Sodium 5-oxo-DL-prolinate

Endpoint summary

Description of key information

Skin irritation: Not irritating (in vitro), OECD 439, EU Method B. 46, Kiss 2012c. 
Eye irritation: Not irritating (in vivo), OECD 405, EU Method B. 5 and EPA OPPTS 870. 2400, Török-Bathó 2012.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 March 2012 to 30 March 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

A valid study is available on the read across substance, sodium 5-oxo-L-prolinate, which is a structural analogue of the registered substance. Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.. Read-across is considered to be suitable based on the structural similarities between the read across substance (sodium 5-oxo-L-prolinate) and the substance to be registered (sodium 5-oxo-DL-prolinate).

Justification for type of information:

A valid study is available on the read across substance, sodium 5-oxo-L-prolinate, which is a structural analogue of the registered substance. Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.. Read-across is considered to be suitable based on the structural similarities between the read across substance (sodium 5-oxo-L-prolinate) and the substance to be registered (sodium 5-oxo-DL-prolinate).

Reason / purpose for cross-reference:

other: Target substance

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Details on test animals or test system and environmental conditions:

EPISKIN-SM
Supplier: SkinEthic, France

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 20 mg

Duration of treatment / exposure:

15 ± 0.5 minutes

Number of animals:

Not applicable

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean

Value:

95

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

not specified

Remarks on result:

no indication of irritation

Other effects:

CELL VIABILITY
- Cell viability of treated disks was 95 %, see Table 1 for results.

VALIDITY OF THE TEST
The mean OD value of the three negative control tissues was 0.641. The positive control result showed 26 % viability. Each standard deviation value (SD) of the % viability was below 18. All validity criteria were within acceptable limits and therefore the study can be considered as valid.

INDICATOR FOR POTENTIAL FALSE VIABILITY
> Possible direct MTT reduction with the test material: No colour change was observed after three hours of incubation of the test item in MTT solution. The test material did not interact with the MTT, therefore additional controls and data calculations were not necessary. A false estimation of viability can be precluded.
> Colouring potential of the test material: As the test material has an intrinsic colour, one additional chemical-treated tissue was used for the non specific OD evaluation. Mean OD (measured at 540 nm) of this tissue was determined as 0.025, Non Specific Colour % was calculated as 4 %. Therefore additional data calculation was not necessary.

Table 1: Optical Density (OD) and the Calculated % Viability

Substance	Optical Density (OD)		Viability (%)
Negative Control	1	0.639	100
	2	0.607	95
	3	0.676	105
	Mean	0.641	100
	Standard Deviation (SD)		5.00
Positive Control	1	0.150	23
	2	0.191	30
	3	0.155	24
	Mean	0.165	26
	Standard Deviation (SD)		3.79
Test Material	1	0.555	87
	2	0.627	98
	3	0.633	99
	Mean	0.605	95
	Standard Deviation (SD)		6.66

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of the study, exposure to the test material resulted in a mean relative cell viability of 95 %. Since the cell viability was determined to be > 50 % of the negative control the test material was considered to be non-irritating.

Executive summary:

The potential for the test material to cause skin irritation was predicted in an in vitro study using the EPISKIN Model. The study was conducted under GLP conditions and in line with OECD 439 and EU Method B.46. The test material was applied topically to the surface of the skin for 15 minutes, which was terminated by rinsing with PBS 1 x solution (0.9 %). Epidermis units were then incubated at 37°C for 42 hours in an incubator with 5 % CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37 °C in 5 % CO2 protected from light. The formazan extract in acidified isopropanol was then spectrophotometrically evaluated for optical density (OD) and quantified. Positive and negative controls were run concurrently and tissue viability was expressed as a % relative to the negative control.

Under the conditions of the test, exposure to the test material resulted in a mean relative cell viability of 95 %. Since the cell viability was determined to be > 50% of the negative control the test material was considered to be non-irritating.

This study was conducted on the the read across substance, sodium 5-oxo-L-prolinate.

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:

26 April 2012 to 29 April 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

A valid study is available on the read across substance, sodium 5-oxo-L-prolinate, which is a structural analogue of the registered substance. This is a non GLP study performed to sound scientific principles with a sufficient level of detail to assess the quality of the submitted data. The study followed a methodology similar to OECD 406. The report was redrafted several years after the study period. Read-across is considered to be suitable based on the structural similarities between the read across substance (sodium 5-oxo-L-prolinate) and the substance to be registered (sodium 5-oxo-DL-prolinate).

Justification for type of information:

A valid study is available on the read across substance, sodium 5-oxo-L-prolinate, which is a structural analogue of the registered substance. This is a non GLP study performed to sound scientific principles with a sufficient level of detail to assess the quality of the submitted data. The study followed a methodology similar to OECD 406. The report was redrafted several years after the study period. Read-across is considered to be suitable based on the structural similarities between the read across substance (sodium 5-oxo-L-prolinate) and the substance to be registered (sodium 5-oxo-DL-prolinate).

Reason / purpose for cross-reference:

other: Target substance

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

yes

Remarks:

(the eyes of the test animals were washed 1 hour after administration of the test material).

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

(the eyes of the test animals were washed 1 hour after administration of the test material).

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Deviations:

yes

Remarks:

(the eyes of the test animals were washed 1 hour after administration of the test material).

GLP compliance:

no

Remarks:

The report was redrafted several years after the study period. At the time of the study period GLP compliance was not a requirement.

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Age at study initiation: Approximately 14 weeks old (adults).
- Weight at study initiation: Males ranged from 3251 to 3518 g.
- Housing: Individually in open wire cages.
- Diet: rabbit diet, ad libitum.
- Water: Municipal tap water, ad libitum.
- Acclimation period: 29 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 3 °C
- Humidity (%): 30 to 70%
- Air changes (per hr): 15 to 20 per hour.
- Photoperiod (hrs dark / hrs light): 12 hours light per day between 06:00 and 18:00 hours.

IN-LIFE DATES: From: 26 April To: 29 April

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 g.

Duration of treatment / exposure:

1 hour

Observation period (in vivo):

72 hours.

Number of animals or in vitro replicates:

Three

Details on study design:

DOSING
- Procedure: An initial test was performed using one animal to assess the initial pain reaction. After consideration of the ocular responses produced in the first animal, one hour after the treatment of the first animal, two additional animals were treated.
- Administration: The test material was instilled into the conjunctival sac of the left eye. The eyelids were held closed for a few seconds to prevent the loss of the test item.

SCORING SYSTEM
- Initial pain reaction: Immediately after the administration of the test material, an assessment of the initial pain reaction was made according to the six point scale shown in Table 1.
- Ocular response: The eyes were examined at 1, 24, 48 and 72 hours after treatment. The duration of the observation period was sufficient to identify reversibility or irreversibility of changes. The eye irritation scores were evaluated according to the scoring system by Draize (1977) shown in Table 2.

OTHER OBSERVATIONS:
- Clinical: Any clinical signs of toxicity or signs of ill-health during the study were recorded.
- Bodyweight: Individual body weight was recorded at the beginning and end of the experiment.
- Necropsy: Performed at termination of the observation period.

Irritation parameter:

cornea opacity score

Remarks:

opacity

Basis:

animal #1

Time point:

other: Mean at 24, 48 and 72 hours

Score:

0

Max. score:

4

Irritation parameter:

cornea opacity score

Remarks:

opacity

Basis:

animal #2

Time point:

other: Mean at 24, 48 and 72 hours

Score:

0

Max. score:

4

Irritation parameter:

cornea opacity score

Remarks:

opacity

Basis:

animal #3

Time point:

other: Mean at 24, 48 and 72 hours

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

animal #1

Time point:

other: Mean at 24, 48 and 72 hours

Score:

0

Max. score:

2

Irritation parameter:

iris score

Basis:

animal #2

Time point:

other: Mean at 24, 48 and 72 hours

Score:

0

Max. score:

2

Irritation parameter:

iris score

Basis:

animal #3

Time point:

other: Mean at 24, 48 and 72 hours

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal #1

Time point:

other: Mean at 24, 48 and 72 hours

Score:

0

Max. score:

3

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal #2

Time point:

other: Mean at 24, 48 and 72 hours

Score:

0

Max. score:

3

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal #3

Time point:

other: Mean at 24, 48 and 72 hours

Score:

0.33

Max. score:

3

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

other: Mean at 24, 48 and 72 hours

Score:

0

Max. score:

4

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

other: Mean at 24, 48 and 72 hours

Score:

0

Max. score:

4

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

other: Mean at 24, 48 and 72 hours

Score:

0

Max. score:

4

Irritant / corrosive response data:

- Initial Pain Reaction (IPR): A score of 2 was observed in all animals.
- One hour after the application: Conjunctival redness (score 1 or 2) and discharge (score 1 or 2) were seen in all rabbits. One animal showed conjuctival chemosis (score 1).
- At 24 hours after treatment: Conjunctival redness (score 1) was seen in one rabbit.
- At 48 and 72 hours after treatment: No signs of eye irritation were observed.
- Reversibility: As all signs of eye irritation had fully reversed the study was terminated after a period of 72 hours observation.

Other effects:

- Mortality: None observed during the study.
- Bodyweight: The body weight and body weight change were considered to be normal with no indication of a treatment related effect. Bodyweights ranged from 3420 to 3553 g at termination representing a weight gain of between 35 and 169 g.
- Clinical: There were no clinical signs observed that could be related to treatment.

Table 3: Individual Scores for Ocular Irritation at 1 Hour

Animal No.	Score of Irritation								IPR
	Conjunctivae			Opacity of Cornea		Iris	Control Eye	Other Signs
	R	CH	D	OD	OE	R
1	2	1	1	0	0	0	0	-	2
2	1	0	1	0	0	0	0	-	2
3	2	0	2	0	0	0	0	-	2

 

Table 4: Individual Scores for Ocular Irritation at 24 Hour

Animal No.	Score of Irritation
	Conjunctivae			Opacity of Cornea		Iris	Control Eye	Other Signs
	R	CH	D	OD	OE	R
1	0	0	0	0	0	0	0	-
2	0	0	0	0	0	0	0	-
3	1	0	0	0	0	0	0	-

 

Table 5: Individual Scores for Ocular Irritation at 48 Hour

Animal No.	Score of Irritation
	Conjunctivae			Opacity of Cornea		Iris	Control Eye	Other Signs
	R	CH	D	OD	OE	R
1	0	0	0	0	0	0	0	-
2	0	0	0	0	0	0	0	-
3	0	0	0	0	0	0	0	-

 

Table 6: Individual Scores for Ocular Irritation at 72 Hour

Animal No.	Score of Irritation
	Conjunctivae			Opacity of Cornea		Iris	Control Eye	Other Signs
	R	CH	D	OD	OE	R
1	0	0	0	0	0	0	0	-
2	0	0	0	0	0	0	0	-
3	0	0	0	0	0	0	0	-

 

R = redness, CH = chemosis; D = discharge; OD = opacity degree of density; OE = extent of opaque area; IPR = initial pain reaction.

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of the test, the test material was determined to be non-irritating. Incidences of conjunctival redness and discharge were observed in all three animals one hour after administration, and one animal also showed evidence of chemosis. All ocular reactions were fully reversed by 48 hours post exposure. These reactions were slight and below the limit of classification.

Executive summary:

The potential for the test material to cause eye irritation was assessed in an in vivo irritation study in rabbits. The study was performed under GLP conditions in line with standardised guidelines OECD 405, EU Method B.5 and EPA OPPTS 870.2400. The test material was administered to three male New Zealand white rabbits, in a single 1 hour application. Rabbits were assessed over 72 hours for signs of eye irritation, clinical sign of toxicity, mortality and bodyweight gain. Application of the test material to the rabbit’s eye caused evidence of conjunctival redness and discharge in all three animals, one hour after administration. One animal also showed evidenece of chemosis at one hour. These signs of irritation were fully reversed within 48 hours, and subsequently the study was terminated after 72 hours. Under the conditions of the study, the test material was determined to be non-irritating.

This study was conducted on the the read across substance, sodium 5-oxo-L-prolinate.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin Irritation:

Two studies are available to address this endpoint, one key and one supporting.

 

Kiss (2012c) has been provided as the key study, where the potential for the test material to cause skin irritation was assessed in vitro using the EPISKIN Model. The study was performed under GLP and in accordance with OECD 439 and EU Method B. 46. The study was performed to a high standard and being a read-across study; has been assigned a reliability score of 2 in line with the principles for assessing data quality defined in Klimisch (1997).

Under the conditions of the test, exposure to the test material resulted in a mean relative cell viability of 95 %. Since the cell viability was determined to be > 50 % of the negative control the test material was considered to be non-irritating. This study has been used as read across and is considered to be suitable based on the structural similarities between read across substance, sodium 5 -oxo-L-prolinate and substance to be registered, sodium 5 -oxo-DL-prolinate.

 

The supporting study Hanzawa (1989) was conducted in vivo in accordance with generally accepted scientific principles, possibly with incomplete reporting or methodological deficiencies which do not affect the quality of the relevant results. This study has been assigned a reliability score of 2 in line with the principles for assessing data quality defined in Klimisch (1997). 

During the study, the test material was soaked into an adhesive plaster for patching which was then patched onto each of the hair clipped areas and fixed by wrapping each rabbit with a body cover. After closed patching for 24 hours, each body cover and adhesive plaster was peeled off from the rabbits and then they were subjected to judgement in accordance with Draize's acceptance criteria. Observations were made in the same manner at 24 hours, 48 hours and 72 hours.

Under the conditions of the study, the test material exhibited no irritant action.

 

Eye Irritation:

Three studies are available to address this endpoint, one key and two supporting, furthermore, two of these studies have been used as read across and are considered to be suitable based on the structural similarities between read across substance, sodium 5 -oxo-L-prolinate and substance to be registered, sodium 5 -oxo-DL-prolinate.

 

The key study Török-Bathó (2012) was performed using a read across substance, sodium 5-oxo-L-prolinate, which is the L- isomeric form of the substance. Read-across is considered to be suitable based on the structural similarities between the read across substance (sodium 5-oxo-L-prolinate) and the substance to be registered (sodium 5-oxo-DL-prolinate), which is a racemic mixture of the D- and L- isomeric forms of the substance. As such the difference in isomeric forms of the substance is unlikely to affect the chemical properties.

Török-Bathó (2012) was conducted under GLP conditions in line with standardised guidelines OECD 405, EU Method B.5 and EPA OPPTS 870.2400. The study was assigned a reliability score of 2 in line with the principles for assessing data quality as defined by Klimisch (1997).

In this key study, the potential for the test material to cause eye irritation was assessed in vivo using rabbits. The test material was administered to three male New Zealand white rabbits, in a single 1 hour application. Rabbits were assessed over 72 hours for signs of eye irritation, clinical sign of toxicity, mortality and bodyweight gain. Application of the test material to the rabbit’s eye caused slight conjunctival redness and discharge in all three animals, one hour after administration. One animal also showed slight chemosis at one hour. These slight signs of irritation were fully reversed within 48 hours, and subsequently the study was terminated after 72 hours. Under the conditions of the study, the test material was determined to be non-irritating.

 

The first supporting study, and second to be used as read across, Kiss (2012d) was performed in vitro using a read across substance, sodium 5-oxo-L-prolinate. An in vitro eye irritation study conducted in isolated chicken eyes was performed under GLP conditions and according to the standardised guidelines OECD 438 and EU Method B.48., to determine if treatment with the test material caused corrosion or severe irritation. Three chicken eyes were exposed to the test material (30 mg) for 10 seconds, rinsed with isotonic saline, and then assessed for irritation at intervals for up to 240 minutes. Toxic effects to the cornea are measured by a qualitative assessment of opacity, damage to epithelium based on application of fluorescein to the eye (fluorescein retention), and measurement of cornea thickness (swelling). Damage was assessed individually and then combined to derive an Eye Irritancy Classification. Positive and negative controls were run concurrently to assess the viability of the test system.

Under the conditions of the test, exposure to the test material resulted in an overall ICE class of 1 x I and 2 x II, which corresponds to an EU classification of non-irritating in accordance with the OECD guideline.

 

The second supporting study has been performed on the test material. Hanzawa (1989) is a non GLP study performed to sound scientific principles with a sufficient level of detail to assess the quality of the submitted data. The report was redrafted several years after the study period.The study was assigned a reliability score of 4 in line with the principles of assessing data as defined by Klimisch (1997).

During the study, the test material was administered to four male New Zealand rabbits and these rabbits were assessed over 72 hours for signs of irritation. Under the conditions of the study, no abnormalities were observed; therefore the test material was determined to be non-irritating.

Justification for selection of skin irritation / corrosion endpoint:
Two studies to address this endpoint; Hanzawa and Fujimoto (1989) was performed in accordance with generally accepted scientific principles, possibly with incomplete reporting or methodological deficiencies which do not affect the quality of the relevant results. It was been assigned a reliability score of 2 in line with the principles for assessing data quality defined in Klimisch (1997).
Kiss (2012c) has been performed in line with GLP and standardised guidelines. As the study has not been performed on the test material, but is considered to be a more robust study, it has also been assigned a reliability score of 2 in accordance with the criteria outlined in Klimisch (1997). It is considered to be the key study for this endpoint due to the superior qulaity of the study.

Justification for selection of eye irritation endpoint:
The key study was performed in vivo, in line with GLP and standardised guidelines. It was assigned a reliability score of 2 in accordance with the criteria outlined in Klimisch (1997). It was therefore considered suitable to be the key study for this endpoint.

Justification for classification or non-classification

Skin Irritation:

According to the criteria outlined in Regulation (EC) No. 1272/2008, the test material does not meet the criteria for classification as a skin irritant.

Eye Irritation:

According to the criteria outlined in Regulation (EC) No. 1272/2008, the test material does not meet the criteria for classification as an eye irritant.