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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 22, 1992 – November 5, 1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report date:
1992

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
mammalian bone marrow chromosome aberration test

Test material

Constituent 1
Reference substance name:
1,3-Propanediol, 2,2-bis(hydroxymethyl)-, allyl ether
EC Number:
293-883-9
EC Name:
1,3-Propanediol, 2,2-bis(hydroxymethyl)-, allyl ether
Cas Number:
91648-24-7
IUPAC Name:
2,2-bis(hydroxymethyl)propane-1,3-diol; 3-(prop-2-en-1-yloxy)prop-1-ene
Test material form:
liquid
Specific details on test material used for the study:
- Storage condition: stored at ambient temperature in the container received from the sponsor
- Stability under test conditions: no apparent change in its physical state during storage
-Test item solutions were prepared fresh prior to dosing.
- No treatment of test material prior to testing.
- No preliminary purification step.
- Aliquots of allyl pentaerythritol was weighed and diluted with corn oil.

Test animals

Species:
mouse
Strain:
CD-1
Details on species / strain selection:
Historically, mice have been used in the MNT and have been shown to exhibit micronuclei indicative of chromosome breakage or lagging chromosomes (Heddle et al., 1983; Salamone et al., 1980; Schmid, 1975; Von Ledebur and Schmid, 1973). Intraperitoneal injection was chosen to maximize the bioavailability of allyl pentaerythritol to the target cells.
Sex:
male/female
Details on test animals or test system and environmental conditions:
August 4, 1992 - November 5, 1992 TEST ANIMALS
- Source: CD-1® mice from Charles River Laboratories, Wilmington, MA01887
- Age at study initiation: 10 weeks of age
- Weight at study initiation: At the time of dosing, body weights ranged between 32- 43 grams for males and 26-35 grams for females.
- Assigned to test groups randomly: Mice were randomized by body weight and assigned to groups by use of a computer generated random number list and individually identified with an ear tag.
- Fasting period before study: no
- Housing: Mice were housed in stainless steel wire mesh cages with a maximum of five per cage according to sex and dose group. Waste material was removed twice per week.
- Diet (e.g. ad libitum): Harlan Teklad Rodent Diet
- Water (e.g. ad libitum): fresh tap water was available ad libitum
- Acclimation period: All mice were acclimated to laboratory conditions for 26 days prior to initiation of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°c ± 3°C
- Humidity (%): 50% ± 20
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle

IN-LIFE DATES: From: To: Aug 4 – Aug 7, 1992

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle:corn oil
- Concentration of test material in vehicle: 10, 20 and 40 mg/ml.
Details on exposure:
Single doses of allyl pentaerythritol (100, 200 and 400 mg/kg) was administered by ip injection to 9 groups of mice (5/sex/group).
Concurrently, the negative control, corn oil, was administered to three groups of mice, and a group of these mice was included in each sacrifice time.
The positive control, TEM (Triethylenemelamine) at 0.5 mg/kg, was administered to one group of mice and sacrificed 24 hours post-dose.
Duration of treatment / exposure:
A single intraperitoneal dose of allyl pentaerythritol (100, 200 and 400 mg/kg) and a group of mice from each dose level was sacrificed at 24, 48 and 72 hours post-dose.
Frequency of treatment:
A single intraperitoneal dose.
Post exposure period:
A group of mice from each dose level was sacrificed at 24, 48 and 72 hours post-dose
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
200 mg/kg bw/day (nominal)
Dose / conc.:
400 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Five/Sex/group
Control animals:
yes, concurrent vehicle
Positive control(s):
Triethylenemelamine (TEM);
- Route of administration: by ip injection
- Doses / concentrations: TEM was dissolved in 0.9% Saline, dose level was 0.5 mg/kg

Examinations

Tissues and cell types examined:
1000 PCEs per mouse were scored for the presence of MPCEs, as well as for the number of MNCEs present in the optical field containing these 1000 PCEs. The data were expressed as the total number of mean percentage of MPCEs for 10,000 PCEs per group (1000 PCEs/mouse, whenever possible). An additional 1000 erythrocytes (PCE and NCE) were also scored per mouse and the proportion was expressed as the PCE/NCE ratio.
Details of tissue and slide preparation:
All mice were sacrificed by cervical dislocation and their femurs removed. One end of each femur (one proximal, one distal to the iliac end) was opened with scissors until a small opening to the marrow canal was visible. A 1 ml tuberculin syringe, fitted with a 5/8” 25 gauge needle containing approximately 0.2 ml fetal bovine serum, was inserted into the marrow cavity and the marrow gently flushed (to assure maximal dispersion) into a 5 ml round bottom culture tube containing 1.0 ml of fetal bovine serum. Each femur was flushed with fetal bovine serum until the bone appeared almost translucent. The cell suspensions were centrifuged for 5 minutes at 1000 rpm. The supernatant was removed leaving a small amount of fetal bovine serum with the cell pellet. With a pasteur pipette, the cell pellet was resuspended into a homogenous cell suspension. A small drop of the cell suspension was spread immediately onto an ethanol pre-cleaned glass slide. The smears were quickly dried on a slide warmer (~56°C), dipped in absolute methanol (~2 seconds) and air-dried. Slides were stained with a Modified Wrights Stain Pak (4481) containing polychrome methylene blue-eosin in an Ames Hema-Tek® 1000 automatic slide stainer. Slides were cleared in xylene,air-dried and coverslipped with Permaslip®.
Evaluation criteria:
Although micronuclei are uniformly round bodies in the cytoplasm of erythrocytes, they may appear almond-, ring-, or teardrop-shaped. Cytoplasmic inclusions which were light reflective, improperly shaped or stained, or which were not in the focal plane of the erythrocyte were considered artifacts and were not scored as micronuclei. Erythrocytes containing one or more than one micronucleus were scored as one micronucleated erythrocyte.

A test article is considered positive if it produced a statistically significant increase in the number of MPCEs as compared to the negative control.
Statistics:
One-tailed-tests were used to make pairwise comparisons between each treatment group and its concurrent negative control for statistically significant increases in the number of MPCEs. The proportion of PCEs per 1000 erythrocytes per mouse was evaluated by pairwise two-tailed-tests after an arcsin transformation was performed. Statistical significance was judged at p ≤ 0.05 and p ≤ 0.01 levels. All comparisons were made for each sacrifice time separately, comparing the treated group versus the concurrent negative control group.

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 100, 250, 500, 750 and 1000 mg/kg of body weight
- Solubility: Aliquots of allyl pentaerythritol was weighed and diluted with corn oil. Apparent homogeneous solutions were obtained and maintained on a magnetic stirplate at all dose levels evaluated.
- Clinical signs of toxicity in test animals: Pharmacotoxic signs (endpoints examined including abnormal gait, ataxia, body drop, decreased activity, decreased body tone, loss of righting, tonic convulsions, writhing) were observed at the dosage of 500, 750 and 1000 mg/kg dose groups.
- Rationale for exposure: Intraperitoneal injection was chosen to maximize the bioavailability of allyl pentaerythritol to the target cells.
- Harvest times: 24, 48 and 72 hours post-dose

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): there was not a statistically significant increase in the frequency of MPCEs in any group of mice treated with allyl pentaerythritol when compared to the concurrent negative control group.
- Ratio of PCE/NCE (for Micronucleus assay): Statistically significant increases in the PCE/NCE ratios were detected at the 48 hour sacrifice time in the 100 and 400 mg/kg group of mice treated with allyl pentaerythritol, but not at 24 nor 72 hour sacrifice time. The biological significance of this increase (if any) is unknown. No statistically significant changes were observed in other groups.

Any other information on results incl. tables

Pharmacotoxic Signs in the dose range fining study:

Mice were observed for death and pharmacotoxic signs immediately, 24, 48 and 72 hours post-dose and the surviving mice were sacrificed at the last observation time.

Table 1. Pharmacotoxic signs observed in mice dosed with allyl pentaerythritol at dosages of 100, 250, 500, 750 and 1000 mg/kg of body weight.

Immediate

24 hrs

48 hrs

72 hrs

Pharmacotoxic Signs 100 mg/kg

M

F

M

F

M

F

M

F

Decreased body tone

0

1

0

1

0

1

 

Immediate

24 hrs

48 hrs

72 hrs

Pharmacotoxic Signs 250 mg/kg

M

F

M

F

M

F

M

F

Abnormal gait

2

2

1

1

 

 

 

 

Decreased body tone

 

 

1

1

1

1

1

1

Piloerection

2

0

2

0

2

0

1

0

Writhing

2

2

 

 

 

 

 

 

Immediate

24 hrs

48 hrs

72 hrs

Pharmacotoxic Signs 500 mg/kg

M

F

M

F

M

F

M

F

Abnormal gait

2

2

1

 

 

 

 

 

Ataxia

1

1

 

 

 

 

 

 

Decreased activity

1

1

 

 

 

 

 

 

Decreased body tone

2

2

 

 

 

 

 

 

Piloerection

 

 

2

2

1

1

1

1

Writhing

2

2

 

 

 

 

 

 

 

Within 7 minutes

24 hrs

48 hrs

72 hrs

Pharmacotoxic Signs 750 mg/kg

M

F

M

F

M

F

M

F

 

Abnormal gait

2

2

2

2

 

 

 

 

 

Ataxia

2

2

 

 

 

 

 

 

 

Body drop

2

2

 

 

 

 

 

 

 

Decreased activity

2

2

 

 

 

 

 

 

 

Decreased body tone

2

2

2

2

2

1

2

1

 

Piloerection

 

 

2

1

1

1

1

0

 

Tonic convulsions

2

2

 

 

 

 

 

 

 

Writhing

2

2

 

 

 

 

 

 

 

 

Within 15 minutes

24 hrs

48 hrs

72 hrs

Pharmacotoxic Signs 1000 mg/kg

M

F

M

F

M

F

M

F

Abnormal gait

2

2

2

2

 

 

 

 

Ataxia

2

2

 

 

 

 

 

 

Body drop

2

2

 

 

 

 

 

 

Decreased activity

2

2

 

 

 

 

 

 

Decreased body tone

2

2

2

2

2

2

2

1

Loss of righting

2

2

 

 

 

 

 

 

Tonic convulsions

2

2

 

 

 

 

 

 

Writhing

2

2

 

 

 

 

 

 

 

 

Pharmacotoxic Signs in the main study:         

Table 2. Pharmacotoxic signs observed in the MNT in mice treated with allyl pentaerythritol:           

A) 100 mg/kg group, B) 200 mg/kg group and C) 400 mg/kg group:

Pharmacotoxic Signs

Immediate

24 hrs

48 hrs

72 hrs

M (15)

F (15)

M (15)

F (15)

M (15)

F (15)

M (15)

F (15)

A)

Piloerection

Writhing

 

2

4

 

1

2

 

5

 

4

 

2

 

 

2

 

 

2

 

1

 

B)

Abnormal gait

Decrease body tone Piloerection

Writhing

 

15

 

10

15

 

15

 

3

15

 

7

3

7

 

 

4

0

7

 

9

2

8

 

 

4

0

7

 

 

1

1

1

 

 

0

0

2

 

C)

Abnormal gait

Body drop

Decrease body tone

Decrease activity

Increase activity Piloerection

Writhing

 

15

14

1

15

 

 

15

 

15

12

0

15

 

 

15

 

11

 

2

1

2

14

 

12

 

0

0

1

13

 

8

 

2

1

1

9

 

7

 

0

0

1

9

 

 

 

 

1

 

 

5

 

 

 

 

0

 

 

5

 

( ) = Number of mice per group in each observation time.

Applicant's summary and conclusion

Conclusions:
1,3-propanediol, 2,2- bis(hydroxymethyl) -, allyl ether at dosages of 100, 200 and 400 mg/kg was considered negative (non-clastogenic) in the mouse micronucleus test at all the time intervals (24, 48 and 72 hours) evaluated under the criteria and the experimental conditions.
Executive summary:

The purpose of this study was to evaluate the clastogenic potential of 1,3-propanediol, 2,2- bis(hydroxymethyl) -, allyl ether to induce micronuclei in the mouse bone marrow erythropoietic cells.

Method:

A range-finding test was performed to find suitable dose levels of the test item. Each mouse received a single intraperitoneal dose of 1,3-propanediol, 2,2- bis(hydroxymethyl) -, allyl ether (100,250,500,750and1000mg/kg of bodyweight). Based on the severe pharmacotoxic signs observed at the dosages of 500, 750 and 1000 mg/kg dose groups, 100, 200 and 400 mg/kg were selected as the doses for the main study. Thirteen groups of CD-1® mice (10 weeks of age) were used in the main study. Each group of mice was comprised of ten animals (five/sex). Each 3 groups mice received a single intraperitoneal dose of 1,3-propanediol, 2,2- bis(hydroxymethyl) -, allyl ether (100, 200 and 400 mg/kg) and a group of mice from each dose level was sacrificed at 24, 48 and 72 hours post-dose. Concurrently, the negative control, corn oil, was administered to three groups of mice, and a group of these mice was included in each sacrifice time. The positive control, TEM at 0.5 mg/kg, was administered to one group of mice and sacrificed 24 hours post-dose. Bone marrow extracted and smear preparation made and stained. One thousand PCEs per mouse were scored for the presence of MPCEs,as well as for the number of MNCEs present in the optical fields containing these 1000 PCEs. The data were expressed as the total number of mean percentage of MPCEs for 10,000 PCEs per group (1000 PCEs/mouse,wheneverpossible). An additional 1000 erythrocytes (PCE and NCE) were also scored per mouse and the proportion was expressed as the PCE/NCE ratio.The cytotoxicity is determined from the PCE/NCE ratio which shifts in favor of NCEs after treatment with a cytotoxic compound. Statistical analyses were performed and a test article is considered positive if it produced a statistically significant increase in the number of MPCEs as compared to the negative control.

Results:

There was no premature death seen in any of the dose groups, the following pharmacotoxic signs were observed: piloerection, writhing, abnormal gait, decreased body tone, activity changes, body drop. No

pharmacotoxic signs were observed in mice administered the negative or positive controls. Analysis of the data indicated there was not a statistically significant increase in the frequency of MPCEs in any group of mice treated with 1,3-propanediol, 2,2- bis(hydroxymethyl) -, allyl ether when compared to the concurrent negative control group. Statistically significant increases in the PCE/NCE ratios were detected at the 48 hour sacrifice time in the 100 and 400 mg/kg group of mice. The biological significance of this increase (if any) is unknown, since there is no dose response and not obsered at 24 nor 72h groups. Mice treated with the positive control produced a statistically significant increase (p ≤ 0.01) in the frequency of MPCE and a statistically significant depression (p ≤0.01) in the PCE/NCE ratio. In the MNT for 1,3-propanediol, 2,2- bis(hydroxymethyl) -, allyl ether both the negative and positive controls are within the range of this historical data.

Conclusion

The test item, 1,3-propanediol, 2,2- bis(hydroxymethyl) -, allyl ether at dosages of 100, 200 and 400 mg/kg was considered negative (non-clastogenic) in the mouse MNT at all the time intervals evaluated under the criteria and the experimental conditions of the test protocol.