[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

Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information

All the results were negative with the exception of a statistically significant increase in micronuclei at the high dose sampled at 24h, which was mainly attributable to males. However this was insufficient to give overall test significance.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

The study was conducted between 16/09/83 and 18/11/83.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted in accordance with generally accepted scientific principles, possibly with incomplete or methodological deficiencies, which do not affect the quality of relevant results.

Qualifier:

according to guideline

Guideline:

other: The test was based on the method proposed by Schmid (1975) with adjustments to dosing and sampling times as suggested in the Report of the UKEMS Sub-Committee on Guidelines for Mutagenicity Testing (Feb. 1983).

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

Mouse CDl, 7-12 weeks old were obtained from Charles River, Manston, Kent.

Animals were housed according to sex in polypropylene cages. Food and water were readily available (Diet: CRMX, Labsure Ltd.). The study was initiated after a 5 day acclimatization period. 85 animals were used in the test.

Route of administration:

oral: gavage

Vehicle:

distilled water

Details on exposure:

BRL 14151K was dosed as a solution in distilled water.
The dosing volume for the agent and controls was 1.0 ml/100g throughout.

Duration of treatment / exposure:

2 hours in experiment 1 and 48 hours in experiment 2

Frequency of treatment:

All animals were given a single dose of the vehicle or compound.

Remarks:

Doses / Concentrations:
1500
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
3000
Basis:
nominal conc.

No. of animals per sex per dose:

3 males and 3 females in the positive control
5 males and 5 females in the negative control
5 males and 5 females in the 1500 mg pfa/kg
5 males and 5 females in the 3000 mg pfa/kg

Control animals:

yes, concurrent vehicle

Positive control(s):

The negative control was distilled water and the positive control was cyclophosphamide (Endoxana, Wellcome) made up as a solution in distilled water and dosed at 75mg/kg.

Details of tissue and slide preparation:

Animals were killed and the femurs dissected out. Adherent tissue was removed from the bone and the epiphyses cut off. The bone marrow was washed out of each shaft with 0.2ml foetal calf serum into a 15ml centrifuge tube containing 4ml (final volume) of serum and mixed well.

Each cell suspension was centrifuged at 1000rpm for 5 minutes and the supernatant discarded. The cells were resuspended in the remaining liquid by gentle aspiration with a glass Pasteur pipette.

Smears were prepared by placing a small drop of the suspension on a clean, grease-free slide about 1-2cm from the end. The spreading slide was placed at an angle of 45° to the slide and moved back to make contact with the drop. The film was spread by a rapid smooth forward movement of the sp.reader and then air-dried. Four to six slides were prepared per animal.

Evaluation criteria:

To be considered genetically significant treated sample must be stochastically significant at p < 0.005 (as analysed above) and must also exceed the negative control by > 2.5 fold.

Statistics:

Comparison between the negative control group and treatment group(s) was made for the following parameters, using suitable statistical methods:
i) polychromatic cells as a proportion of the total cells scored.
ii) micronucleated polychromatic cells as a proportion of total polychromatic cells.
iii) micronucleated mature cells as a proportion of total mature cells.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

There were three mortalities during the study. One male died in the high dose group, Cage 11 (Sample I) and two males died in the low dose group: one from Cage 7 (Sample I) and one from Cage 9 (Sample II).

Vehicle controls validity:

other: the results for the male negative control was atypically low viz. one out of 5000 compared with a median value of 5.5 out of 5000.

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

There were three mortalities during the study. One male died in the high dose group, Cage 11 (Sample I) and two males died in the low dose group: one from Cage 7 (Sample 1) and one from Cage 9 (Sample II). Sufficient animals remained in each group thus it was not necessary to dose further animals.

Analysis of results (Sample 1):

At the high dose of BRL 14151K (3000mg pfa/kg) there was a 3 fold increase in the number of micronuclei observed in polychromatic cells, mainly attributable to the 4-5 fold increase over the controls in the male group. The result was not significant when the data for males and females were analysed separately by the Kolmogorov-Smirnov two sample test. However when the data for males and females were combined, the result was just significant at p < 0.05 (P> 0.025).

Using an alternative parametric analysis (the Behrens-Fisher test) the result was significant at p = 0.005 (p > 0.0005) for the combined male and female results (and at p < 0.025, > 0.010 for males only).

At the low dose of BRL 14151K (1500mg pfa/kg) there were no significant increases in micronuclei relative to the control; this included the small (approximately 2 fold) increase observed in the males.

Analysis of results: Sample 2 (48 hour post treatment):

The number of micronuclei observed at the high dose of BRL 14151K (3000 mg pfa/kg) was marginally above the negative control. Statistical analysis showed that the result was not significant. At the low dose of BRL 14151K (1500mg pfa/kg) there were no significant increases in micronuclei for either or both sexes compared to the negative control.

Discussion:

The results from the 24h sample (combined males and females) at the high dose (BRL 14151K, 3000mg pfa/kg) were statistically significant at p < 0.05, P > 0.025 using the kolmogorov-smirnov two sample test, and at P = 0.005, P > 0.0005 using the Behrens-Fisher test.

For theoretical reasons the Kolmogorov-Smirnov test under-estimates significance and the Behrens-Fisher test overestimates significance. Taking these considerations into account the individual sample significance probably lies between p = 0.005 and

p = 0.05. This is not sufficient to give the test overall significance after the application of the Bonferroni correction (for multi-comparisons

Thus, despite the size of the numerical increase in micronuclei in this group the result in the high dose group at 24h could be a type I (a) error i.e. a statistical artefact.

Please see tables A and B in the any other information on results section:

the result for the male negative control was atypically low viz. one out of 5000 compared with a median value of 5.5 out of 5000, see table A for more info.

Also an increase in the number of micronuclei was observed for males treated at the high dose group Sample I (24h), see table B for more details. All tables have been attached in the attached background material section.

Conclusions:

Interpretation of results (migrated information): negative
All the results were negative with the exception of a statistically significant increase in micronuclei at the high dose sampled at 24h, which was mainly attributable to males. However this was insufficient to give overall test significance.

Executive summary:

A test was conducted according to the following guideline:

The test was based on the method proposed by Schmid (1975) with adjustments to dosing and sampling times as suggested in the Report of the UKEMS Sub-Committee on Guidelines for Mutagenicity Testing (Feb. 1983).

All the results of the investigation were negative with the exception of a statistically significant increase in micronuclei at the high dose sampled at 24h, which was mainly attributable to males. However this was insufficient to give overall test significance.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

In total eight studies have been used to address this endpoint. Of these, six of the studies have been carried out on the analogue material potassium clavulanate and two on the anion Tert-butylamine. The studies carried out on Tert-butylamine were intended to investigate the effects of the TBA component of the substance of interest.

Four of the studies were in vitro, the remaining four studies being in vivo.

Brice: in vivo mouse micronucleus (potassium clavulanate) (Key study)

The test was based on the method proposed by Schmid (1975) with adjustments to dosing and sampling times as suggested in the Report of the UKEMS Sub-Committee on Guidelines for Mutagenicity Testing (Feb. 1983).

All the results of the investigation were negative with the exception of a statistically significant increase in micronuclei at the high dose sampled at 24h, which was mainly attributable to males. However this was insufficient to give overall test significance.

In vitro information

Mcgregor: Ames (potassium clavulanate)

The ability of the bacterial strains to respond to low concentrations of a known mutagen was demonstrated. Also, the ability of the liver preparations to activate 2-aminoanthracene was shown. In spite of these demonstrations of the correct functioning of the test system, no mutagenic property could be found in the preparation submitted.The high toxicity of the compound to bacteria in the presence of liver preparations is worthy of further investigation.

Takeda: Ames (potassium clavulanate)

In the Ames test the number of revertant colonies showed no major differences from the negative controls for potassium clavulanate. No mutations were induced in any of the test strains. There were also no effects for the compound in the metabolic activation method by adding S9 mix. Therefore. it is assumed that tBA clavulanate does not induce genetic mutations either in the presence or absence of metabolic activation.

In vivo information: Potassium clavulanate

Takeda: Mouse micronucleus (potassium clavulanate)

In the micronucleus test, there were no significant increases in the number of cells with micronuclei in the polychromatic erythrocytes 6 and 30 hours after the administration of potassium clavulanate. Injection of potassium clavulanate did not increase the incidence of micronuclei to polychromatic erythrocytes of Jcl-ICR mice (8-9 weeks old) 6 hours or 30 hours after treatment.

Takeda: Gene mutation (potassium clavulanate)

Potassium clavulanate did not cause dominant lethality in Jcl-ICR mice (male 10 weeks old). Therefore, we can conclude that potassium clavulanate and its possible metabolites has no mutagenic activities.

Mitchell, Carlton & Brice: Chromosome aberration(potassium clavulanate)

SRL 14151K did not produce an increase in the incidence of micronuclei at any level tested. Thus BRL 14151K showed no genetic activity in thisin vivo assay.

As the only difference between the surrogate material potassium clavulanate and the test material tBA clavulanate is a different salt, testing has also been conducted on the anion tert butylamine. A chromosome aberration study and an AMES study were conducted on the anion, please see the results below:

In Vitro studies conducted on tert-butylamine:

Chromosome aberration (Tert-butylamine)

There were no significant increases in the frequency of cells with structural aberrations (excluding gaps) in any of the test concentrations analysed compared with the concurrent vehicle controls, indicating a negative result.

Gene mutation (Tert-butylamine)

At the maximum concentration tested (365.7 ug/mL) the Relative Total Growth (RTG) was 115% and 110% for the 3 hour treatment in the presence and absence of S9-mix, respectively and 79% for the 24 hour treatment in the absence of S9-mix. The mutant frequencies of the concentrations plated were all less than the sum of the mean vehicle control mutant frequency plus the global evaluation factor (GEF), indicating a negative result.

The negative results obtained in the in vitro studies conducted on potassium clavulanate and tert butylamine are considered to represent both parts of the test substance, and gives a good indication that tBA Clavulanate is not mutagenic. It was concluded that based on the two negative results obtained for the in vitro studies on tert-butylamine, that no further in vivo testing was required on the anion.

Justification for selection of genetic toxicity endpoint
The study was conducted on the analogue material potassium clavulanate. The analogue material is considered to be similar enough to the substance of interest for results to be used for the purpose of Health and Hazard assessment.
The study conducted by Brice in 1984 has been considered to be the main study of interest.The study was conducted in accordance with generally accepted scientific principles, possibly with incomplete or methodological deficiencies, which do not affect the quality of relevant results and as a result was assigned the Klimisch rating of 2 (reliable with restrictions).

Justification for classification or non-classification

A number of studies (in vitro and in vivo) were carried out on the analogue material potassium clavulanate and the anion Tert-butylamine.

Although the majority of the studies were carried out on the analogue material, the analogue is considered to be sufficiently similar to the substance of interest (please see attached data matrix and justification in Section 13 for additional details) for it to be used for the purposes of health and environmental risk assessments.

In all cases conclusions were negative for mutagenic and clastogenic effects. Based on the results of these studies the test material is considered to be not classified for genotoxicity.