[2R-(2α,3Z,5α)]-3-(2-hydroxyethylidene)-7-oxo-4-oxa-1-azabicyclo[3.2.0]heptane-2-carboxylic acid, compound with tert-butylamine (1:1)

Endpoint summary

Description of key information

Skin Irritation
The test material produced a primary irritation index of 0.3 and was classified as Mild Irritant to rabbit skin according to the draize classification scheme. No corrosive effects were noted.
The test material did not produced positive criteria in any rabbit according to the EEC labelling regulations and was classified as Non-irritant to rabbit skin. No symbol and risk phrase are therefore required.
Eye Irritation
The test material produced a maximum group mean score of 7.0 and was classified as a mild irritant (Class 4 on a 1 - 8 scale) to the rabbit eye according to a modified Kay and Clandra classification system. However the test material did not produce positive criteria in any rabbit according to the EEC labelling regulations and was classified as non-irritant to the rabbit eye.

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

Study period:

2 October 1991 and 5 October 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: 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 relevant results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Qualifier:

according to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

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: supplied by David Percival Ltd., Moston, Sandbach, Cheshire, U.K.
- Age at study initiation:12-16 weeks
- Weight at study initiation: 2.31-2.68 kg
- Housing:suspended metal cages.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-22°C
- Humidity (%): 59-69%
- Air changes (per hr): 15 changes/hour
- Photoperiod (hrs dark / hrs light): 12 light / 12 dark

Type of coverage:

semiocclusive

Preparation of test site:

abraded

Vehicle:

water

Controls:

not required

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5g of substance moistened with 0.5ml of distilled water.

VEHICLE
- Amount(s) applied (volume or weight with unit): 0.5g of substance moistened with 0.5ml of distilled water.

Duration of treatment / exposure:

4 hours

Observation period:

72 hours

Number of animals:

3

Details on study design:

TEST SITE
- Area of exposure: 2.5 x 2.5 cm
- Type of wrap if used: gauze patches secured with surgical adhesive tape, the trunk of each animal was wrapped in an elasticated corset (Turbigrip)

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

SCORING SYSTEM:
Evaluation of skin Reactions
Erythema and Eschar Formation
no erythema ...............................................................................................................................................0
very slight erythema (barely perceptible) .............................................................................................1
Well-defined erythema .............................................................................................................................2
Moderate to severe erythema .................................................................................................................3
Severe erythema (beet redness) to slight eschar formation (injuries in depth) .............................4

Oedema Formation
No oedema..................................................................................................................................................0
Very Slight oedema (barely perceptible).................................................................................................1
Slight oedema ( edges of area well-defined by definite raising).........................................................2
Moderate oedema (raised approximately 1 mm)...................................................................................3
Severe oedema (raised more than 1 mm and extending beyond the area of exposure)................4

Interpretation of results
The scores for erythema and oedema for each intact and abraded skin site at the 24 and 72 hour readings were totalled for the three test rabbits (24 values) and this total was divided by 12 to give the primary irritation index of the test material.
The test material was classified based on the criteria layed out by Regulation (EC) 1272/2008 on classification, labelling and packaging of substances and mixtures (CLP) and 67/548/EEC Dangerous Substances Directive.

Irritation parameter:

primary dermal irritation index (PDII)

Basis:

mean

Time point:

other: 72 hours

Score:

0.3

Max. score:

2

Reversibility:

fully reversible within: 72 hours

Irritant / corrosive response data:

The individual scores for erythema /eschar and oedema, are given in Table 1. Mean Values Required for EEC labelling regulations are given in Table 2.
Yellow-coloured staining was commonly noted during the study. this did not affect evaluation of the skin responses.
Very slight erythema was noted at one intact treated skin site one hour after patch removal. very slight erythema was noted at two intact and two abbraded treated skin sites at the 24 hour observation and persisted at one abraded skin site at the 48 hour observation.
No signs of skin irritation were noted 72 hours after treatment.

Table 1 Individual Skin Reactions

Skin Reaction	Skin Site	Observation Time Hour(s)	Rabbit Number and Sec (Bodyweight Kg)
			40 Male (2.38)	41 Male (2.38)	42 Female (2.31)	Total
Erythema & Eschar Formation	Intact	1	0 STA	0 STA	0 STA	( 1 )
	Intact	24	0 STA	0 STA	0 STA	2
	Intact	48	0 STA	0 STA	0 STA	( 0 )
	Intact	72	0 STA	0 STA	0 STA	0
	Abraded	1	0 STA	0 STA	0 STA	( 0 )
	Abraded	24	0 STA	0 STA	0 STA	2
	Abraded	48	0 STA	0 STA	0 STA	( 1 )
	Abraded	72	0 STA	0 STA	0 STA	0
Oedema Formation	Intact	1	0	0	0	( 0 )
	Intact	24	0	0	0	0
	Intact	48	0	0	0	( 0 )
	Intact	72	0	0	0	0
	Abraded	1	0	0	0	( 0 )
	Abraded	24	0	0	0	0
	Abraded	48	0	0	0	( 0 )
	Abraded	72	0	0	0	0
Sum of 24 and 72 hour readings (5)  :         4
Primary Irritation Index ( 5/12 )          :         4/12 = 0.3
() = total values not used for calculation of primary irritation index                         STA = yellow-coloured staining

Table 2: Individual Daily and Idividual Mean Scores for Dermal Irritation 4 -hour exposure Required for EEC labelling regulations

Skin Reaction	Reading (hours)	Rabbit Number and Sex (Bodyweight Kg)
		40 Male (2.68)	41 Male (2.38)	42 Female (2.31)
Erythema/Eschar Formation	24	1	1	0
	48	0	0	0
	72	0	0	0
Total		1	1	0
Mean Score		0.3	0.3	0.0
Oedema Formation	24	0	0	0
	48	0	0	0
	72	0	0	0
Total		0	0	0
Mean Score		0.0	0.0	0.0

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test material, BRL 14151C, produced a primary irritation index of 0.3. No corrosive effects were noted during the study.
The test material did not produce positive criteria in any rabbit according to the EEC labelling regulations and was determined to be not irritating to rabbit skin. No symbol and risk phrase are therefore required.

Executive summary:

This study was performed to assess the irritancy potential of the test material to the skin of the New Zealand White rabbit.

A single 4 hour, semi-occluded application of tet material to intact and abraded skin of three rabbits produced incidents of very slight erythema. No signs of skin irritation were noted 72 hours after treatment.

No Corrosive effects were noted during the study. The test material was determined to be not irritating to rabbit skin according to criteria layed out by Regulation (EC) 1272/2008 on classification, labelling and packaging of substances and mixtures (CLP) and 67/548/EEC Dangerous Substances Directive.

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:

7 October 1991 and 12 October 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: 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 relevant results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: supplied by David Percival ltd., Moston, Sandbach, Cheshire, U.k.
- Age at study initiation: 12-16 weeks
- Weight at study initiation: 2.73-2.94 kg
- Housing: suspended metal cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23°C
- Humidity (%): 57-66%
- Air changes (per hr): 15 changes/hour
- Photoperiod (hrs dark / hrs light): 12 light / 12 dark

Vehicle:

unchanged (no vehicle)

Controls:

other: left eye untreated for control purposes.

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit):
A volume of 0.1 ml of the test material, which was found to weigh approximately 77 mg (as measured by gently compacting the required volume into an adapted syringe) was placed into the conjunctival sac of the right eye, formed by gently pulling the lower lid away from the eyeball.

Duration of treatment / exposure:

single application, no washing

Observation period (in vivo):

72 hours

Number of animals or in vitro replicates:

3

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): no washing

SCORING SYSTEM: Assessment of occular damage/irritation was made approximately 1 hour and 24, 24 and 72 hours following treatment, according to the numerical evaluation from Draize J.H. 1959, Association of Food and Frug Officials of the United States, Austin, Texas, "The Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics"

TOOL USED TO ASSESS SCORE: Examination of the eye was facilitated by use of the light source from a standard opthalmoscope.

Irritation parameter:

cornea opacity score

Remarks:

199 male

Basis:

mean

Time point:

other: average for 24,48 and 72 hours

Score:

0

Max. score:

4

Reversibility:

other: no effects seen

Irritation parameter:

iris score

Remarks:

199 male

Basis:

mean

Time point:

other: average for 24, 48 and 72 hours

Score:

0.3

Max. score:

2

Reversibility:

fully reversible

Irritation parameter:

chemosis score

Remarks:

199 male

Basis:

mean

Time point:

other: average for 24, 48 and 72 hours

Score:

1

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

cornea opacity score

Remarks:

194 female

Basis:

mean

Time point:

other: average

Score:

0

Max. score:

4

Reversibility:

other: no effects seen

Irritation parameter:

iris score

Remarks:

194 female

Basis:

mean

Time point:

other: average for 24, 48 and 72 hours

Score:

0

Max. score:

2

Reversibility:

other: no effects seen

Irritation parameter:

chemosis score

Remarks:

194 female

Basis:

mean

Time point:

other: average for 24, 48 and 72 hours

Score:

0

Max. score:

4

Reversibility:

other: no effects seen

Irritation parameter:

cornea opacity score

Remarks:

197 female

Basis:

mean

Time point:

other: average of 24, 48 and 72 hours

Score:

0

Max. score:

4

Reversibility:

other: no effects seen

Irritation parameter:

iris score

Remarks:

197 female

Basis:

mean

Time point:

other: average of 24, 48 and 72 hours

Score:

0.3

Max. score:

2

Reversibility:

fully reversible

Irritation parameter:

chemosis score

Remarks:

197 female

Basis:

mean

Time point:

other: average of 24, 48 and 72 hours

Score:

0.3

Max. score:

4

Reversibility:

fully reversible

Irritant / corrosive response data:

Individual and group mean scores for ocular irritation are given in Tables 1 and 2. the Individual mean scores as required for the EEC labelling regulations are presented in Table 3.
Residual Test material was noted around the treated eyes of all anmals during the study.
No adverse corneal effects were noted during the study.
Iridial Inflamation was noted in one treated eye at the 24-hour observation. No other Adverse iridial effects were noted.
Minimal conunctival irritation was noted in all treated eyes one hour after treatment and in two treated eyes at the 24-hour observation. The reaction increased to moderate conjunctival irritation in one treated eye at the 24 hour observation with minimal conjunctival irritation ath the 48-hour observation.
All treated eyes appeared normal 72 hours after treatment.

Table 1: Individual Scores and Individual Total Scores for Occular Irritation

Rabbit Number and Sex (Bodyweight kg)	199 Male (2.76) (IPR = 3)				194 Female (2.94) (IPR = 3)				197 Female (2.73) (IPR= 2)
Time After Treatment	1hr	24hr	48hr	72hr	1hr	24hr	48hr	72hr	1hr	24hr	48hr	72hr
Cornea
E = Degree of Opacity	0	0	0	0	0	0	0	0	0	0	0	0
F = Area of Opacity	0	0	0	0	0	0	0	0	0	0	0	0
Score = (E x F) x 5	0	0	0	0	0	0	0	0	0	0	0	0
Iris
D	0	1	0	0	0	0	0	0	0	0	0	0
Score = (Dx5)	0	5	0	0	0	0	0	0	0	0	0	0
Conjunctivae
A = Redness	1	2	1	0	1	1	0	0	1	1	0	0
B = Chemosis	1	2	1	0	1	0	0	0	1	1	0	0
C = Discharge	0 Re	1	0	0	0 Re	0 Re	0 Re	0 Re	0 Re	0 Re	0	0
Score = (A+B+C)x2	4	10	4	0	4	2	0	0	4	4	0	0
Total Score	4	15	4	0	4	2	0	0	4	4	0	0
Key:  hr = hour(s)               IPR = initial pain reaction               Re = residual test material around the treated eye

Table 2 : Individual Total Scores and Group Mean Scores for Occular irritation

Rabbit Number and Sex	Individual Total Scores At:
	1 hour	24 hours	48 hours	72 hours
199 Male	4	15	4	0
194 Female	4	2	0	0
197 Female	4	4	0	0
Group Total	12	21	4	0
Group Mean Score	4	7	1.3	0

Table 3 : Individual & Mean Scores for Cornea, Iris & Conjunctivae Required for EEC labelling regulations

Rabbit Number & Sex (Bodyweight Kg)	Time After Treatment	Corneal Opacity	Iridial Inflamation	Conjunctival Redness	Conjunctival Chemosis
199 Male (2.76)	24 hours	0	1	2	2
	48 hours	0	0	1	1
	72 hours	0	0	0	0
Total		0	1	3	3
Mean		0	0.3	1	1
194 Female (2.94)	24 hours	0	0	1	0
	48 hours	0	0	0	0
	72 hours	0	0	0	0
Total		0	0	1	0
Mean		0	0	0.3	0
197 Female (2.73)	24 hours	0	0	1	1
	48 hours	0	0	0	0
	72 hours	0	0	0	0
Total		0	0	1	1
Mean		0	0.3	0.3	0.3

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test material did not produce positive criteria in any rabbit according to the EEC labelling regulations and was classified as non-irritant to the rabbit eye.

Executive summary:

A single application of the test material to the non-irrigated eye of three rabbits produced transient minimal to moderate conjunctival irritation. All trreated eyes appeared normal eye at the 24 -hour observation. All treated eyes appeared normal 72 hours after treatment.

However the test material did not produce positive criteria in any rabbit according to the EEC labelling regulations and was classified as non-irritant to the rabbit eye according to the EEC labelling regulations. No symbol and risk phrase are required.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin Irritation

This study was performed to assess the irritancy potential of the test material to the skin of the New Zealand White rabbit.

A single 4 hour, semi-occluded application of tet material to intact and abraded skin of three rabbits produced incidents of very slight erythema. No signs of skin irritation were noted 72 hours after treatment.

The test material was also classified as a mild irritant to rabbit skin according to the Draize Classification scheme. No Corrosive effects were noted. The test material was not classified as an irritant to rabbit skin according to EEC labelling regulations.

In addition, a study carried out on the analogue substance potassium clavulanate (Klimisch rating 2) concluded that the analogue substancewas not irritating to rabbit skin according to EEC labelling regulations.

Eye Irritation

A single application of the test material to the non-irrigated eye of three rabbits produced transient minimal to moderate conjunctival irritation. Iridial inflammation was confined to one treated eye at the 24 -hour observation. All treated eyes appeared normal 72 hours after treatment.

The test material did not produce positive criteria in any rabbit according to the EEC labelling regulations and was classified as non-irritant to the rabbit eye according to the EEC labelling regulations. No symbol and risk phrases are required.

In addition, a study carried out on the analogue substance potassium clavulanate (Klimisch rating 2) concluded that the analogue substance to be also classified as non-irritant to the rabbit eye according to EEC labelling regulations.

Justification for selection of skin irritation / corrosion endpoint:
The study has been carried out on the substance of interest, furthermore the study has been 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 relevant results. The study has been assigned a Klimisch reliability rating of 1 (reliable without restriction).

Justification for selection of eye irritation endpoint:
The study has been carried out on the substance of interest, furthermore the study has been 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 relevant results. The study has been assigned a Klimisch reliability rating of 1 (reliable without restriction).

Justification for classification or non-classification

In a study to assess the skin irritancy potential of the test material there were no signs of skin irritation noted 72 hours after treatment.

In a study to assess the eye irritancy potential of the test material, all treated eyes appeared normal 72 hours after treatment.

Based on these results the test material was shown to be not irritating to both rabbit skin and eyes according to EEC labelling regulations.