Reaction products of dimethyl phosphonate with 2-alkyl-2-alkylpropanediol and alkanediol

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 April 2017 to 27 June 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: damage to chromosomes and/or aneuploidy

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test material form:

liquid: viscous

Details on test material:

- Appearance/physical state: Clear viscous liquid
- Storage conditions: Room temperature in the dark

Test animals

Species:

mouse

Strain:

other: Hsd: ICR (CD-1)

Remarks:

albino

Sex:

male

Details on test animals or test system and environmental conditions:

ANIMALS AND ANIMAL HUSBANDRY
- Sufficient albino Hsd: ICR (CD-1) strain mice were obtained from Harlan Laboratories UK Ltd, Oxon, UK.
- At the start of the main test, the mice weighed 23.5 to 30.9 g and were approximately six to ten weeks old.
- After a minimum acclimatisation period of five days, the animals were selected at random and given a number unique within the study by tail marking and a number written on a colour coded cage card.
- The animals were housed in groups of up to seven in solid-floor polypropylene cages with wood-flake bedding.
- Free access to mains drinking water and food (Harlan Teklad 2014C Global Certified Rodent diet supplied by Harlan Laboratories Ltd, Oxon, UK) was allowed throughout the study. Representative analyses of food and water quality are retained in the laboratory archive.
- Temperature and relative humidity were set to achieve limits of 19 to 25 °C and 30 to 70 % respectively.
- The rate of air exchange was approximately 15 changes per hour.
- Lighting was controlled by a time switch to give twelve hours light and twelve hours darkness.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

VEHICLE
- Identification: Arachis oil
- Label number: ZZZ08613
- Envigo serial number: V-6498
- Purity: Treated as 100 %
- Expiry date: 12 May 2018
- Storage conditions: Room temperature

Details on exposure:

PURPOSE OF THE TEST
- The micronucleus test is a mammalian in vivo test that detects damage to the chromosomes induced by chemicals. In addition, numerical changes due to chromosome loss during cell division can be detected by the test. The results are believed to be of value in predicting the mutagenic potential of the test item to man. The test system was chosen because the mouse has been shown to be a suitable model for this type of study and is recommended in the test method.
- In mitotic cells in which chromosome damage has been caused by the test item or its metabolites, fragments (centric or acentric) or whole chromosomes tend to lag behind in the anaphase stage of cell division. After telophase a large proportion of the fragments are not included in the nuclei of the daughter cells and hence form a single or multiple micronuclei (Howell-Jolly bodies) in the cytoplasm of these cells. These micronuclei are seen in a wide variety of cell types but erythrocytes are chosen since micronuclei are easily detected in these cells.
- A few hours after the last mitosis is completed, erythrocytes expel their nuclei. Immature erythrocytes, less than 24 hours old, stain blue with May-Grünwald/Giemsa due to the presence of minute fragments of nuclear material in the cytoplasm. This material is mainly ribonucleic acid (RNA), which gradually disappears so that more mature erythrocytes (normochromatic erythrocytes) stain pink with May-Grünwald/Giemsa. The immature blue-staining cells are known as polychromatic erythrocytes and mauve-stained micronuclei are easily detected in this cell type. If scoring is restricted to polychromatic erythrocytes, all the chromosomal damage detected will have been caused during the final cell cycle of the nucleated precursor cells. Thus by examining polychromatic cells at various periods after administration, the effect of the test item over the previous 30 hours can be monitored.
- Any toxic effects of the test item on the immature nucleated cells may lead to a reduction in cell division and cell death. This in turn leads to a reduction in cell volume and, to compensate for this, peripheral blood is shunted into the bone marrow. If the ratio of polychromatic to normochromatic erythrocytes is scored and found to be significantly lower than the control value, this is taken to be indicative of cytotoxicity.

PREPARATION OF TEST ITEM
- When required, the test item was freshly prepared as a solution at the appropriate concentration in arachis oil.
- No analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation. The test item was formulated within two hours of it being applied to the test system and it was assumed the formulation was stable over that duration. This is an exception with regard to GLP and was reflected in the GLP compliance statement.

PREPARATION OF POSITIVE CONTROL ITEM
- When required, the positive control item was freshly prepared as a solution at the appropriate concentration in distilled water (Laboratoire Aguettant batch number 3012436).

RANGE-FINDING TOXICITY TEST
- A range-finding toxicity test was performed to determine a suitable dose level and route of administration for the main test.
- The dose level selected should ideally be the maximum tolerated dose level, or that which produces some evidence of toxicity up to a maximum recommended dose of 2000 mg/kg.
- The range-finding test was also used to determine if the main test should be performed using both sexes or males only.
- Bone marrow samples were taken from the range-finding animals. Slides were then prepared and qualitatively assessed to ensure that any bone marrow toxicity observed was within acceptable limits for the main test.
- Groups of mice were dosed as shown in the table below.
- All animals were dosed once only at the appropriate dose level.
- Dosing was by gavage using a metal cannula or with a hypodermic needle attached to a graduated syringe.
- The volume administered to each animal was calculated according to its bodyweight at the time of dosing.
- Animals were observed one hour after dosing and subsequently once daily for two days.
- Any deaths and evidence of overt toxicity were recorded at each observation.
- No necropsies were performed.

MICRONUCLEUS TEST
- Groups of seven mice were dosed with the test item once only via the oral route. Doses received were 2000, 1000 or 500 mg/kg.
- One group of mice from each dose level was killed by cervical dislocation 24 hours following treatment and a second group dosed with test item at 2000 mg/kg was killed after 48 hours.
- In addition, two further groups of mice were included in the study; one group (five mice) was dosed via the oral route with the vehicle alone (arachis oil) and second group (five mice) was dosed orally with cyclophosphamide, which is a positive control item known to produce micronuclei under the conditions of the test.
- The vehicle controls and positive control group animals were killed 24 hours after dosing.
- Experimental design is summarised in the table below.
- All animals were observed one hour after dosing and then once daily and immediately prior to termination.

Duration of treatment / exposure:

24 or 48 hours

Frequency of treatment:

Single dose

Post exposure period:

Not applicable

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

500 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Seven

Control animals:

yes, concurrent vehicle

Positive control(s):

POSITIVE CONTROL
- Identification: Cyclophosphamide monohydrate
- Supplier: Acros Organics
- Lot number: A0373263
- Envigo serial number: R-6737
- Purity: 97 %
- Expiry date: 22 February 2019
- Storage conditions: Approximately 4 °C in the dark

Examinations

Tissues and cell types examined:

Both femurs were dissected from each animal immediately following sacrifice.

Details of tissue and slide preparation:

SLIDE PREPARATION
- Immediately following termination (24 or 48 hours after dosing), both femurs were dissected from each animal, aspirated with foetal bovine serum and bone marrow smears prepared following centrifugation and re-suspension.
- The smears were air-dried, fixed in absolute methanol, stained in May-Grünwald/Giemsa, allowed to air-dry and a cover slip was applied using mounting medium.

SLIDE EVALUATION
- Stained bone marrow smears were coded and examined blind using light microscopy at x1000 magnification.
- The incidence of micronucleated cells per 2000 polychromatic erythrocytes (PCE-blue stained immature cells) per animal was scored.
- Micronuclei are normally circular in shape although, occasionally, they may be oval or half-moon shaped. The micronuclei have a sharp contour with even staining. In addition, the number of normochromatic erythrocytes (NCE-pink stained mature cells) associated with 1000 erythrocytes was counted. These cells were also scored for incidence of micronuclei.
- The ratio of polychromatic to normochromatic erythrocytes was calculated together with appropriate group mean values and standard deviations.

Evaluation criteria:

DATA EVALUATION
- Comparison was made between the number of micronucleated polychromatic erythrocytes occurring in each of the test item groups and the number occurring in the vehicle control group.
- A positive mutagenic response is demonstrated when a statistically significant, dose-responsive, toxicologically relevant increase in the number of micronucleated polychromatic erythrocytes is observed for either the 24 or 48-hour kill times when compared to the vehicle control group.
- If these criteria were not fulfilled, then the test item was considered to be non-genotoxic under the conditions of the test.
- A positive response for bone marrow toxicity was demonstrated when the dose group mean polychromatic to normochromatic ratio was shown to be statistically significantly lower than the vehicle control group.

Statistics:

STATISTICAL ANALYSIS
- All data were statistically analysed using appropriate statistical methods as recommended by the UKEMS Sub-committee on Guidelines for Mutagenicity Testing Report, Part III (1989).
- The data was analysed following a transformation using Student's t-test (two tailed).

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING TOXICITY TEST
- Mortality data are summarised in the table below.
- Clinical signs were not observed in any of the animals dosed at 1000 mg/kg, or the maximum recommended dose of 2000 mg/kg. Qualitative analysis of the bone marrow slides indicated that there was evidence of very modest toxicity to the bone marrow at 2000 mg/kg.
- The micronucleus test was therefore conducted using the oral route in groups of seven mice (males) at the maximum recommended dose of 2000 mg/kg, with 1000 and 500 mg/kg as the two lower dose levels. There was no marked difference in toxicity of the test item between the sexes; therefore the main test was performed using only male mice.

MICRONUCLEUS TEST
- There were no premature deaths or clinical signs observed in any of the dose groups in the main test.

EVALUATION OF BONE MARROW SLIDES
- A summary of results of the micronucleus test is given in Table 1 (attached).
- Historical control data for studies performed between 2014 and 2017 is summarised in Appendix 1 (attached).
- Individual and group mean data are presented in Tables 2 to 7 (attached).
- There were no statistically significant decreases in the PCE/NCE ratio in the 24 or 48-hour test item groups when compared to the vehicle control group. However, it should be noted that the vehicle control PCE/NCE ratio was quite low.
- There were no statistically significant increases in the frequency of micronucleated PCEs in any of the test item dose groups when compared to the vehicle control group.
- The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test.

Any other information on results incl. tables

MORTALITY DURING RANGE-FINDING TOXICITY TEST

Dose level (mg/kg)	Sex	Number of animals treated	Route	Deaths on day 0	Deaths on day 1	Deaths on day 2
1000	Male	1	Oral	0	0	0
1000	Female	1	Oral	0	0	0
2000	Male	1	Oral	0	0	0
2000	Female	1	Oral	0	0	0
2000	Male	2	Oral	0	0	0

Applicant's summary and conclusion

Conclusions:

The test item was considered to be non-genotoxic under the conditions of the test.

Executive summary:

GUIDELINE

The study was performed to assess the potential of the test item to produce damage to chromosomes or aneuploidy when administered to mice. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals No. 474 “Mammalian Erythrocyte Micronucleus Test” (adopted 29 July 2016), Method B.12 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPA (TSCA) OPPTS 870.5395, EPA 712-C-98-226, August 1998 guidelines, and be acceptable to the Japanese METI/MHLW/MAFF guidelines for testing of new chemical substances.

 

METHODS

A range-finding test was performed to find suitable dose levels of the test item and to investigate if there was a marked difference in toxic response between the sexes. There was no marked difference in the toxicity of the test item between the sexes; therefore, the main test was performed using only male mice using the single administration treatment schedule for test items. The micronucleus test was conducted using the oral route in groups of seven mice (males) at the maximum recommended dose of 2000 mg/kg, with 1000 and 500 mg/kg as the two lower dose levels. Animals were killed 24 or 48 hours after dosing, the bone marrows were extracted, and smear preparations made and stained. Polychromatic (PCE) and normochromatic (NCE) erythrocytes were scored for the presence of micronuclei and PCE/NCE ratio was calculated as an indicator for toxicity. Two additional groups of five mice were given a single oral dose of arachis oil, or dosed orally with cyclophosphamide, to serve as vehicle and positive controls respectively. Vehicle and positive control animals were euthanised 24 hours after dosing.

 

RESULTS

There were no premature deaths or clinical signs observed in any of the dose groups in the main test. There were no marked decreases in the PCE/NCE ratio observed in the 24 or 48-hour test item dose groups when compared to the vehicle control group. There was no evidence of any significant increases in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test item when compared to the vehicle control group. The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test.

 

CONCLUSION

The test item was considered to be non-genotoxic under the conditions of the test.