benzhydryl (6R,7R)-7-[[(2Z)-2-(5-amino-1,2,4-thiadiazol-3-yl)-2-trityloxyiminoacetyl]amino]-3-formyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate

Acute Toxicity: oral

Administrative data

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 August 2017 to 13 September 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

OECD GUIDELINES FOR TESTING OF CHEMICALS (423, adopted at 17th Dec. 2001)

Deviations:

yes

Remarks:

Due to technical reason, relative humidity values (maximum of 80 %) outside the expected range of 30-70 % were recorded during the acclimation period. This deviation has no presumed impact on the outcome or integrity of the study.

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Regulation (EC) No 440/2008 of 30 May 2008, B.1.tris

Deviations:

yes

Remarks:

Due to technical reason, relative humidity values (maximum of 80 %) outside the expected range of 30-70 % were recorded during the acclimation period. This deviation has no presumed impact on the outcome or integrity of the study.

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1100 (Acute Oral Toxicity)

Version / remarks:

EPA Health Effects Test Guidelines (OPPTS 870.1100), United States, EPA 712-C-98-190 (1998)

Deviations:

yes

Remarks:

Due to technical reason, relative humidity values (maximum of 80 %) outside the expected range of 30-70 % were recorded during the acclimation period. This deviation has no presumed impact on the outcome or integrity of the study.

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

diphenylmethyl (6R,7R)-7-[(2Z)-2-(5-amino-1,2,4-thiadiazol-3-yl)-2-[(triphenylmethoxy)imino]acetamido]-3-formyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate

EC Number:

609-462-3

Cas Number:

376653-37-1

Molecular formula:

C44H34N6O6S2

IUPAC Name:

diphenylmethyl (6R,7R)-7-[(2Z)-2-(5-amino-1,2,4-thiadiazol-3-yl)-2-[(triphenylmethoxy)imino]acetamido]-3-formyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate

Test material form:

solid: particulate/powder

Details on test material:

Name: BAL0001024
CAS number: 376653-37-1
Batch/Lot number: 013
Appearance: Brown powder
Purity: 63.1% (content by qNMR)
Expiry date: 31 May 2020
Storage conditions: Under inert gas, protected from light and humidity (tight closed container), frozen (≤-15 °C)
Safety precautions: Enhanced safety precautions were applied considering the supplied safety datasheet to assure personnel health and safety.

Specific details on test material used for the study:

No further details specified in the study report.

Test animals

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

Species and strain: CRL:(WI) Wistar rats
Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633 Sulzfeld
Hygienic level at arrival: SPF
Hygienic level during the study: Standard housing conditions
Number of animals: 6 animals
Sex: Female, nulliparous and non-pregnant
Age of animals at dosing: Young healthy adult rats, 9 weeks old
Body weight at treatment: 210 – 244 g
Acclimatization period: at least 12 days

Husbandry
Animal health: Only healthy animals were used for the test. The staff Veterinarian certified health status.
Number of animal room: 522/1
Housing: 3 animals / cage
Cage type: Type II polypropylene/polycarbonate
Bedding/Nesting: “Lignocel 3/4-S Hygienic Animal Bedding” and “Arbocel crinklets natural” nest building material produced by J. Rettenmaier & Söhne GmbH & Co.KG (D-73494 Rosenberg, Germany) were available to animals during the study.
Lighting period: 12 hours daily, from 6.00 am to 6.00 pm
Temperature: 22 ± 3 °C
Relative humidity: 33-80%
Ventilation: 15 – 20 air exchanges/hour
Enrichment: Animals were housed by group to allow social interaction and with deep wood sawdust bedding to allow digging and other normal rodent activities.
The room temperature and relative humidity were recorded twice daily during the study.

Food and Water Supply
Animals received ssniff® SM R/M "Autoclavable complete diet for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest, Germany (Batch no.: 262 21592, expiry date: 31 January 2018), ad libitum, and tap water from the municipal supply, as for human consumption from 500 ml bottles, ad libitum. The food is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
Water quality control analysis is performed once every three months and microbiological assessment is performed monthly, by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József A.u.36., Hungary).

Animal Identification
Animals were individually identified using numbers written on the tail with an indelible marker pen. The numbers were given on the basis of Citoxlab Hungary Ltd.' s Master File, for each animal allocated to the treatment groups. The cages were identified by cards, with information about study code, sex, dose group, cage number and individual animal numbers.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

PEG 400

Details on oral exposure:

Formulation
The test item was freshly formulated at a concentration of 200 mg/mL in the vehicle, in the Pharmacy of Citoxlab Hungary Ltd. on the day of administration. The formulation container was stirred continuously up to finishing the treatment.

Vehicle Selection
The selection of the vehicle was made during trial formulations with the test item. The final choice of vehicle was approved by the Sponsor.
On the basis of the trial formulations with the test item, the vehicle used was PEG 400.
Vehicle: PEG 400 (Poly (ethylene glycol))
Batch number: BCBS1795V
Expiry date: 31 May 2018

Doses:

A limit of 2000 mg/kg bw dose was selected by the Sponsor in line with the guidelines.

No. of animals per sex per dose:

Initially, three females (assigned to Group 1) were treated at a dose level of 2000 mg/kg bw. As the test item did not cause mortality in this group a second group (Group 2) was treated at the same dose level.

Control animals:

no

Details on study design:

Procedure
A single oral gavage administration (dose administered at 10 mL/kg bw) was followed by a 14-day observation period. The day before treatment, food was removed at the end of the working day and the animals therefore fasted for a period of approximately 17 hours. Water was not withheld during this period. Animals were weighed before treatment. The test item was administered by oral gavage in the morning and food made available 3 hours after the treatment.

OBSERVATIONS
Clinical Observations
Clinical observations were performed at 30 minutes, 1, 2, 3, 4 and 6 hours after dosing and daily for 14 days thereafter. Individual observations were performed on the skin, fur, eyes, mucous membranes, respiratory, circulatory, autonomic and central nervous system, somatomotor activity and behaviour pattern. Particular attention was directed to observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

Body Weight Measurement
The body weight was recorded the day before treatment (Day -1), on the day of the treatment (Day 0), Day 7 and Day 14.

NECROPSY
All animals were subjected to a necropsy and a macroscopic examination at the end of the study on Day 14. The animals were exsanguinated after verification of narcosis following an injection of pentobarbital sodium (Release; Lot No.: 106075, Expiry Date: July 2018, Produced by: Wirtschaftsgenossenschaft deutscher Tierärzte eG, Siemensstr. 14, 30827 Garbsen, Germany). After examination of the external appearance, the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed. All gross macroscopic changes were recorded for each animal.

Statistics:

Not specified

Results and discussion

Effect levels

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

BAL0001024 did not cause mortality at a dose level of 2000 mg/kg bw.

Clinical signs:

Clinical signs were observed in all animals treated at the dose level of 2000 mg/kg bw with BAL0001024 and included decreased activity (6/6), hunched back (6/6) and eyelids partially closed (6/6).
All symptoms had fully reversed in all animals by the 6 hours and/or Day 1 observation time points

Body weight:

Two animals at dose level 2000 mg/kg bw (No: 630, 634) showed a reduced body weight gain (0.39 and 2.61 %) in the second week of the observation period. This change was considered incidental and minimal and not ascribed to treatment. There were no treatment related effects on body weight or body weight gain during the observation period.

Gross pathology:

There was no evidence of treatment-related macroscopic changes at necropsy at a dose level of 2000 mg/kg bw.

Any other information on results incl. tables

INDIVIDUAL CLINICAL OBSERVATIONS

DOSE LEVEL: 2000 mg/kg bw, Treatment on Day 0     SEX: FEMALE

Cage No.	Animal Number	Observations	Observation days													Frequency
			0						1	2	3	4	5	6	7-14
			30’	1h	2h	3h	4h	6h
1	630	Symptom Free	+	-	-	-	-	+	+	+	+	+	+	+	+	16/20
		Activity decreased	-	1	1	1	-	-	-	-	-	-	-	-	-	3/20
		Hunched back	-	-	+	-	-	-	-	-	-	-	-	-	-	1/20
		Eyelids partially closed	-	-	+	+	+	-	-	-	-	-	-	-	-	3/20
	631	Symptom Free	+	-	-	-	-	-	+	+	+	+	+	+	+	15/20
		Activity decreased	-	1	1	1	-	-	-	-	-	-	-	-	-	3/20
		Hunched back	-	+	-	-	-	-	-	-	-	-	-	-	-	1/20
		Eyelids partially closed	-	-	+	+	+	+	-	-	-	-	-	-	-	4/20
	632	Symptom Free	+	-	-	-	-	-	+	+	+	+	+	+	+	15/20
		Activity decreased	-	1	1	1	-	-	-	-	-	-	-	-	-	3/20
		Hunched back	-	-	+	-	-	-	-	-	-	-	-	-	-	1/20
		Eyelids partially closed	-	-	+	+	+	+	-	-	-	-	-	-	-	4/20
2	633	Symptom Free	+	-	-	-	-	-	+	+	+	+	+	+	+	15/20
		Activity decreased	-	1	1	1	-	-	-	-	-	-	-	-	-	3/20
		Hunched back	-	+	+	-	-	-	-	-	-	-	-	-	-	2/20
		Eyelids partially closed	-	+	+	+	+	+	-	-	-	-	-	-	-	5/20
	634	Symptom Free	+	-	-	-	-	-	+	+	+	+	+	+	+	15/20
		Activity decreased	-	1	1	1	-	-	-	-	-	-	-	-	-	3/20
		Hunched back	-	+	-	-	-	-	-	-	-	-	-	-	-	1/20
		Eyelids partially closed	-	+	+	+	+	+	-	-	-	-	-	-	-	5/20
	635	Symptom Free	+	-	-	-	-	-	+	+	+	+	+	+	+	5/20
		Activity decreased	-	1	1	1	-	-	-	-	-	-	-	-	-	3/20
		Hunched back	-	+	-	-	-	-	-	-	-	-	-	-	-	1/20
		Eyelids partially closed	-	+	+	+	+	+	-	-	-	-	-	-	-	5/20

Remarks:              + = present           - = absent

                               h = hour‘ = minute

                               Frequency of observation = number of occurrence of observation / total number of observations

                               Severities: 1 = Slight/Small/Few; 2 = Moderate/Medium; 3 = Marked/Large/Many

 

INDIVIDUAL BODY WEIGHT AND BODY WEIGHT GAIN

DOSE LEVEL: 2000 mg/kg bw, Treatment on Day 0                 SEX: FEMALE

Cage No.	Animal Number	Bodyweight (g) Days				Body Weight Gain (g)
		-1	0	7	14	1—0	0-7	7-14	-1-14
1	630	236	226	258	257	-10	32	-1	21
	631	244	226	254	266	-18	28	12	22
	632	255	244	273	284	-11	29	11	29
2	633	228	210	239	256	-18	29	17	28
	634	245	229	268	261	-16	39	-7	16
	635	235	222	243	247	-13	20	5	12
Mean:		240.5	226.2	255.7	261.8	-14.3	29.5	6.2	21.3
Standard deviation:		9.5	11.0	13.6	12.5	3.5	6.2	9.0	6.6

 

INDIVIDUAL INTERNAL AND EXTERNAL MACROSCOPIC OBSERVATIONS

DOSE LEVEL: 2000 mg/kg bw, Treatment on Day 0                             SEX: FEMALE

Cage No.	Animal Number	Necropsy Date/ Necropsy Day	External Observations	Internal Observations	Organ/Tissue
1	630	12 September 2017 Day 14	No external observations recorded	No internal observations recorded	Not applicable
	631	12 September 2017 Day 14	No external observations recorded	No internal observations recorded	Not applicable
	632	12 September 2017 Day 14	No external observations recorded	No internal observations recorded	Not applicable
2	633	13 September 2017 Day 14	No external observations recorded	No internal observations recorded	Not applicable
	634	13 September 2017 Day 14	No external observations recorded	No internal observations recorded	Not applicable
	635	13 September 2017 Day 14	No external observations recorded	No internal observations recorded	Not applicable

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In the absence of any acute toxicity, BAL0001024 can be ranked as "Category 5" or "Unclassified" for acute oral toxicity according to the GHS criteria. Although this study was not designed to determine the acute oral LD50, under the conditions of this study, it is assumed for BAL0001024 to be above 2000 mg/kg bw in female CRL:(WI) rats.

Executive summary:

The single-dose oral toxicity of BAL0001024 was performed according to the acute toxic class method (OECD 423 and Commission Regulation (EC) No 440/2008 of 30 May 2008, B.1. tris) in CRL:(WI) rats.

 

Initially, three females (Group 1) were treated at a dose level of 2000 mg/kg bw. The test item was administered formulated in Poly (ethylene glycol) 400 (PEG 400) at a concentration of 200 mg/mL at a dosing volume of 10 mL/kg bw. A single oral treatment was carried out by gavage for each animal after food had been withdrawn overnight. Food was made available again 3 hours after the treatment. Clinical observations were performed at 30 minutes, 1, 2, 3, 4 and 6 hours after dosing and daily for 14 days thereafter. Body weight was measured on Days -1, 0 and 7 and Day 14. All animals were subjected to a necropsy and a macroscopic examination.

 

As no mortality was observed in group 1 a second group (Group 2) of 3 animals was treated at the same dose level using the methods described for group 1.

 

 

Results

 

Mortality

BAL0001024 did not cause mortality at a dose level of 2000 mg/kg bw.

 

Clinical Observations

Clinical signs were observed in all animals treated at the dose level of 2000 mg/kg bw with BAL0001024 and included decreased activity (6/6), hunched back (6/6) and eyelids partially closed (6/6). All symptoms had fully reversed in all animals by the 6 hours and/or Day 1 observation time points.

 

Body Weight and Body Weight Gain

Two animals at dose level 2000 mg/kg bw (No: 630, 634) showed a reduced body weight gain (0.39 and 2.61 %) in the second week of the observation period. This change was considered incidental and minimal and not ascribed to treatment. There were no treatment related effects on body weight or body weight gain during the observation period.

 

Macroscopic Findings

There was no evidence of treatment-related macroscopic changes at necropsy in animals given 2000 mg/kg bw.

 

Conclusion:

In the absence of any acute toxicity, BAL0001024 can be ranked as "Category 5" or "Unclassified" for acute oral toxicity according to the GHS criteria. Although this study was not designed to determine the acute oral LD50, under the conditions of this study, it is assumed for BAL0001024 to be above 2000 mg/kg bw in female CRL:(WI) rats.