Amines, di-C12-18-alkylmethyl

Endpoint summary

Description of key information

There is no information of Amines, di-C12-18-alkylmethyl (CAS No.: 68439-75-8; EC No.: 270-418-8) itself but there is information available for 1-Decanamine, N-decyl, N-methyl (CAS No.: 7396-58-9; EC No.: 230-990-1) which is structurally similar to Amines, di-C12-18-alkylmethyl.

The dermal irritant potential of the substance was assessed using two in vivo acute dermal irritation/corrosion tests performed in rabbits according to OECD 404 guideline and Good Laboratory Practices (Jones & Guest, 1987 and Guest, 1991) and was found to be irritating to the skin.

The ocular irritant potential of the substance was assessed using an in vivo acute eye irritation/corrosion test performed in rabbits according to OECD 405 guideline and Good Laboratory Practices (Hoechst 1988c) and the substance was found to be not irritating to the eyes.

A read-across document has been constructed to validate conclusions drawn from these structurally similar chemicals and is present in Section 13.2 'Other assessment reports'.

 

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 September 1989 - 4 January 1990

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Justification for type of information:

There is no information of Amines, di-C12-18-alkylmethyl (CAS No.: 68439-75-8; EC No.: 270-418-8) itself but there is information available for 1-Decanamine, N-decyl, N-methyl (CAS No.: 7396-58-9; EC No.: 230-990-1) which is structurally similar to Amines, di-C12-18-alkylmethyl.

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: David Percival Ltd., Moston, Sandbach, Cheshire, U.K.
- Age at study initiation: 12-16 weeks
- Weight at study initiation: 2.00 - 3.00 kg
- Housing: individually housed in suspended metal cages.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: minimal 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 13-21
- Humidity (%): 47-69
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: no data

Type of coverage:

semiocclusive

Preparation of test site:

shaved

Vehicle:

water

Controls:

other: untreated flank

Amount / concentration applied:

0.5 ml

Duration of treatment / exposure:

4 hours

Observation period:

14 days

Number of animals:

6, 4 females and 2 males

Details on study design:

TEST SITE
- Area of exposure: 2.5 cm x 2.5 cm gauze patch
- % coverage: -
- Type of wrap if used: The patch was secured in position with a strip of surgical adhesive tape (BLENDERM: approximate s i r e 2.5 crn x 4.0 cm) . To
prevent the animals interfering with the patches and ingesting the test material the trunk of each rabbit was wrapped in an elasticated corset (TUBIGRIP)

REMOVAL OF TEST SUBSTANCE
- Washing (if done): test material removed by gentle swabbing with cotton wool soaked in water
- Time after start of exposure: 4 hours

SCORING SYSTEM: Approximately one hour following the removal of the patches, and 24, 48 and 72 hours later, the test sites were examined for evidence of primary irritation and scored according to the following scale i.e. Draize J.H. (1959) Association o f Food and Drug Officials of the United States, Austin, Texas, "The Appraisal o f the Safety of Chemicals in Foods, Drugs and Cosmetics". See below:

Irritation parameter:

erythema score

Basis:

mean

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

not fully reversible within: 14 days

Irritation parameter:

edema score

Basis:

mean

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 14 days

Irritant / corrosive response data:

Severe dermal responses were produced. The adverse skin reaction sometimes precluded accurate evaluation of erythema and oedema. Very slight to well-defined erythema with or without very slight oedema was noted one hour after patch removal. The responses increased in severity and at subsequent observations were identified as well -defined erythema, very slight to moderate oedema, blanching of the skin, haemorrhage of the dermal capillaries, a white exudate, white raised areas of skin, thickening of the skin, scabbing, fissuring, a dry straw-coloured crust (possible hyperkeratinisation), reduced re-growth of fur, glossy skin and desquamation. No corrosive effects were noted.

Results table:

Rabbit No and sex	Region of the eye	Hours after instillation						Average 24-48-72 hours
		1	24	48	72	7	14
2 m	erythema	1R	2 RBl	2 HdRBlW	2 HdRBlW	?eThF iRSp	?eHyRSs	2
	oedema	0	3 Oe	2 R	2 R	?0d	0	2.3
5 f	erythema	2R	2R	2 RBlWEx	2 RBlWEx	?eThSkSp	?eHyRSs	2
	oedema	1	2	2 R	2 R	?0d	0	2
9 f	erythema	2R	2R	2 RBlWEx	?eBlWE	?eThFiRSp	?eHyRFr	2
	oedema	1	1	1 R	1 R	?0d	0	1
12 f	erythema	1R	2RBl	?eRBIWWe	?eRBlWExWe	?eThSkRSpp	?eDRFr	2
	oedema	0	2	2 R	2 R	?0d	0	2
21 m	erythema	2R	2HdR	2 HdBlWR	2 HdBlWR	?eThRSp	?eDRFr	2
	oedema	1	3 Oe	2 R	2 R	?0d	0	2.3
22 f	erythema	1R	2R	2 RBlW	2 RBlW	?eThFiRSp	?eRHyGFr	2
	oedema	0	3 Oe	2 R	2 R	?0d	0	2.3

Hd - General ised areas of slight haemorrhage of the dermal capillaries

(release of small amounts of blood into surrounding dermal tissue)

R - Dermal reactions extend beyond sit e of test material application

Bl - Blanching of the skin

W - White raised areas of skin

We - Well-defined erythema surrounding other adverse dermal reactions

Th  - Thickening of the skin

Ex - Exudate

St - Hardened, dark brown/black-colored scab

?e -  Unable to evaluate degree of erythema due to presence of other adverse dermal responses

Hy - Dry, straw-coloured crust, sometimes flaking from the skin

(possible hyperkeratinisation)

G - Skin glossy in appearance (keratinolysis)

Fr - Reduced re-growth of fur

Sp - Light brown-coloured scab

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Remarks:

Migrated information

Conclusions:

According to the results from this study 2 animals had a erythema or oedema score ≥ 2.3 and after 14 days the erythema score could not be read. The oedema score was fully reversible. Therefore "Cesio 28" is classified as irritating to skin (Category 2) under GHS.

Executive summary:

An acute dermal irritation/corrosion study was performed in rabbits (4 females and 2 males) according to OECD 404 and under GLP. Severe dermal responses were produced. The adverse skin reaction sometimes precluded accurate evaluation of erythema and oedema. Very slight to well-defined erythema with or without very slight oedema was noted one hour after patch removal. The responses increased in severity and at subsequent observations were identified as well -defined erythema, very slight to moderate oedema, blanching of the skin, haemorrhage of the dermal capillaries, a white exudate, white raised areas of skin, thickening of the skin, scabbing, fissuring, a dry straw-coloured crust (possible hyperkeratinisation), reduced re-growth of fur, glossy skin and desquamation. No corrosive effects were noted. According to the results from this study 2 animals had a erythema or oedema score ≥ 2.3 and after 14 days the erythema score could not be read. The oedema score was fully reverible. Therefore "Cesio 28" is classified as irritating to skin (Category 2) under GHS.

Reference 2

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30-09-1987 until 24-11-1987

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Justification for type of information:

There is no information of Amines, di-C12-18-alkylmethyl (CAS No.: 68439-75-8; EC No.: 270-418-8) itself but there is information available for 1-Decanamine, N-decyl, N-methyl (CAS No.: 7396-58-9; EC No.: 230-990-1) which is structurally similar to Amines, di-C12-18-alkylmethyl.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

yes

Remarks:

see remarks below

Principles of method if other than guideline:

Three test animals were exposed for 3 min and 1 hour durations. Since no corrosive effects were observed another group of three rabbits was exposed for 4 hours.

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Nottingham University, School of Agriculture, Sutton Bonington, Leicestershire, United Kingdom
- Age at study initiation: 12 to 16 weeks
- Weight at study initiation: 2.21 - 3.16 kg
- Housing: individually in suspended metal cage
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: minimum five days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16 - 21
- Humidity (%): 45 - 70
- Air changes (per hr): 15 changes/hour
- Photoperiod (hrs dark / hrs light): 12 / 12

Type of coverage:

semiocclusive

Preparation of test site:

other: clipped

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

0.5 ml

Duration of treatment / exposure:

3 minutes, 1 hour and 4 hours

Observation period:

1 hr after removal of patches and 24, 48 and 72 hours later. Additional observations were made after 7 and 14 days.

Number of animals:

3

Details on study design:

TEST SITE
- Area of exposure: 2.5cm x 2.5 cm
- Type of wrap if used: elasticated corset (tubigrip)

REMOVAL OF TEST SUBSTANCE
- Washing (if done): gentle swabbing with cotton wool soaked in 3% (v/v) aqueous acetic acid followed by cotton wool soaked in distilled water.
- Time after start of exposure: 3 min, 1 hr and 4 hours

SCORING SYSTEM:
- According to Draize (1959) Association of Food and Drug Officials of the United States, Austin, Texas, "The appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics", see table below.

Irritation parameter:

erythema score

Basis:

mean

Remarks:

all animals 4 hrs exposure

Time point:

24/48/72 h

Score:

2.5

Max. score:

4

Reversibility:

not fully reversible within: 21 days

Irritation parameter:

edema score

Basis:

mean

Remarks:

all animals 4 hrs exposure

Time point:

24/48/72 h

Score:

2.4

Max. score:

4

Reversibility:

fully reversible within: 14 days

Irritant / corrosive response data:

Three-mi nute Exposure
Very slight erythema was noted at two treated s k i n sites one hour after removal of the patches. Well-defined erythema had developed at all treated skin sites a t the 24-hour observation and well-defined or moderate t o severe erythema continued to be noted a t the 48 and 72-hour observations. Flaking, dry, - straw-col oured s k i n (possi ble hyperkeratinisation) was noted a t all treated skin s i t e s on days seven and fourteen. The reactions extended approximately 8 cm beyond one of the treated skin sites from the 72-hour observation onwards.
Very slight oedema was confined to one treated skin s i t e one hour after patch removal. Slight or moderate oedema had developed at all treated skin sites a t t h e 24-hour observation and slight oedema con-tinued to be noted at the 48 and 72-hour observations. The oedema extended beyond one treated s k i n s i t e a t the 24, 48 and 72-hour observations. No oedema was noted on days seven and fourteen.

One-hour Exposure
Very slight or well-defined erythema was noted at all treated skin sites one hour after removal of the patches. Well -defined or moderate to severe
erythema, w i t h areas of brown discolouration of the s k i n , were noted a t all treated s k i n sites at the 24, 48 and 72-hour observations. A brown dis-colouration of the skin and loss of skin suppleness were noted a t two treated s k i n sites on day seven. Flaking, dry, straw-coloured skin (possible hyperkeratinisation) was noted at the remaining treated skin site a t this time and a t all treated skin sites on day fourteen. The reactions extended approximately 8 to 10 cm beyond all treated s k i n sites during the observati on period.
Oedema, ranging from very slight to moderate was noted a t all treated skin sites one hour after patch removal. Severe oedema had developed a t all
treated s k i n sites at the 24-hour observation b u t the oedema decreased and was only slight a t the 72-hour observation. The oedema extended beyond all treated s k i n sites at the 24, 48 and 72-hour observations. No oedema was noted on days seven and fourteen.

Four-hour Exposure
The dermal reactions extended up to 8 cm beyond all treated skin sites during the observation period. Well-defined erythema was noted at at 7 treated skin sites one and 24 hours after removal of the patches. A slight haemorrhage of the dermal capillaries was noted at one treated skin site a t these observations. A brown discolouration of the s k i n was also noted at one treated skin site at the 24-hour observation. Well-defined or moderate to severe erythema was noted at all treated skin sites at the 48 and 72-hour observations. A green or brown discolouration of the skin was noted at two treated skin sites at these times, with transverse folding of the skin apparent at one treated skin site. On day seven dry, straw-coloured, flaking skin
( possi ble hyperkeratinisation) was noted at all treated skin sites. Small scattered scabs were also noted at two treated skin sites. Fissuring of the
skin, with signs of dried blood i n the fissures, was apparent at one treated skin site.
On day fourteen, glossy skin and reduced fur growth, w i t h or without desquamation, was noted at two treated s k i n sites. Fissuring, w i t h
associated small scattered scabs, and reduced fur growth were noted at the remaining treated skin site.
To establish reversibil ity of the dermal reactions an additional observation was made on day twenty-one. Glossy skin and reduced fur growth were noted at all treated skin sites. No evidence of corrosion was noted.
Slight or severe oedema was noted at all treated skin sites one hour after patch removal. Oedema ranging from very slight to moderate was noted at all treated skin sites at the 24, 48 and 72-hour observations. Very slight or slight oedema was noted at two treated skin sites on day seven. No oedema
was noted at the remaining treated skin site at this time or at any treated skin site on days fourteen or twenty-one.

Summary of dermal lesions (following 4-h application)

no.	Effect	Hour	Days after application						Mean score erythema 24/48/72 h	Mean score oedema 24/48/72 h
		1	1	2	3	7	14	21
48	Erythema/ eschar Oedema	2 R 4 R	2 BrR 3 R	3 GrRTf 3 R	3 GrRTf 3 R	HyScR 2 R	GsFrDR 0	GsFrR 0	2.7	3.0
92	Erythema/ eschar Oedema	2 H 2 R	2 HR 3 R	3 BrR 2 R	3 BrR 2 R	HyFiScR 1 R	FrFiScR 0	GsFrR 0	2.7	2.3
110	Erythema/ eschar Oedema	2 2	2 R 3 R	2 R 2 R	2 R 2 R	HyR 0	GsFrR 0	GsFrR 0	2.0	2.0
Mean									2.5	2.4

Fa = abnormal fur growth; Fr = reduced fur growth; D = desquamation; T = thickening of skin; R= reactions extending beyond treatment site; Hy = hyperkeratinisation; Br = brown discolourisation of skin; L = loss of skin suppleness; H = haemorrhage of dermal capillaries; Gr = green discoulorisation of skin; Tf = transverse folding of skin; Fi = fissuring of skin; Sc = small scattered scabs; Gs = glossy skin

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Remarks:

Migrated information

Conclusions:

The mean erythema and oedema score after 4 hr exposure was between 2.3 and 4.0 for two out of three animals.
Reversibility not completely shown within 21 days.
No signs of necrosis.

Executive summary:

A study was performed to assess the effects of Amine M210D on the irritancy potential to the skin of the New Zealand White rabbit. The method used was based on that described in the OECD Guidelines for Testing of Chemicals (1981) No. 404 "Acute Dermal Irritation/Corrosion".

A three minute, 1 hour and 4 hours, semi-occluded dermal application of the test material to the intact skin of three rabbits followed by a decontamination procedure with 3% (v/v) aqueous acetic acid followed by cotton wool in distilled water, produced irritation scores directly after patch removal and 1,2 and 3 days afterwards. The erythema/eschar effects were not fully reversible within 21 days, but the oedema effects were within 14 days. There was no evidence of visible necrosis.

The test material produced positive criteria for skin irritation following a four-hour exposure period in all rabbits.

The test material produced no evidence of visible necrosis following exposure periods of up to four hours.

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:

From 1988-03-15 to 1988-03-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

There is no information of Amines, di-C12-18-alkylmethyl (CAS No.: 68439-75-8; EC No.: 270-418-8) itself but there is information available for Amines, di-C16-18-alkylmethyl (CAS No.: 1227096-04-9; EC No.: 627-132-7) which is structurally similar to Amines, di-C12-18-alkylmethyl.

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

Reliability scoring based on 1987 guideline for test n°405

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Hoechst AG, Kastengrund, Konventionelle zucht
- Age at study initiation: 3-5 months
- Weight at study initiation: 2.5-2.9 kg
- Diet and water : ad libitum (diet: Altromin 2123)

ENVIRONMENTAL CONDITIONS
- Temperature: 17-23 °C
- Humidity: 30-70
- Photoperiod : 12 hrs dark / 12 hrs light

Vehicle:

unchanged (no vehicle)

Controls:

other: each animal served as its own control

Amount / concentration applied:

- Amount applied :0.1 ml

Duration of treatment / exposure:

Single exposure

Observation period (in vivo):

1, 24 48 and 72 hours after application

Number of animals or in vitro replicates:

3

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing : no

SCORING SYSTEM: according to guideline OECD 405

TOOL USED TO ASSESS SCORE: fluorescein

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

: 3 animals

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

other: All individual scores were 0

Irritation parameter:

conjunctivae score

Remarks:

(redness)

Basis:

mean

Remarks:

: 3 animals

Time point:

24/48/72 h

Score:

0.7

Max. score:

2

Reversibility:

fully reversible within: 72 hours

Remarks on result:

other: Individual mean scores for each animal: 0.7; 1.0 and 0.3

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

: 3 animals

Time point:

24/48/72 h

Score:

0.3

Max. score:

1

Reversibility:

fully reversible

Remarks:

72 hours

Remarks on result:

other: Individual mean scores for each animal: 0.3; 0.7 and 0.0

Irritation parameter:

iris score

Basis:

mean

Remarks:

: 3 animals

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

other: All individual scores were 0

Irritant / corrosive response data:

At the 24-hour reading, slight (grade 1) and moderate (grade 2) rednesses of the conjunctivae were observed in 1 and 2 rabbits respectively. A slight chemosis (grade 1) was present in 2 out of 3 rabbits. The redness of the conjunctivae and the chemosis (grade 1) were still present at the 48-hour reading in 1 animal but the recovery was complete at the 72-hour reading time.
No lesions of cornea or iris were reported. (See Table 1 for complete raw data)

Individual scores for eye irritation:

	Time after application	1 hr			24 hrs			48 hrs			72 hrs
	Rabbit	1	2	3	1	2	3	1	2	3	1	2	3
Scores	Chemosis	1	1	1	1	1	0	0	1	0	0	0	0
	Conjunctivae	3	1	2	2	2	1	0	1	0	0	0	0
	Iris	0	0	0	0	0	0	0	0	0	0	0	0
	Cornea opacity	0	0	0	0	0	0	0	0	0	0	0	0

Mean scores calculated for each animal over 24, 48 and 72 hours were (0.3) (0.7) and (0) for chemosis, (0.7) (1) and (0.3) for redness of the conjunctivae, (0) (0) and (0) for iris lesions and (0) (0) and (0) for corneal opacity.

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: other: CLP (Reg. 1272/2008/EC) and Directive 67/548/EEC

Conclusions:

Under these experimental conditions, the tested substance Genamin SH 301 is not classified according to CLP (Reg. n° 1272/2008/EC) and directive 67/548/EEC.

Executive summary:

The potential of Genamin SH 301 to induce eye irritation was assessed in 3 rabbits according to the OECD guideline 405 and in compliance with the principles of Good Laboratory Practice regulations.

A single dose of 0.1ml of the test item was instilled into one eye, the other eye was not treated and served as control. The eyes were not rinsed after administration of the test item.

Ocular reactions were observed 1, 24, 48 and 72 hours. The values of the scores for chemosis, redness of the conjunctiva, iris lesions and corneal opacity were noted for each animal.

Mean scores calculated over 24, 48 and 72 hours for the 3 animals were 0.3 for chemosis, 0.7 for redness of the conjunctiva, 0 for iris lesions and 0 for corneal opacity. Redness and chemosis were fully reversible within 72 hours.

Under these experimental conditions, Genamin SH 301 was considered non- irritant when administered by ocular route to rabbits.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2010-07-01 to 2010-07-16

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

There is no information of Amines, di-C12-18-alkylmethyl (CAS No.: 68439-75-8; EC No.: 270-418-8) itself but there is information available for 1-Decanamine, N-decyl, N-methyl (CAS No.: 7396-58-9; EC No.: 230-990-1) which is structurally similar to Amines, di-C12-18-alkylmethyl.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

not specified

Qualifier:

equivalent or similar to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Deviations:

not specified

Principles of method if other than guideline:

The method used is adapted from that described by Gautheron P. & al. (1992) Fundam. Appl. Toxicol. 18 442-449.
The principle of this evaluation is based on the measurement of two factors: the opacity and the permeability of the treated corneas. The changes in these parameters correspond to the damages induced to the tissues.

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Strain:

other: bovine calf

Details on test animals or tissues and environmental conditions:

Origin: calf eyes were obtained from freshly slaughtered calves at the abattoir SOCAVIA, Cany Barville, France.

Reason for choice: calf corneas are adapted for the evaluation of potential ocular irritants since they are part of the target organ.

Transport from supplier to CIT: the eyes were transported to CIT at ambient temperature, immerged in buffered Hanks medium containing antibiotics (Hank’s Balanced Salts Solution (HBSS) plus penicillin/streptomycin).

Preparation of the corneas: The corneas were prepared as quickly as possible after receipt. Each step was carried out avoiding to touch the corneas in order to not injure them.

Selection: upon arrival at CIT, all eyes were carefully examined macroscopically for defects (opacity, scratches, pigmentation, etc) and those
exhibiting any defect were discarded. The too large eyes were also discarded in order to avoid the formation of folds at the assembly of corneas in theholder. The examination was performed under a lamp and using HBSS in order to maintain the corneas moistened and shiny. Each cornea was
observed with attention, while making swivel the eye in order to see any less refringent areas under the light or any scratches.

Preparation of the selected corneas: the tissue surrounding the eyeball was carefully pulled away and the cornea was dissected such that
approximately 2 to 3 mm of sclera was present around the cornea. The isolated corneas were stored in HBSS until all corneas were dissected.

As the corneas were not used extemporaneously for a treatment, they were washed three times of 15 minutes each in HBSS plus
penicillin/streptomycin (100 units/100 µg/mL final) at room temperature, then stored individually in 12 mL of M199 medium containing 5% dextran, plus penicillin/streptomycin, at +4°C for 24 hours maximum before use.

Vehicle:

unchanged (no vehicle)

Controls:

other: not applicable (control corneas were treated with the negative control)

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): a volume of 750 μL ± 8 μL was gently applied to the cornea, as uniformly as possible.

Duration of treatment / exposure:

30 minutes and 4 hours

Observation period (in vivo):

Not applicable

Number of animals or in vitro replicates:

Not applicable (three corneas were used for each treated series)

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): as the dosage form was liquid, the anterior compartment of the holder was emptied using a metal gavage tube attached to a vacuum pump, then the compartment was filled with heated cMEM (32°C). In the second experiment, the dosage form having adhered to the walls of the
compartment was eliminated using a cotton bud. Then the compartment was filled with heated cMEM (32°C).
The rinsing was repeated four times.

TOOL USED TO ASSESS SCORE: opacitometer, fluorescein, spectrophotometer

Irritation parameter:

in vitro irritation score

Remarks:

in vitro score = Corrected Opacity + (15 x Corrected OD490 nm)

Run / experiment:

4 hours exposure

Value:

1.3

Remarks on result:

other: Following the 30-minute treatment, the mean in vitro score was -0.3

Irritation parameter:

other: corrected opacity

Run / experiment:

4 hours exposure

Value:

0.7

Irritation parameter:

other: corrected OD490 nm

Run / experiment:

4 hours exposure

Value:

0.044

Other effects / acceptance of results:

No notable opaque spots or irregularities were observed on negative control corneas, either following the 30-minute treatment or following the 4-hour treatment.
Fluoresceine fixation was observed on test item-treated corneas, following both the 30 minute and 4-hour treatments. Residual test item was also noted on corneas following the 4-hour treatment.

Acceptance criteria:

For each experiment, the acceptance criteria were fulfilled:

. the individual corneal opacity values of negative controls were < 10 in both experiments,

. the individual OD_490nm values of negative control corneas were < 0.100 in both experiments,

. the solution of fluoresceine (at 5 mg/mL in DPBS) diluted 1:1000 in cMEM had OD_490nm values between 0.850 and 0.940 in both experiments,

. following the 30-minute treatment, the positive control mean in vitro score was 90.3, thus demonstrating the sensitivity of the test system under the experimental conditions of this study.

Interpretation of results:

slightly irritating

Remarks:

Migrated information Criteria used for interpretation of results: other: scoring table showed above (any other information on materials and methods)

Conclusions:

Under these experimental conditions, the test item is slightly irritant for the isolated calf cornea and does not required a classification according to according to CLP (Reg. n° 1272/2008/EC) and directive 67/548/EEC.

Executive summary:

The aim of this study was to evaluate the potential irritant properties of the test item for the isolated calf cornea. This study was conducted in compliance with CIT’s standard operating procedures and the principles of Good Laboratory Practices.

The corneas were obtained from the eyes of freshly slaughtered calves at the abattoir. They were mounted in the corneal holders with the endothelial side against the O-ring of the posterior half of the holder. Both compartments of the corneal holder were filled in excesswith Minimal Essential Medium Eagle completed with 1% fetal calf serum plus penicillin/streptomycin (cMEM), then the holders were preincubated for 1 hour at 32°C. Three corneas were used for each treated series (test item, positive control and negative control).

Before the treatment, a first opacity measurement was performed using an opacitometer (determining the light transmission through the center of each mounted cornea). For the treatment, the test item was used in its original form.

 

The test item was tested sequentially in two consecutive experiments.

As the mean in vitro score at the 30-minute treatment was ≤ 10, the second experiment was undertaken using a 4-hour treatment.

 At the completion of the treatment period, the test item was removed from the front opening of the anterior part of the holder and the epithelium was washed.

 Following the 30-minute treatment, the corneas were incubated for 2 hours at. At the completion of the 2-hour incubation period, the second opacity measurement was performed.

Following the 4-hour treatment, the second opacity measurement was performed immediately without any further incubation after the rinsing of the dosage form.

After the second opacity measurement, the medium was removed from both compartments of each holder. The posterior compartment was refilled with cMEM at, while the anterior compartment received 1 mL of a 5 mg/mL fluoresceine solution in Dulbecco's Phosphate-Buffered Saline (DPBS). Then, the holders were incubated vertically for 90 minutes at 32°C.

 At the end of the 90-minute incubation, the optical density of the solution from the posterior compartment of the holder was measured at 490 nm in order to determine the permeability of the cornea. Then the cornea was removed from the holder and observed for opaque spots and other irregularities.

For each experiment, the acceptance criteria were fulfilled and the study was therefore considered to be valid. No notable opaque spots or irregularities were observed on negative control corneas, either following the 30-minute treatment or following the 4 -hour treatment.

Fluoresceine fixation was observed on test item-treated corneas, following both the 30-minute and 4-hour treatments. Residual test item was also noted on corneas following the 4-hour treatment.

Following the 30-minute treatment, the mean in vitro score was -0.3. Then following the 4-hour treatment, the mean in vitro score was 1.3.

 

Under these experimental conditions and according to both mean in vitro scores of the 30‑minute and 4-hour treatments, the test item

is slightly irritant for the isolated calf cornea.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation/corrosion:

Two valid studies were recorded for this endpoint. The Kreiling study (1988) was a reliable without restrictions study and the Jones & Guest study (1987) was reliable with some restrictions due to limited information reported about the tested substance. The Kreiling study (1988) was identified as the key study and supported by the results of the study performed by Jones & Guest (1987).

 

The Hoechst study (1988b) was performed in rabbits according to the OECD guideline 404 and to the EU Method B.4. The study was conducted in compliance with the principles of Good Laboratory Practice regulations.

Three rabbits were dermally exposed to 0.5 ml of the substance for 4 hours. The test substance was held in contact with the skin by means of a semi- occlusive dressing. Skin reactions were observed approximately 0.5 /1 hour, 24, 48 and 72 hours after removal of the dressing and then on day 7, 14 and 21 in order to observe their reversibility. The mean values of the scores for erythema and oedema were calculated for each animal.

The mean scores over 24, 48 and 72 hours for individual animals were 3.0, 1.3, 2.0 for erythema and 1.0, 1.0, and 0.7 for oedema. No ulceration or necrosis was noted. Erythema and edema had reversed by day 14 and by day 7, respectively.

 

The Jones & Guest study (1987) was designed to assess the acute dermal irritation of the substance in rabbits according to OECD guideline 404 and in compliance with the principles of Good Laboratory Practice regulations.

The substance was applied to the skin of 3 rabbits and held in contact for 3 minutes, 1 hour and 4 hours by means of a semi-occlusive dressing.

After 3 minutes of exposure, no skin irritation was observed 1 hour after removal of the patch, but a very slight to well defined erythema and a very slight oedema were observed at 24, 48 and 72 hours in all animals. All reactions cleared within 14 days.

After 1hour of exposure, no skin irritation was observed one hour after patch removal in 1 animal while the 2 remaining animals showed very slight erythema and oedema. 24, 48 and 72 hours after patch removal , a well defined to moderate erythema and a very slight oedema were observed in all animals. All reactions cleared within 14 days.

After 4 hour of exposure, very slight erythema was noted for all animals one hour after patch removaland 2 out of 3 animals showed a very slight oedema. 24, 48 and 72 hours after patch removal, a well defined to moderate erythema and a very slight oedema were observed in all animals except for 1 which showed a severe oedema at the 24 -hour reading. At any dose level, all treatment sites showed desquamation on day 7 but were normal on day 14.

Mean scores over 24, 48 and 72 hours for each animal were 2.0, 1.7 and 2.3 for erythema and 1.0, 1.0 and 2.0 for oedema. All reactions cleared within 14 days.

 

Eye irritation:

One study is recorded for this endpoint and was chosen as a key study (Hoechst, 1988c).

The potential of the substance to induce eye irritation was assessed in 3 rabbits according to the OECD guideline 405 and to the EU method B.5. The study was conducted in compliance with the principles of Good Laboratory Practice regulations.

A single dose of 0.1ml of the test item was instilled into one eye, the other eye was not treated and served as control.The eyes were not rinsed after administration of the test item.

Ocular reactions were observed 1, 24, 48 and 72 hours. The values of the scores for chemosis, redness of the conjunctiva, iris lesions and corneal opacity were noted for each animal.

Mean scores calculated over 24, 48 and 72 hours for the 3 animals were 0.3 for chemosis, 0.7 for redness of the conjunctiva, 0 for iris lesions and 0 for corneal opacity. Redness and chemosis were fully reversible within 72 hours.

 

 

 

Effects on skin irritation/corrosion: irritating

 

Effect level: empty Endpoint conclusion: Adverse effect observed

Justification for classification or non-classification

Skin irritation/corrosion:

 

Based on the results of the Guest (1991) and Jones & Guest (1987) studies and according to the criteria laid down in EU directive 67/548/EEC, the substance is considered irritating to skin and is classified as Skin Irritation Category 2, R38. In each of the above mentioned studies, for 2 of the 3 tested animals

individual mean score for erythema over the 24, 48 and 72-hour readings are equal or greater than the cut-off value of 2.

 

According to the criteria laid down in EC regulation 1272/2008/EC, the substance is not considered as a skin irritant and is not classified.

In each of the above mentioned studies, only 1 of the 3 tested animals has an individual mean score for erythema over the 24, 48 and 72-hour readings equal or greater than the cut-off value of 2.3.

 

Eye irritation:

 

Based on the result of the Hoechst study (1988c) and according to the criteria laid down in EC regulation 1272/2008/EC and EU directive 67/548/EEC, the substance is considered as non-irritant when administered by ocular route and is not classified.