noted in all test eyes during the study.

Moderate fluorescein uptake was noted in the test eyes 240 minutes following test material applicaiton. No fluorescein uptake was noted in the control eyes 240 minutes following treatment.

Interpretation of results:
Category 1 (irreversible effects on the eye)
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
CNSL, Distilled causes severe irritation to eyes.
Executive summary:

In a rabbit enucleated eye study 0.1 ml of CNSL, Distilled was instilled into three eyes of New Zealand White rabbits. The eyes were observed for 240 minutes. Irritation was scored by the method of McDonald-Shadduck.

Severe eye irritation was observed. In this study, CNSL, Distilled is a severe eye irritant.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Additional information

Skin irritation/corrosion:

Key study: A primary dermal irritation study was performed according to FHS Standard 16 CFR 1500.41 (GLP study). Three young adult New Zealand white rabbits were dermally exposed to test item for 24 hours. Animals were observed for 72 hours. Irritation was scored by the method of Draize. Two of the analysed samples were irritating to the skin (erythema and oedema scores > 2.3) and another two were determined to be not irritating to the skin. Based on these results, the substance was determined to a dermal irritant.

Eye irritation:

Key study: An in-house validated in-vitro Enucleated Eye Test study was performed to assess the potential to produce severe effects in eyes. 0.1 ml of test item was instilled into three eyes of New Zealand White rabbits. The eyes were observed for 240 minutes. Irritation was scored by the method of McDonald-Shadduck. Severe eye irritation was observed. The substance was determined to be a severe eye irritant.

Discussion:

Due to data sharing obligation and following ECHA’s indications, the registrant Wave S.L. has been forced to acquire robust study summaries for acute dermal, skin and eye irritation from CNSL Consortium. This consortium didn’t allow the registrant to get access to full study reports for quality evaluation and/or proper substance identification. Consequently the present REACH registration dossier contains the available information owned by CNSL Consortium.

Wave S.L. performed an acute oral toxicity study according to OECD Guideline 423 (GLP study) with its own substance. No mortalities were observed. The substance did not cause any clinical signs during the 14 days observation period. Body weight gains during the study showed no indication of a test item-related effect, and no macroscopic observations were seen at necropsy.

On the other hand, an acute dermal toxicity study was performed by CNSL Consortium according to OECD Guideline 402 (GLP study). No deaths occurred during the study and no systemic toxicity was observed. Nevertheless, changes in the body weight were noticed alongside with local effects. Thickening of the skin, haemorrhage of dermal capillaries, hardened light brown or dark brown/black coloured scab, small superficial scattered scabs, scab undulating, scab cracking and scab lifting at edges to reveal bleeding, dried blood, glossy skin or further deep scabbing were noted. Adverse reactions prevented accurate evaluation of erythema and oedema at all test sites during the study. Although no mortality was seen, the CNSL Consortium classified the substance as Acute Toxicity Category 4, H312, based in these local effects.

The effects observed in both acute toxicity studies are inconsistent. In the acute test performed by oral route no toxicity was observed, even at necropsy. Instead, dermal local effects were observed in rats exposed by dermal route. Therefore, it can be concluded that the test item from CNSL Consortium is toxicologically different from the one of Wave S.L.

Furthermore the studies performed by CNSL Consortium are not coherent among them as well. The local dermal effects seen in the acute dermal toxicity test are however not detected in the skin irritation test. Four different CNSL samples were tested for skin irritation according to FHSA standard 16 CFR 1500.41 (GLP study). Two of the analysed samples were irritating to the skin (mean 24-72h erythema and oedema scores > 2.3) and another two were determined to be not irritating. A difference on test sample composition is a very plausible explanation.

This registrant is not allowed to perform any of these studies in vertebrates without Authorities’ permission under risk to breaching Directive 2010/63/EU of The European Parliament and of The Council of 22nd of September 2010 regarding the protection of animals used for scientific purposes which prohibit duplicate testing. However under the light of acute oral toxicity test, no acute dermal effects can be anticipated for the substance object of the present REACH Registration dossier.

Justification for selection of skin irritation / corrosion endpoint:

Only one study available.

Justification for selection of eye irritation endpoint:

Only one study available.

Effects on skin irritation/corrosion: irritating

Effects on eye irritation: corrosive

Justification for classification or non-classification

Taking into account the available experimental data, the substance was determined to be classified for Skin Irritation Category 2, H315 (based on erythema and edema scores > 2.3) and for Severe Eye Damage Category 1, H318 (based on an in-vitro Enucleated Eye Test), in accordance with CLP Regulation (EU) no. 1272/2008.