Oxazolidine, diheptyl~

Genetic toxicity: in vivo

Some information in this page has been claimed confidential.

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September - December 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

20 July, 2016

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Version / remarks:

August 1998

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

February 14, 2017

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

ANIMALS

Species: Rat (mus musculus)
Strain: Healthy Wistar rats, Crl: WI(Han) (Full Barrier)
Source: Charles River, 97633 Sulzfeld, Germany
Number of animals: 5 of each sex per dose group (7 animals for 1 MTD)
Initial age at start of acclimatisation: 6 - 8 weeks
Age at start of treatment: Minimum 7 weeks
The animals were derived from a controlled full barrier maintained breeding system (spf). According to Art. 9.2, No. 7 of the German Act on Animal Welfare the animals were bred for experimental purposes and underwent an adequate acclimatisation period after arrival (at least five days). The animals were randomly distributed to test groups.

HUSBANDRY

The animals were barrier maintained (semi-barrier) in an air conditioned room. The experiment was conducted under standard laboratory conditions.
Housing: 5 animals of identical sex per cage
Cage type: IVC cage (Polysulphone), Type III H
Bedding: Altromin saw fiber bedding (Batch: 1238)
Feed: Free access to Altromin 1324 (Batch: 1033) maintenance diet for rats and mice
Air change: At least 10 x per hour
Water: Free access to tap water, sulphur acidified to pH value of approx. 2.8 (drinking water, municipal residue control, micro-biologically controlled at frequent intervals)
Environment: Temperature 22 ± 3 °C
Relative humidity 55 ± 10%
Artificial light 6:00 - 18:00
Certificates of food, water and bedding was filed for two years at BSL Munich and afterwards archived at Eurofins Munich.
The animals were randomly distributed into the test groups. The animals of each test group and sex were housed in separate cages that were individually marked (study number, sex, control/test group). The animals of each cage were individually marked for identification by tail and ear drawing.

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

corn oil

Details on exposure:

The test item formulations were prepared freshly one hour prior to administration at the Test Facility. The prepared formulations were transferred to the Test Facility protected from light together with the control formulations under consideration of stability.
The test item was diluted in Corn oil. All animals received a single volume intraperitoneally of 10 mL/kg bw. The solvent was chosen according to its relative non-toxicity for the animals.

Duration of treatment / exposure:

single administration

Frequency of treatment:

single administration

Post exposure period:

48 and 72 h

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

400 mg/kg bw (total dose)

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw (total dose)

Doses / concentrations 3

Dose / conc.:

2 000 mg/kg bw (total dose)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Name: CPA; Cyclophosphamide
CAS No.: 50-18-0
Supplier: Sigma
Catalogue No.: C0768
Batch No.: MKCL2547
Dissolved in: physiological saline
Dosing: 10 mg/kg bw
Route and frequency of administration: ip, single
Volume administered: 10 mL/kg bw
Expiry date: October 2022
The solution was aliquoted and stored at <=-15°C. On the day of administration, the solution was freshly thawed. The stability of CPA at room temperature is quite good (3.5% is hydrolysed per day in aqueous solution). It is acceptable that the positive control can be administered by a route different from the test agent and sampled only at a single time. The sampling time for the positive control was 48 h after treatment

Examinations

Tissues and cell types examined:

Peripheral blood - Erythrocytes

Details of tissue and slide preparation:

Blood cells were immediately fixed in ultracold methanol. For the analysis (at the earliest 72 h after fixation) the MicroFlow Kit (Litron Laboratories, Rochester, New York, USA) was used and the procedures described in the manual were followed for analyzing peripheral blood samples for the presence of micronuclei. Briefly, blood cells were washed in Hank’s Balanced Salt Solution, centrifuged at 400 x g for 5 minutes at 4 °C and the supernatant was discarded. Blood cell populations were discriminated using specific antibodies against CD71 (expressed only at the surface of immature erythrocytes) and CD61 (expressed at the surface of platelets) and DNA content of micronuclei, was determined by the use of a DNA specific stain (propidium iodide, PI). Evaluation of all samples, including those of positive (CPA) and negative controls, were performed using a flow cytometer (FACSLyric, BD Biosciences). Anti-CD71 antibodies were labeled with fluorescein isothiocyanate (FITC), anti-CD61 antibodies were labelled with phycoerythrin (PE). Particles were differentiated using forward scatter (FSC) and side scatter (SSC) parameters of the flow cytometer. Fluorescence intensities were recorded on PMT position E, D and B for FITC, PE and PI, respectively. 10000 immature erythrocytes per animal were scored for the incidence of micronucleated immature erythrocytes. To detect a possibly occurring cytotoxic effect of the test item, the ratio between immature and mature erythrocytes was determined. The result was expressed as relative PCE (rel. PCE = proportion of polychromatic (immature) erythrocytes among total erythrocytes).

Statistics:

Statistical methods applied used the animal as the experimental unit.
Pairwise comparison of the proportion of PCE among total erythrocytes as well as the proportion of micronucleated polychromatic (immature) erythrocytes (PCE) among total PCE between the control groups and each of the treatment groups was performed by means of the non-parametric Mann-Whitney test at a statistical significance level of 5% (p < 0.05, two-tailed).
The y² test for trend at a statistical significance level of 5% (p < 0.05, two-tailed) was used to test whether there is a dose-related increase in the micronucleated cells frequency of the dose groups of the 48 h sampling time.
Statistical methods were performed using the software GraphPad Prism version 6.0.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

other: vehicle control served as negative control

Positive controls validity:

valid

Any other information on results incl. tables

Pre-Experiment

The test item was diluted in Corn oil within 1 h before treatment. 200 mg/mL of the test item was used as highest dose group corresponding to 2000 mg/kg bw according to the OECD guideline 474 [5].

The selection of the highest dose was conducted in accordance with the following current international guidelines for assessment of acute toxicity (OECD 423 and OECD 425), particularly with respect to selection of dose spacing and animal welfare aspects.

In the pre-experiment a concentration of 200 mg/mL of the test item was evaluated. Three male and three female rats received a single dose of 2000 mg/kg bw ip and showed toxicity such as reduction of spontaneous activity, prone position, piloerection, ataxia, opisthotonos, diarrhoea, abnormal breathing and half eyelid closure. In male rats the clinical signs persisted up to 48 h after application. The female rats were fully recovered 4 hours after application (Table 1).

Due to the results obtained in the pre-experiment 2000 mg/kg bw was chosen as maximum tolerated dose (1 MTD) in the main experiment.

Signs of Toxicity observed in the Pre-Experiment (2000 mg/kg bw)

Signs of Toxicity	Time post-application / Sex (3 male and 3 female rats)
	30 min		1 h		2 h		4 h		24 h		48 h		72 h
	m	f	m	f	m	f	m	f	m	f	m	f	m	f
Reduction of spontaneous activity	3	3	3	2	3	0	2	1	2	0	2	0	0	0
Prone position	1	1	0	0	0	0	0	0	0	0	0	0	0	0
Piloerection	0	0	0	0	2	0	2	0	0	0	2	0	0	0
Ataxia	0	2	0	1	1	0	0	0	0	0	0	0	0	0
Opisthotonos	1	1	0	0	0	0	0	0	0	0	0	0	0	0
Diarrhoea	1	0	2	1	2	1	2	1	0	0	0	0	0	0
Abnormal breathing	0	0	0	1	0	0	0	0	0	0	0	0	0	0
Half eyelid closure	0	0	1	0	0	0	0	0	0	0	0	0	0	0

m:     male
f:       female

Toxicity in the Main Experiment

2000 mg/kg bw was tested as the maximum tolerated dose (1 MTD) in the main experiment. The volume administered i.p. was 10 mL/kg bw.

Animals treated with the highest dose (1 MTD) showed moderate toxic effects (Table 2) with reduction of spontaneous activity, prone position, piloerection, ataxia, hunched posture, constricted abdomen, opisthotonos, abnormal breathing and half eyelid closure up to 4 h after application. After 24 h only reduction of spontaneous activity and piloerection were still seen and 48 h after application only reduction of spontaneous activity was still observed for two male animals.

Rats treated with 1000 mg/kg bw (0.5 MTD) showed the same clinical signs as the 1 MTD animals except for abnormal breathing and the clinical signs were less pronounced and the animals recovered faster.

The male animals treated with 400 mg/kg bw (0.2 MTD) showed mild toxic effects after the treatment with the test item up to 4 h after application. Female animals treated with 400 mg/kg bw (0.2 MTD) did not show any clinical signs

Signs of Toxicity observed in the Main Experiment (2000 mg/kg bw)

Signs of Toxicity	Time post-application / Sex (5 male and 5 female rats)
	30 min		1 h		2 h		4 h		24 h		48 h		72 h
	m	f	m	f	m	f	m	f	m	f	m	f	m	f
Reduction of spontaneous activity	5	5	5	5	5	5	5	5	4	1	2	0	0	0
Prone position	5	5	5	2	4	5	0	0	0	0	0	0	0	0
Piloerection	0	0	1	4	1	4	5	4	3	1	0	0	0	0
Ataxia	5	1	0	2	1	2	0	0	0	0	0	0	0	0
Hunched posture	0	0	0	3	0	0	5	5	0	0	0	0	0	0
Constricted abdomen	0	0	2	3	4	2	5	2	0	0	0	0	0	0
Opisthotonos	0	0	3	0	2	0	0	0	0	0	0	0	0	0
Abnormal breathing	0	0	1	1	0	0	0	1	0	0	0	0	0	0
Half eyelid closure	5	4	5	5	4	5	4	4	0	0	0	0	0	0

m:     male
f:       female

Relative PCE

The relative PCE (rel. PCE = proportion of polychromatic (immature) erythrocytes among total erythrocytes) was determined for each animal. The relative PCE is the supportive endpoint to assess cytotoxicity, which helps to demonstrate a target cell exposure with the test item.

The negative controls (48 h, 72 h) were within the historical control limits of the negative control (0.64% - 2.46% for males, 0.30% - 1.81% for females). The mean values noted for the 48 h negative control were 1.14% for males and 1.29 for females. For the 72 h negative control mean values of 1.24% for males and 1.34% for females were observed.

The animal group treated with 0.2 MTD showed mean values of the relative PCE of 1.32% for males and 0.72% for females. The mean value observed in the male group was within the range of the concurrent negative control and within the historical control limits of the negative control. The mean value observed for the female group was statistically significantly decreased compared to the concurrent negative control but was still within the historical control limits.

The dose group, which was treated with 0.5 MTD, showed mean values of the relative PCE of 0.82% for males and 0.67% for females. The mean value observed in the male group was within the range of the concurrent negative control and within the historical control limits of the negative control. The mean value observed for the female group was statistically significantly decreased compared to the concurrent negative control but was still within the historical control limits.

The animals who received 1 MTD (48 h sampling) showed mean values of the relative PCE of 0.71% for males and 0.36% for females. The mean value observed in the male group was within the range of the concurrent negative control and within the historical control limits of the negative control. The mean value observed for the female group was statistically significantly decreased compared to the concurrent negative control but was still within the historical control limits.

The animal group which was treated with 1 MTD (72 h sampling) showed mean values of the relative PCE of 1.09% for males and 0.84% for females (Table 10). The mean values observed in the male and female group were within the range of the concurrent negative control and within the historical control limits of the negative control. (Figure 2).

The decrease of PCE values in treated animals compared to control animals is related to toxicity and therefore also a hint for a target cell exposure of the test item.

Micronucleated polychromatic erythrocytes

For all dose groups, including positive and negative controls, 10000 immature erythrocytes per animal were scored for the incidence of micronucleated immature erythrocytes.

The negative controls (48 h and 72 h) evaluated were within the historical control limits of the negative control (0.03 – 0.15% for males, 0.03 – 0.12% for females). The mean values of micronuclei observed for the negative control were 0.08% for males and 0.04% for females after 48 h and 0.09% for males and 0.09% for females after 72 h.

The mean values of micronuclei observed after treatment with 0.2 MTD were 0.08% for males and 0.07% for females. The mean value observed in the male group was within the range of the concurrent negative control as well as within the historical control limits of the negative control. The mean value observed for the female group was slightly increased compared to the concurrent negative control but this was not statistically significant and the value was within the historical control limits.

The mean values noted for the 0.5 MTD dose groups were 0.10% for males and 0.10% for females. The mean value observed in the male group was within the range of the concurrent negative control as well as within the historical control limits of the negative control. The mean value observed for the female group was statistically significantly increased compared to the concurrent negative control. Since the value was within the historical control limits and no dose-response relationship was observed, this increase was considered as not biologically relevant.

The dose group treated with 1 MTD (48 h sampling) showed mean values of 0.09% for males and 0.05% for females. The mean values observed in the male and female group were within the range of the concurrent negative control as well as within the historical control limits of the negative control.

The mean values observed for the 1 MTD (72 h sampling) were 0.09% for males and 0.07% for females. The mean values observed in the male and female group were within the range of the concurrent negative control as well as within the historical control limits of the negative control.

No biologically relevant increase of micronuclei was found after treatment with the test item in any of the dose groups evaluated.

The nonparametric Mann-Whitney Test was performed to verify the results. No statistically significant increases (p< 0.05) of cells with micronuclei were noted in the dose groups of the test item evaluated except for the female 0.5 MTD dose group that showed a statistically significant increase compared to the concurrent negative control. However, the value was within the historical control limits of the negative control and no dose-response relationship was observed. Based on these data this increase was regarded as not biologically relevant.

Additionally, the c² Test for trend was performed to test whether there is a dose-related increase in the micronucleated cells frequency of the dose groups of the 48 h sampling time. No statistically significant increase in the frequency of micronucleated cells was observed.

Cyclophosphamide (10 mg/kg bw) administered ip was used as positive control, which induced a statistically significant increase in the micronucleus frequency (mean percentage of cells with micronuclei was 0.72% for male and 1.05% for female rats (Table 5)). This demonstrates the validity of the assay.

 

 

 

 

Applicant's summary and conclusion

Conclusions:

In conclusion, it can be stated that during the study and under the experimental conditions reported, the test item Sa 34 did not induce structural and/or numerical chromosomal damage in the immature erythrocytes of the rat.
Therefore, the test item Sa 34 is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in the Mammalian Erythrocyte Micronucleus Test.

Executive summary:

This study was performed to investigate the potential of Sa 34 to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the rat. After expulsion of the main nucleus, PCE are released into peripheral blood. Peripheral blood was sampled and analysed for presence of micronucleated PCE

The test item was diluted in Corn oil. The volume administered ip was 10 mL/kg bw. Peripheral blood samples were collected for micronuclei analysis 48 h and 72 h after a single application of the test item.

A pre-experiment was performed as dose range finding study based on the OECD guideline 474 and other relevant documents (OECD 423 and OECD 425). Based on the outcome of the dose range finding study, a dose of 2000 mg/kg bw was selected as maximum tolerated dose (MTD).

In the main experiment three dose levels were used covering a range from the maximum tolerated dose to little or no toxicity. The following dose groups were selected based on the toxicity observed in the pre-experiment:

Dose Group	Dose [mg/kg bw]
0.2 MTD	400
0.5 MTD	1000
1 MTD	2000

 

No toxicity was observed in female animals during the treatment with 0.2 MTD. Male animals showed mild symptoms after treatment with 0.2 MTD. Animals treated with 0.5 MTD showed mild signs of systemic toxicity. The animals treated with a dose of 1 MTD showed moderate signs of systemic toxicity such as with reduction of spontaneous activity, prone position, piloerection, ataxia, hunched posture, constricted abdomen, opisthotonos, abnormal breathing and half eyelid closure.

For all dose groups, including positive and negative controls, 10000 polychromatic erythrocytes per animal were scored for incidence of micronucleated immature erythrocytes. The negative controls (48 h, 72 h) were within the historical control limits of the negative control. The mean values noted for the dose groups which were treated with the test item were within the range of the concurrent negative control except for the female 0.5 MTD dose group that showed a statistically significant increase. Since the value was within the historical control limits and no dose-response relationship was observed, this increase was considered as not biologically relevant.

No biologically relevant increase of micronuclei was found after treatment with the test item in any of the dose groups evaluated.

The nonparametric Mann-Whitney Test was performed to verify the results. No statistically significant increases (p< 0.05) of cells with micronuclei were noted in the dose groups of the test item evaluated except for the female 0.5 MTD dose group that showed a statistically significant increase compared to the concurrent negative control. However, the value was within the historical control limits of the negative control and no dose-response relationship was observed. Based on these data this increase was regarded as not biologically relevant.

Additionally, the c² Test for trend was performed to test whether there is a dose-related increase in the micronucleated cells frequency of the dose groups of the 48 h sampling time. No statistically significant increase in the frequency of micronucleated cells was observed.

Cyclophosphamide (10 mg/kg bw) administered ip was used as positive control, which induced a statistically significant increase in the micronucleus frequency. This demonstrates the validity of the assay.