[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

Acute toxicity: via oral route
The approximate LD50 was found to be greater than 2000 mg/kg bodyweight. the test mateial was therefore classified as non-toxic if swallowed.
The no-observed effect level was found to be 200 mg/kg bodyweight.
Acute toxicity: via inhalation route
Due to its nature, it proved impossible to generate atmospheres of the test item with a MMAD of less than 4 μm. It was, therefore, considered appropriate to terminate this study prior to the exposure of a group of animals.

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study performed according to method equivalent to OECD and EU Guidlines.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Version / remarks:

Safepharm Standard Method Number SPL 59

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)

Version / remarks:

Safepharm Standard Method Number SPL 59

Principles of method if other than guideline:

Study performed according to Safepharm Standard Method Number SPL 59.

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: supplied by Charles River (UK) Ltd., Manston, Kent.
- Age at study initiation: 6-9 weeks
- Weight at study initiation: Male: 149-172, Female: 143-151
- Fasting period before study: overnight fast
- Housing: solid-floor polypropylene cages with sawdust bedding
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: minimum acclimation period of 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23°C
- Humidity (%): 40-69%
- Air changes (per hr): 15changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours light, 12 hours dark

Route of administration:

oral: gavage

Vehicle:

other: suspension in 0.5% gum tragacanth

Details on oral exposure:

VEHICLE
- Concentration in vehicle: suspension in 0.5% gum tragacanth
- Amount of vehicle (if gavage): as stated above

MAXIMUM DOSE VOLUME APPLIED: no data (however 2000mg/kg @ 172g = 358mg)

Doses:

2000 mg/kg and 200 mg/kg

No. of animals per sex per dose:

3M+3F @ 2000 mg/kg and 1M + 1F @ 200 mg/kg

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: All animals were observed for overt signs of systemic toxicity 1/2, 1, 2 and 4 hours after dosing and then once daily for 14 days. Individual bodyweights were recorded on Day 0 (the day of dosing), on Day 7 and Day 14.
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight,organ weights, histopathology, other:Any Macroscopic findings were recorded and the affected tissues preserved in fixative (10% neutral buffered formaldehyde). The carcases and tissues showing no macroscopic lesions were also maintained in fixative.

Statistics:

Evaluation of data
From the Mortality data obtained, an estimate of the acute oral median lethal dose (LD50) and maximum non-lethal dose level was made, the estimated LD50 was used to classify the test material as follows:
Approximate LD50 > 2000 EEC: Unclassified
Approximate LD50 >200 to 2000 EEC: Harmful
Approximate LD50 >25 to 200 EEC: Toxic
Approximate LD50 <25 EEC: Very Toxic
The clinical observations and necropsy findings were examined and if possible the maximum no-observed effect level was identified.

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Sex:

male/female

Dose descriptor:

other: NOEL

Effect level:

200 mg/kg bw

Based on:

test mat.

Mortality:

For mortality data, see Table 1 (below).

Clinical signs:

other: Individual clinical observations are given in Table 2 (below). lethargy was noted in one male and two femals two or four hours after dosing. Ptosis was also noted in one female two hours after dosing. No Clinical signs of toxicity were noted in animals tr

Gross pathology:

Individual necropsy findings are given in Table 4 (below).
No macroscopic abnormalities were noted at necropsy.

Table 1: Mortality Data:

Dose Level mg/kg	Sex	Number of Animals Treated	Deaths
2000	M	3	0/3
	F	3	0/3
200	M	1	0/1
	F	1	0/1

Table 2: Individual Clinical Observations and Mortality Data

Dose Level mg/kg	Animal Number & Sex	Effects Noted after Dosing (hours)				Effects Noted During Period After Dosing (Days)
		1	1	2	4	1	2	3	4	5	6	7	8	9	10	11	12	13	14
2000	1-0 M	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	3-0 M	0	0	L	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	3-1 M	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	2-0 F	0	0	0	L	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	4-0 F	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	4-1 F	0	0	L Pt	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
200	5-0 M	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	6-0 F	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Where L = lethargy,                     Pt = ptosis,                0 = no signs of systemic toxicity

Table 3: Individual Bodyweights and Weekly Bodyweight Increases

Dose level mg/kg	Animal Number & Sex	Bodyweight (g) at Day			Increment (g) During Week
		0	7	14	1	2
2000	1-0  M	149	220	276	71	56
	3-0  M	152	214	273	62	59
	3-1  M	166	239	305	73	66
	2-0  F	149	195	210	46	15
	4-0  F	151	187	215	36	28
	4-1  F	151	199	233	48	34
200	5-0  M	172	265	334	93	69
	6-0  F	143	201	227	58	26

Table 4: Individual Necropsy Findings

Dose Level mg/kg	Animal Number & Sex	Macroscopic Observations
2000	1-0  M	No abnormalities detected
	3-0  M	No abnormalities detected
	3-1  M	No abnormalities detected
	2-0  F	No abnormalities detected
	4-0  F	No abnormalities detected
200	4-1  F	No abnormalities detected
	5-0  M	No abnormalities detected
	6-0  F	No abnormalities detected

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The approximate LD50 was found to be greater than 2000 mg/kg bodyweight. the test mateial was therefore classified as non-toxic if swallowed.
The no-observed effect level was found to be 200 mg/kg bodyweight.

Executive summary:

This study was performed to assess the acute oral lethality of the test material in the rat and to identify an approximate no-observed effect level. The results may be used as a basis for classification.

The acute oral toxicity of the test material was assessed by treatment of a group of fasted animals (three males and three females) at a dose level of a group of fasted animals (three males and three females) at a dose level of 2000 mg/kg bodyweight. The test material was administered as a suspension in 0.5% gum tragacanth.

There were no deaths. Lethargy was noted in three animals two or four hours after dosing.

All animals showed expected gain in bodyweight during the study.

No macroscopic abnormalities were noted at necropsy.

The no-observed effect level was determined by treatment of a pair of animals (one male and one female) at a lower dose level of 200 mg/kg bodyweight.

There were no deaths or clinical signs of toxicity. No macroscopic abnormalities were noted at necropsy.

The approximate LD50 was found to be greater tha 2000 mg/kg bodyweight.

The no-observed effect dose level was found to be 200 mg/kg bodyweight.

The test material was therefore classified as non-toxic if swallowed, according to 67/548/EEC Dangerous Substances Directive (DSD) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures (CLP)

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

Quality of whole database:

The key study has been assigned a Klimisch reliability rating of 1 (reliable without restriction).
Additional data for this endpoint includes an endpoint study report and two investigative brochure reports on the analogue material potassium clavulanate. The endpoint study report has been assigned a Klimisch rating of 2 whilst due to the age, number of studies discussed and the limited documentation of methods used, the investigative brochure information has been assigned a Klimisch rating of 4.
A further clinical brochure documented studies carried out orally and parenteral. Again, due to the age, number of studies discussed and the limited documentation of methods used, this information has been assigned a Klimisch rating of 4.

Acute toxicity: via inhalation route

Link to relevant study records

Reference

Endpoint:

acute toxicity: inhalation

Type of information:

other: Due to its nature, it proved impossible to generate atmospheres of the test item with a MMAD of less than 4 μm. It was, therefore, considered appropriate to terminate this study prior to the exposure of a group of animals.

Adequacy of study:

key study

Study period:

testing was performed on the 11th March 2013.

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:

no guideline required

Principles of method if other than guideline:

In order to comply with regulatory requirements, work was carried out in an attempt to generate atmospheres of the test item. Had this work been successful, the study would have proceeded such that it would have complied with the method described in the OECD Guidelines for Testing of Chemicals (2009) No. 436 “Acute Inhalation Toxicity – Acute Toxic Class Method”.

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

no

Species:

other: not applicable as no actual testing was conducted

Strain:

other: not applicable as no actual testing was conducted

Sex:

not specified

Details on test animals or test system and environmental conditions:

not applicable as no actual testing on animals was conducted

Route of administration:

other: not applicable as no actual testing on animals was conducted

Type of inhalation exposure:

other: not applicable as no actual testing on animals was conducted

Vehicle:

other: not applicable as no actual testing on animals was conducted

Details on inhalation exposure:

not applicable as no actual testing on animals was conducted

Analytical verification of test atmosphere concentrations:

not specified

Duration of exposure:

0 d

Concentrations:

not applicable as no actual testing on animals was conducted

No. of animals per sex per dose:

not applicable as no actual testing on animals was conducted

Control animals:

other: not applicable as no actual testing on animals was conducted

Details on study design:

not applicable as no actual testing on animals was conducted

Remarks on result:

other: It proved impossible to generate atmospheres of the test item with a MMAD of less than 4 μm. It was, therefore, considered appropriate to terminate this study prior to the exposure of a group of animals, therefore no actual testing was conducted.

Mortality:

Not applicable as no actual testing was conducted

Clinical signs:

other: Not applicable as no actual testing was conducted

Body weight:

Not applicable as no actual testing was conducted

Gross pathology:

Not applicable as no actual testing was conducted

Due to its nature, it proved impossible to generate atmospheres of the test item with a MMAD of less than 4 μm. It was, therefore, considered appropriate to terminate this study prior to the exposure of a group of animals.

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: expert judgment

Conclusions:

Due to its nature, it proved impossible to generate atmospheres of the test item with a MMAD of less than 4 μm. It was, therefore, considered appropriate to terminate this study prior to the exposure of a group of animals.

The dust generation properties of tBA Clavulanate with a particle size of less than 4 μm were shown to be low. It was, therefore, considered not to be possible to generate an atmosphere with a satisfactory particle size from the test item, for use in an inhalation study.

In view of the physical nature of the test item, its apparent low volatility and its’ tendancy to clump, it is considered unlikely to represent a significant hazard by the inhalation route.

Executive summary:

Due to its nature, it proved impossible to generate atmospheres of the test item with a MMAD of less than 4 μm. It was, therefore, considered appropriate to terminate this study prior to the exposure of a group of animals.

The dust generation properties of tBA Clavulanate with a particle size of less than 4 μm were shown to be low. It was, therefore, considered not to be possible to generate an atmosphere with a satisfactory particle size from the test item, for use in an inhalation study. In view of the physical nature of the test item, its apparent low volatility and its’ tendancy to clump, it is considered unlikely to represent a significant hazard by the inhalation route.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Quality of whole database:

The key study has been assigned a Klimisch reliability rating of 1 (reliable without restriction).
The study was conducted recently (2013) as there was potential for inhalation due to the high vapour pressure of the substance, tBA Clavulanate. Testing of the substance according to OECD guideline was not possible, as it was not possible to generate a particle size which could be inhaled by the test subjects. Testing was therefore cancelled before and animals were dosed.

Acute toxicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Acute toxicity: via oral route

Assessment of the test material was based mainly on the following two studies as the clinical brochures (with a Klimisch rating of 4) were considered to be the least reliable information, due to the age of the data and the lack of information regarding the methods used.

Acute toxicity: oral

The acute oral toxicity of the test material was assessed by treatment of a group of fasted animals (three males and three females) at a dose level of 2000 mg/kg bodyweight. The test material was administered as a suspension in 0.5% gum tragacanth.

There were no deaths. Lethargy was noted in three animals two or four hours after dosing.

All animals showed expected gain in bodyweight during the study.

No macroscopic abnormalities were noted at necropsy.

The no-observed effect level was determined by treatment of a pair of animals (one male and one female) at a lower dose level of 200 mg/kg bodyweight.

There were no deaths or clinical signs of toxicity. No macroscopic abnormalities were noted at necropsy.

The approximate LD₅₀ was found to be greater tha 2000 mg/kg bodyweight.

The no-observed effect dose level was found to be 200 mg/kg bodyweight.

Acute toxicity: oral - read-across

Among 4 day old rats, mortality rates in excess of 50% were observed at all dosages examined in both the initial and second neonate studies. The acute median lethal dose (LD₅₀) of orally administered BRL 14151 was therefore less than 500 mg of pure free acid per kilogram bodyweight for 4 day old rats.

Among 22 day old rats, there were twelve mortalities - 1 female at 4220 mg/kg, 3 males at 5620 mg/kg, 4 males and 4 females at 7500 mg/kg. The acute median lethal dose (LD50) and its 95% confidence limits for orally administered BRL 14151 was calculated to be 6200 (5300 to 7200) mg of pure free acid per kilogram bodyweight for 22 day old rats.

Among 33 day old rats, there was one mortality - 1 male at 7500 mg/kg. The acute median lethal dose (LD50) of orally administered BRL 14151 was therefore in excess of 7500 mg of pure free acid per kilogram bodyweight for 33 day old rats.

Acute toxicity: oral - read-across (Clinical Brochure)

LD₅₀ Mouse: subcutaneous: 3000 - 4000 mg/kg; intravenous: 3000 - 5000 mg/kg (collapse and convulsions).

LD₅₀ Rat: subcutaneous: > 3000 mg/kg; intravenous: 5000 mg/kg the following effects were observed throughout the study(collapse, hindlimb paralysis. Pale livers, kidneys at post-mortem).

Acute toxicity: via inhalation route

Testing through the inhalation route for tBA Clavulanate was thought to be necessary due to the high vapour pressure. However the dust generation properties of tBA Clavulanate with a particle size of less than 4 μm were shown to be low. It was, therefore, considered not to be possible to generate an atmosphere with a satisfactory particle size from the test item, for use in an inhalation study. In view of the physical nature of the test item, its apparent low volatility and its’ tendancy to clump, it is considered unlikely to represent a significant hazard by the inhalation route.

Justification for selection of acute toxicity – oral endpoint
The study has been carried out on the substance of interest, furthermore the study is a GLP study performed according to a method equivalent to OECD and EU Guidelines. The study has been assigned a Klimisch reliability rating of 1 (reliable without restriction).

Justification for selection of acute toxicity – inhalation 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

Acute toxicity: via oral route

The approximate LD₅₀ was found to be greater than 2000 mg/kg bodyweight. The test mateial was therefore classified as non-toxic if swallowed.

The No-Observed Effect Level was found to be 200 mg/kg bodyweight.

Based on the above conclusions the test material is considered to be not classified according to CLP (the EU version of GHS) or to the Dangerous Substances Directive (DSD).

Acute toxicity: via inhalation route

Due to its nature, it proved impossible to generate atmospheres of the test item with a MMAD of less than 4 μm. It was, therefore, considered appropriate to terminate this study prior to the exposure of a group of animals.

The dust generation properties of tBA Clavulanate with a particle size of less than 4 μm were shown to be low. Based on expert judgement it was, therefore, considered not to be possible to generate an atmosphere with a satisfactory particle size from the test item, for use in an inhalation study. In view of the physical nature of the test item, its apparent low volatility and its’ tendancy to clump, it is considered unlikely to represent a significant hazard by the inhalation route.

Based on the above conclusions the test material is considered to be not classified according to CLP (the EU version of GHS) or to the dangerous Substances Directive (DSD).