Dibenzoyl-L-tartaric acid monohydrate

Endpoint summary

Description of key information

Skin irritation/corrosion: Key study. In vivo study. Method according to OECD 404 (non-GLP). The test item is not irritant to the skin.

Eye irritation: Weight of evidence. Based on the available, information, the test item causes damage to the eyes.

- In vitro interlaboratory study. Method according to OECD 437 (GLP study). The test item causes damage to the eyes.

- Two in vivo studies. Method according to EU B.5 / OECD 405 (GLP studies). The test item was moderately/severely irritant to the eyes (MAS = 36.7 and 33.7).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin corrosion: in vitro / ex vivo

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

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

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
In accordance with REACH Annex XI, the study does not appear scientifically necessary, as an in vivo study was available (1.1. Use of existing data).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Aldrich.

OTHER SPECIFICS: To ensure blind experimental conditions, in vitro as well as in vivo, chemicals were sampled, coded, and shipped by an independent Company (MCS-Pharma, Erstein, France).

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Bovine eyes were collected from a commercial abattoir.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): eyes were collected in a plastic jar containing 1 litre HBSS for approximately 25 eyes. Buffer storage and eyes transportation to the laboratories were performed at ambient temperature, and the eyes were used within 2 hr of the killing of the animals.
- Time interval prior to initiating testing: within 2h after collection.
- indication of any existing defects or lesions in ocular tissue samples: no.
- Indication of any antibiotics used: no.

Vehicle:

other: Minimum essential medium (MEM)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 ml was applied on the epithelium with an appropriate syringe and needle.
- Concentration: Solids were prepared as an approximate 20% solution or suspension (200mg + 1ml) in MEM using a mortar and pestle for homogeneous preparations.

VEHICLE
- Concentration (if solution): Minimum essential medium (MEM) was prepared from powder supplemented with glutamine and sodium bicarbonate as indicated by the supplier, the pH was adjusted to 7.4 and the medium was freshly used or stored refrigerated (1-wk stock).
- Lot/batch no.: Sigma M-3024

Duration of treatment / exposure:

Corneas were incubated in a horizontal position for 4 h at 32ºC.

Number of animals or in vitro replicates:

The number of corneas per experiment was 15, including a control group of 3 corneas treated with MEM, and two groups of 6 corneas treated with test compound.

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
During dissection great care was taken to avoid damage of corneal surfaces (epithelial and endothelial). Each cornea was carefully examined, and those presenting defects, such as neovascularization, pigmentation, opacity or scratches were discarded. Globes were first dissected free of surrounding tissues and placed in a jar containing fresh HBSS. Selected corneas were dissected with a 2-3-mm rim of sclera for easier handling, and stored in a petri dish containing HBSS until use. Corneas were then mounted in holders filled with MEM, and incubated for 1 hr in a water-bath at 32ºC.

NUMBER OF REPLICATES : 6

NEGATIVE/SOLVENT CONTROL USED : MEM

POSITIVE CONTROL USED : 750 μL of 20% (w/v) imidazole solution in MEM

APPLICATION DOSE AND EXPOSURE TIME : 750 μL of 20% (w/v) test item solution in MEM, 4h exposure.

TREATMENT METHOD: [closed chamber / open chamber]

POST-INCUBATION PERIOD: no.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: at least 3 times (until the medium was clear) with approximately 4 ml MEM.
- POST-EXPOSURE INCUBATION: 2h at 32ºC.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: opacitometer (Electro-Design, Riom, France) previously calibrated with standardized opaque sheets of polyester.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of UV/VIS spectrophotometry (490 nm)
- Others: pertinent visual observations.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS) = opacity value + 15 times O.D. value

DECISION CRITERIA: score 0 - 25, mild irritant; 25.1 - 55, moderate irritant; ≥ 55.1, severe irritant..

Irritation parameter:

in vitro irritation score

Run / experiment:

mean of results from 11 laboratories, mean of 6 corneas each

Value:

120.5

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: not specified.

DEMONSTRATION OF TECHNICAL PROFICIENCY: not applicable (100% agreement within all laboratories).

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Table 1. In vitro scores from individual laboratories for BCOP assay (n=6)

	Score in laboratory no.:													Mean score
Compound	Physical form	1	2	3	4	5	6	7	8	9	10	11	12
Test item 2743-38-6	S	104	134	82	118	110	66	88	193	82	-	213	135	120.5
Imidazole 288-32-4	S	75	73	140	81	96	62	82	122	64	81	114	65	87.9

 

Table 2. Agreement between laboratories.

Compound	Physical form	Mean IVIS	Agreement(%)	Result
Test item 2743-38-6	Solid	120.5	100	Severe irritant
Imidazole 288-32-4	Solid	87.9	100	Severe irritant

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Remarks:

EU criteria.

Conclusions:

The test item is severely irritant to the eye (IVIS = 120.5)

Executive summary:

An in vitro (ex vivo) Bovine Corneal Opacity and Permeability study was conducted by 12 laboratories in an interlaboratory validation study, in order to determine the potential severe eye damaging effects of the test item. The study was performed according to the OECD guideline 437 under GLP conditions. Sets consisting of 6 corneas each were exposed to either 750 μL of 20% (w/v) of test item suspension in MEM or 750 μL of 20% (w/v) imidazole in MEM (positive control) for 4h at 32ºC; a negative control was carried out in parallel with 3 corneas, treated with MEM. After exposure, opacity and permeability of the corneas was measured, and an in vitro irritancy score was calculated combining both parameters. As a protocol variation, four of the laboratories performed the study with preserved corneas. The mean value from 11 laboratories and the % agreement were calculated and compared to in vivo data. The mean IVIS score values obtained were 87.9 and 120.5 for imidazole and the test item, respectively. Therefore, the test item causes damage to the eye (Cat.1).

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 100 mg

Observation period (in vivo):

14 days

Number of animals or in vitro replicates:

3

Irritation parameter:

maximum mean total score (MMTS)

Basis:

mean

Time point:

24/48/72 h

Score:

36.67

Max. score:

110

Reversibility:

fully reversible within: 14 days

Remarks on result:

other: SD: 10.02 (n = 3)

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2

Reversibility:

fully reversible within: 14 days

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

3

Reversibility:

fully reversible within: 14 days

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

3

Reversibility:

not fully reversible within: 14 days

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1.3

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

2.7

Table 1. Summary of results. MMAS (Modified Maximum Average Score) = (36 + 27 + 47) / 3 = 36.7

Animal No.	Parameter			Observation period (days)
				1h	4h	1	2	3	4	7	9	10	12	14	21
1	Cornea	Opacity	A	1	-	3	3	0	0	0	-	-	-	0	-
		Area involved	B	1	-	1	1	0	0	0	-	-	-	0	-
		(AxB) x 5		5	-	15	15	0	0	0	-	-	-	0	-
	Iris		C	1	-	1	1	0	0	0	-	-	-	0	-
		C x 5		5	-	5	5	0	0	0	-	-	-	0	-
	Conjunctiva	Redness	D	2	-	2	2	1	1	0	-	-	-	0	-
		Chemosis	E	2	-	3	2	1	1	0	-	-	-	0	-
		Discharge	F	2	-	3	1	0	0	0	-	-	-	0	-
		(D+E+F) x 2		12	-	16	10	4	4	0	-	-	-	0	-
	TOTAL			22	-	36	30	4	4	0		-	-	0	-
2	Cornea	Opacity	A	1	-	3	3	3	0	0	-	-	-	0	-
		Area involved	B	1	-	1	1	1	0	0	-	-	-	0	-
		(AxB) x 5		5	-	15	15	15	0	0	-	-	-	0	-
	Iris		C	1	-	0	0	0	0	0	-	-	-	0	-
		C x 5		5	-	0	0	0	0	0	-	-	-	0	-
	Conjunctiva	Redness	D	2	-	2	2	1	1	0	-	-	-	0	-
		Chemosis	E	3	-	2	1	1	1	0	-	-	-	0	-
		Discharge	F	2	-	2	0	0	0	0	-	-	-	0	-
		(D+E+F) x 2		14	-	12	6	4	2	0	-	-	-	0	-
	TOTAL			24	-	27	21	19	2	0		-	-	0	-
3	Cornea	Opacity	A	1	-	3	3	3	3	3	-	-	-	2	-
		Area involved	B	4	-	2	2	1	1	1	-	-	-	2	-
		(AxB) x 5		20	-	30	30	15	15	15	-	-	-	20	-
	Iris		C	1	-	1	1	1	1	1	-	-	-	1	-
		C x 5		5	-	5	5	5	5	5	-	-	-	5	-
	Conjunctiva	Redness	D	2	-	2	3	3	3	3	-	-	-	2	-
		Chemosis	E	3	-	2	3	3	3	3	-	-	-	2	-
		Discharge	F	2	-	2	2	2	2	2	-	-	-	0	-
		(D+E+F) x 2		14	-	12	16	16	16	16	-	-	-	8	-
	TOTAL			39	-	47	51	36	36	36		-	-	33	-

 

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Remarks:

EU criteria.

Conclusions:

Under test conditions, the test substance was found to be irritant to the eyes, with a MAS = 36.7.

Executive summary:

A study to investigate possible irritation or corrosion of the test substance on the eyes of rabbits was performed according to OECD Guideline 405 (GLP study). The right eye of 3 New Zealand White rabbits received single application of 0.1 mg test item, while the left eye was used as a control. The treated eyes of all rabbits remained unwashed for 24h: Ocular lesions (cornea, iris, conjunctiva) were scored using hand-held lenses 1h after application and then 1, 2, 3, 4, 7, 14 and 21 days after to determine reversibility. Modified Maximum Average Score (MMAS) was calculated. Under test conditions, the test substance was found to be irritant to the eyes, with a MAS = 36.7.

Reference 3

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

according to EEC (1984 and 1991) and french (1984 and 1991) directives

Deviations:

yes

Remarks:

Any mass of material present in the conjunctival sac was removed after the 1-h observation.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Principles of method if other than guideline:

- Parameters analysed / observed: ocular lesions (cornea, iris and conjunctiva) were scored (Draize et al., 1944) at 1h, then at 1, 2, 3, 4, 7 and 14 days. In the case of positive score, eyes were also examined at 21 days. Any mass of material present in the conjunctival sac was removed after the 1-h observation. Also, a fluorescein solution was used for observation of corneal lesions. Maximal average score (MAS) as well as score at day 1 were calculated. Raw data were also used to classify chemicals according to the rating system described by Kay and Calandra (1962), and the EC criteria.

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

100 mg

Observation period (in vivo):

21 days

Number of animals or in vitro replicates:

3

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing: yes, any mass of material present in the conjunctival sac was removed after the 1-h observation.
- Time after start of exposure: 1h

SCORING SYSTEM: ocular lesions (cornea, iris and conjunctiva) were scored (Draize et al., 1944) at 1h, then at 1, 2, 3, 4, 7 and 14 days. In the case of positive score, eyes were also examined at 21 days. Also, a fluorescein solution was used for observation of corneal lesions. Maximal average score (MAS) as well as score at day 1 were calculated. Raw data were also used to classify chemicals according to the rating system described by Kay and Calandra (1962), and the EC criteria.

TOOL USED TO ASSESS SCORE: hand-slit lamp / fluorescein

Irritation parameter:

maximum mean total score (MMTS)

Basis:

mean

Time point:

24/48/72 h

Score:

33.7

Max. score:

110

Reversibility:

fully reversible within: 21 days

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

24/48/72 h

Reversibility:

fully reversible

Remarks on result:

positive indication of irritation

Irritation parameter:

chemosis score

Basis:

mean

Time point:

24/48/72 h

Reversibility:

fully reversible

Remarks on result:

positive indication of irritation

Irritation parameter:

iris score

Basis:

mean

Time point:

24/48/72 h

Reversibility:

fully reversible

Remarks on result:

positive indication of irritation

Irritation parameter:

conjunctivae score

Basis:

mean

Time point:

24/48/72 h

Reversibility:

fully reversible

Remarks on result:

positive indication of irritation

Irritant / corrosive response data:

Not specified.

Table 1. Summary of results.

Compound (physical form)	In vivo score		Reversibility (d)	Class
	MAS	Day 1		K-C*	EEC
Dibenzoyl-L-tartaric acid (S)	33.7	33.7	21	Mod: moderately irritant	R41: severely irritant

 *K-C: Kay and Calandra (1962)

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Remarks:

EU criteria.

Conclusions:

Under test conditions, the test substance was found to be a moderate/severe irritant to the eyes, with a MAS = 33.7.

Executive summary:

A study to investigate possible irritation or corrosion of the test substance on the eyes of rabbits was performed according to EU method B.5 / OECD Guideline 405 (GLP study). The right eye of 3 New Zealand White rabbits received single application of 0.1 mg test item, while the left eye was used as a control. Any mass of material present in the conjunctival sac was removed after 1h. Ocular lesions (cornea, iris and conjunctiva) were scored (Draize et al., 1944) at 1h, then at 1, 2, 3, 4, 7, 14 and 21 days. Also, a fluorescein solution was used for observation of corneal lesions. Maximal average score (MAS) as well as score at day 1 were calculated. Raw data were also used to classify chemicals according to the rating system described by Kay and Calandra (1962), and the EC criteria. Under test conditions, the test substance was found to be moderately/severely irritant to the eyes, with a MAS = 33.7.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation:

In vivo: The dermal irritation/corrosion of the test item was studied in vivo acorrding to OECD 404 (GLP study, certificate not available). 1 female and 2 male Vienna White rabbits were exposed to a 50% aqueous solution of the test item for 4h, and the responses were observed 4, 24, 48 and 72h after patch removal. Animals were necropsied at the end of the study. Erythema and edema scores were 0/4 at all time points, and no abnormalities were observed at necropsy. Therefore, the test item is not irritant to the skin.

In vitro: Data waiving (study scientifically not necessary / other information available): In accordance with REACH Annex XI, the study does not appear scientifically necessary, as an in vivo study was available (1.1. Use of existing data).

Eye irritation: Weight of evidence.

An in vitro (ex vivo) Bovine Corneal Opacity and Permeability study was conducted by 12 laboratories in an interlaboratory validation study, in order to determine the potential severe eye damaging effects of the test item. The study was performed according to the OECD guideline 437 under GLP conditions. Sets consisting of 6 corneas each were exposed to either 750 μL of 20% (w/v) of test item suspension in MEM or 750 μL of 20% (w/v) imidazole in MEM (positive control) for 4h at 32ºC; a negative control was carried out in parallel with 3 corneas, treated with MEM. After exposure, opacity and permeability of the corneas was measured, and an in vitro irritancy score was calculated combining both parameters. As a protocol variation, four of the laboratories performed the study with preserved corneas. The mean value from 11 laboratories and the % agreement were calculated and compared to in vivo data. The mean IVIS score values obtained were 87.9 and 120.5 for imidazole and the test item, respectively (100% agreement). Therefore, the test item causes damage to the eyes.

Two in vivo studies to investigate possible irritation or corrosion of the test substance on the eyes of rabbits were performed according to EU B.5/ OECD 405 (GLP studies). The right eye of 3 New Zealand White rabbits received single application of 0.1 mg test item, while the left eye was used as a control. Ocular lesions (cornea, iris, conjunctiva) were scored 1h after application and then 1, 2, 3, 4, 7, 14 and 21 days after to determine reversibility. Maximum Average Scores (MAS) were calculated. Under test conditions, the test substance was found to be a severe irritant to the eyes, with a MAS = 36.7 and 33.7, respectively.

Justification for classification or non-classification

Based on the available information, the substance is not classified for skin irritation, but is classified as Eye Damage 1 in accordance with CLP, Regulation (EC) No 1272/2008.