Hydroxyprogesterone

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:

March - July 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

other:

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March - July 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

adopted 28 July 2015

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:WI (Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Margate, UK
- Age at study initiation: 10 -11 weeks (males), 9 - 10 weeks (females)
- Weight at start of dosing: 280.4 - 409.2 g (males), 186.4 - 242.4 g (females)
- Fasting period before study: not applicable
- Housing: housed in groups (up to four animals/cage by sex [both sexes pre-pairing and males post-pairing] or with one female and one male [pairing]), individually (mated females), or with their litter (lactation)
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: up to 20 days

DETAILS OF FOOD AND WATER QUALITY:
No contaminants were present in diet and water at levels which might have interfered with achieving the objective of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): within 19 - 25°C
- Humidity (%): 30 - 70%
- Air changes (per hr): 15 - 20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To: 11 April - 9 June 2016

Route of administration:

oral: gavage

Vehicle:

other: 0.1 % Myrj S-50-PA, 1.0 % Klucel LF add 100 % with 0.9 % physiological saline solution

Details on oral exposure:

Formulations were prepared weekly.
The test item formulations were formulated as a suspension in 0.1% Myrj S-50-PA, 1.0% Klucel LF add 100% with 0.9% physiological saline solution. The formulations were stored at room temperature (15 to 25°C) in a sealed container, protected from light.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Suspensions of 0.1 and 100 mg/mL were previously found to be homogenous and stable for 10 days at 15 to 25°C.

Formulations prepared for use in Week 1 were analysed to determine homogeneity. Formulations are normally considered to be homogeneous if the coefficient of variation (CV) of the results is ≤ 6.0% and the homogeneity results are within ± 10% of the mean. The results were within these criteria.

Formulations prepared for use in Weeks 1, 3 and 6 of the study were analysed to determine achieved concentration. The target range for the preparation of the
formulations was 90 to 110% of nominal. Results were within this range.

Test article was not detected in the Group 1 control samples.

Duration of treatment / exposure:

Males: 42 days
Females: up to 64 days

Frequency of treatment:

once daily

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Control

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

Low dose

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Remarks:

Intermediate dose

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

High dose

No. of animals per sex per dose:

Males: 10
Females: 13 (control), 11 (dosing groups); more than 10 female rats per group were included to ensure 10 females/group showed a regular estrous
cycle before dosing commenced

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
High dose: A high dose level of 1000 mg/kg/day was considered a suitable dose level as it would be well tolerated, although an effect on reproduction was expected, based on the findings of the dose range finder study.
Intermediate dose: An intermediate dose level of 300 mg/kg/day was expected to be a no observed adverse effect level (NOAEL) for systemic toxicity, and no effects on reproduction were expected.
Low dose: A low dose level of 100 mg/kg/day was anticipated to be the NOEL for systemic toxicity.

Positive control:

Not applicable.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes, for signs of ill health or overt toxicity
- Time schedule: at the beginning and end of working day (in addition, post dosing observations, upon return to the home cage and approximately 2 hours after dosing

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily

BODY WEIGHT: Yes
- Time schedule for examinations:
males: prior to dosing, on the first day of dosing, afterwards weekly, and prior to necropsy at day 43;
females: prior to dosing, on the first day of dosing, afterwards weekly prior to pairin; until confirmation of mating; on gestation day (GD) 0, 7, 14, 20; and on lactation day (LD) 1, 4, 7, and 13; and prior to necropsy on lactation day 14;

FOOD CONSUMPTION: Yes
- males: twice weekly
- females: twice weekly prior to pairing, then daily from gestation day 0 to 20 and from lactation day 1 to 13

OPHTHALMOSCOPIC EXAMINATION: Not specified

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, overnight
- How many animals: all adults
- Parameters checked in table [No.1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at necropsy
- Animals fasted: Yes, overnight
- How many animals: all adults
- Parameters checked in table [No.2] were examined.

URINALYSIS: Yes (5 selected males/group)
- Time schedule for collection of urine: Week 6
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Not specified
- Parameters checked in table [No.3] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: males: prior to dosing and once weekly thereafter; females: prior to dosing, once weekly during the pre-pairing and pairing phases; on GD 0, 7, 14, and 20; and on LD 1, 7, and 13.
- Dose groups that were examined: all groups
- Battery of functions tested: sensory activity / grip strength / motor activity
- Subjective and quantitative assessments: potential effects on behavior, gait, posture, respiration, secretion, excretion, involuntary movements, skin, tail, eyes, pelage, and activity;

IMMUNOLOGY: Yes, (total and differential white cell count; see table [No.1].

OTHER:

Estrous cycle determination:
- during predose phase, from one week after arrival until the day prior to dosing and daily vaginal lavage samples were taken from females from the start of dosing until
the confirmation of mating

Thyroid hormones:
- Time schedule for examinations: at necropsy
- How many animals: all adults
- Parameters checked: total T4 (thyroxine), TSH (thyroid stimulating hormone)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table [No.4])

HISTOPATHOLOGY: Yes (see table [No.4])

Statistics:

Data for each sex was analyzed separately, unless stated otherwise. Except when otherwise stated, tests were performed using a two-sided risk and were considered significant when p≤0.05.
Body weight, body weight gains, food consumption (gestation and lactation phases), absolute organ weights, organ:terminal body weight ratios, and terminal body weights were analyzed using analysis of variance (ANOVA).
Mean numbers of estrous cycles and mean cycle length were analyzed using the Kruskal-Wallis and Wilcoxon rank sum test.

Clinical signs:

no effects observed

Description (incidence and severity):

The clinical observations noted included isolated instances of physical injury to the tip of the tail, teeth pallor, sores, lesions and staining of the skin and/or fur, and thin fur; these were noted throughout dose groups, including the control group; as such, they
were considered low incidence findings observed in this species and were unrelated to test item toxicity.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

All parameters, including those statistically significant, were considered unrelated to test item administration as they were small in magnitude or lacked a dose-dependent response.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

- Two females administered 1000 mg/kg/day test substance showed higher than expected creatinine and urea values, when compared with controls.
- elevated cholesterol levels for all test substance-treated females compared to controls (p<0.01 - p<0.05), without dose-related response
Cholesterol females [mmol/L]: (Control: 1.9 +- 0.37; Low dose: 2.5 +- 0.32***; Intermediate dose: 2.6 +- 0.3***; High dose: 2.4 +- 0.3***)
*** P<=0.001; ANOVA and Dunnett's

Urinalysis findings:

no effects observed

Description (incidence and severity):

All parameters, including those statistically significant, were considered unrelated to test item administration as they were small in magnitude or lacked a dose-dependent response.

Behaviour (functional findings):

no effects observed

Immunological findings:

no effects observed

Description (incidence and severity):

All parameters, including those statistically significant, were considered unrelated to test item administration as they were small in magnitude or lacked a dose-dependent response.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

- Considerable variation in organ weights occurred, and apparent decreases in prostate weights were noted for test item-treated animals. However, the lack of any microscopic correlate or inter- or intra-group variation in weights made relations to administration of Hydroxyprogesterone unlikely.
- Organ weight and/or organ weight ratio changes, including those statistically significant, were attributed to normal biological variation and were considered not related to administration of test substance as they were small in magnitude, not dose-dependent, inconsistent between sexes, due to normal inter-animal variability, and/or lacked a microscopic correlate.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Macroscopically, in the kidney, uni- or bilateral depressed foci were noted in two females administered 1000 mg/kg/day.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

An increased incidence and/or severity of tubular basophilia and/or cortical scar were present in females administered 100, 300, or 1000 mg/kg/day. Cortical scar was correlated with depressed foci, noted at necropsy. Basophilia, tubule was characterized by focal or multifocal groups of tubules in the renal cortex, with basophilic epithelium, crowding of nuclei, more intense staining of the cells, and occasionally thickened basement membranes, and often with an inflammatory cell infiltrate. Cortical scar was characterized by focal lesions in the cortex, with fibroblast proliferation and collagen deposition, generally inconspicuous tubular elements, and occasionally cystic tubules, with pigment and few inflammatory cells.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

- TSH levels were elevated for all test item-treated females compared with controls (p<0.01), without dose-related response. Further, no effects on organ weight or histopathological changes were observed.
TSH µlU/mL (Control: 0.46 +- 0.295; Low dose: 1.38 +- 1.135**; Intermediate dose: 1.16 +- 0.621**; High dose: 1.71 +- 2.536**)
**P<=0.01; ANOVA und Dunnett's

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: no effects observed

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

clinical biochemistry

histopathology: non-neoplastic

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (actual dose received)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Table 5:

Incidence of Selected Kidney Findings - Terminal Sacrifice               Males           Females  Tissue and finding Level (mg/kg bw/day)  0   100  300  1000  0  100  300  1000  Kidney  No. examined: 5   5  5  5  5  5  5  5  basophilia, tubule  Grade -  3  4  3  4  4  2  1  -    1  2  1  2  1  1  3  2  -    2  -  -  -  -  -  -  2  4    3  -  -  -  -  -  -  -  1  cortical scar  Grade -  5  5  5  5  5  5  5  3    1  -  -  -  -  -  -  -  1    2  -  -  -  -  -  -  -  1

- = Finding not present; 1 = Minimal; 2 = Slight; 3 = Moderate.

Conclusions:

The repeated dose study was performed in accordance to OECD 422. The objective was to provide information on the effects of the test item following daily oral (gavage) administration to the male and female rat for at least 42 days and to screen for potential adverse effects on reproductive performance in the rat, including offspring development (see 7.8.1).
Once daily oral gavage administration of 100, 300, or 1000 mg/kg/day test substance to male rats for 42 days and to female rats for up to 64 days (pre-pairing, throughout gestation, and during the first 2 weeks of lactation) did not result in any test item-related effects in males; as such, 1000 mg/kg/day is considered the no observed effect level (NOEL) for males for systemic toxicity.
In females, microscopically, an increased incidence and/or severity of tubular basophilia and/or cortical scar of kidneys was present in test item-treated groups. Slightly elevated urea and creatinine, together with an increased incidence and severity of tubular basophila and/or scarring, observed for females administered 1000 mg/kg/day were indicative of renal injury, and as such, was considered adverse. In the low and intermediate dose group slightly lower grades of severity in regard to microscopic kidney changes compared to high dose group were observed. In the absence of any notable changes in blood creatinine and urea they were considered as not adverse. 300 mg/kg/day is considered the no observed adverse effect level (NOAEL) for females for systemic toxicity.

Executive summary:

In a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test according to OECD TG 422 Hydroxyprogesteron was administered to 10 Wistar rats/sex/dose by gavage at dose levels of 100, 300, or 1000 mg/kg bw/day for at least 42 days at a constant dose volume of 10 mL/kg.

The control article (vehicle) was 0.1% Myrj S-50-PA, 1.0% Klucel LF add 100% with 0.9% physiological saline solution. The test item was formulated as a suspension. Formulations prepared for use in Weeks 1, 3, and 6, including the vehicle control were analysed for accuracy.

Before the start of dosing, all females were screened for regular estrous cycles; only females with  regular estrous cycles were included in the study. On Day 3 of dosing, additional females were added to the study because not all females initially included showed a regular estrous cycle during the predose phase. Therefore, the number of females on study increased to 13, 11, 11, and 11 (0, 100, 300, or 1000 mg/kg/day, respectively).

In accordance with the test guideline, males were dosed once daily for 42 consecutive days (two weeks prior to mating, during the mating period and approximately two weeks post-mating) and sent to necropsy on Day 43. Females were dosed for up to 64 days (two weeks prior to mating, during the mating period and until Day post-partum) and sent to necropsy on Lactation Day (LD) 14.

For pups, clinical observations, litter size, sex, and body weight were recorded.

Ano-genital distance was recorded on Postnatal Day (PND) 4, and nipple retention was recorded for male pups on PND 13. One pup/sex/litter from each dose group was selected for collection of thyroid weights and processing for microscopic examinations.

Assessment of toxicity in adults was based on clinical observations, neurobehavioral assessments, body weights, food consumption, estrous cycling, mating, fertility and pregnancy indices, offspring development, and anatomic pathology. On the day of necropsy, blood samples were withdrawn for clinical pathology (adults) and thyroid hormone assessments (adults and offspring). Complete necropsies were performed on all animals, and any macroscopic abnormalities were noted. Organ weights were recorded for all adults and microscopic examinations were conducted for selected animals.

Accuracy of formulations was demonstrated by analyses.

 

No unscheduled deaths occurred, and no test item-related clinical observations or changes in neurobehavior were noted. Furthermore, no Hydroxyprogesterone-related effects on body weight change or food consumption were noted.

No toxicologically significant effects of Hydroxyprogesterone were noted on hematology or urinalysis parameters.

Clinical chemistry assessments showed slightly elevated urea and creatinine for females administered 1000 mg/kg/day, and elevated cholesterol and thyroid stimulating hormone (TSH) levels for all Hydroxyprogesterone-treated females, compared with controls. No such effect was noted for males.

No macroscopic findings, organ weight and/or organ weight ratio changes considered related to administration of Hydroxyprogesterone were noted. Microscopically, in the kidney, an increased incidence and/or severity of tubular basophilia and/or cortical scar were present for females administered 100, 300, or 1000 mg/kg/day.

In conclusion, once daily oral gavage administration of 100, 300, or 1000 mg/kg/day Hydroxyprogesterone to male rats for 42 days and to female rats for up to 64 days (pre-pairing, throughout gestation, and during the first 2 weeks of lactation) did not result in any test item-related effects in males; as such, 1000 mg/kg/day is considered the no observed effect level (NOEL) for males for systemic toxicity.

Microscopic kidney changes noted for females administered 100 or 300 mg/kg/day were confined to slightly lower grades of severity compared with high dose females, and in the absence of any notable changes in blood creatinine and urea, were considered not adverse; as such, 300 mg/kg/day is considered the no observed adverse effect level (NOAEL) for females for systemic toxicity.

For reproductive toxicity see chapter 7.8

 

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted 26 September 2014

GLP compliance:

yes

Type of assay:

other: micronucleus test in vivo

Test material

Test material information

Constituent 1

Reference substance name:

Hydroxyprogesterone

EC Number:

200-699-4

EC Name:

Hydroxyprogesterone

Cas Number:

68-96-2

Molecular formula:

C21H30O3

IUPAC Name:

17-hydroxypregn-4-ene-3,20-dione

Test animals

Species:

rat

Strain:

other: Crl:WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Margate, UK
- Age at study initiation: 10 -11 weeks (males), 9 - 10 weeks (females)
- Weight at start of dosing: 280.4 - 409.2 g (males), 186.4 - 242.4 g (females)
- Fasting period before study: not applicable
- Housing: housed in groups (up to four animals/cage by sex [both sexes pre-pairing and males post-pairing] or with one female and one male [pairing]), individually (mated females), or with their litter (lactation)
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: up to 20 days

DETAILS OF FOOD AND WATER QUALITY:
No contaminants were present in diet and water at levels which might have interfered with achieving the objective of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): within 19 - 25°C
- Humidity (%): 30 - 70%
- Air changes (per hr): 15 - 20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To: 11 April - 9 June 2016

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

0.1% Myrj S-50-PA, 1.0% Klucel LF add 100% with 0.9% physiological saline solution

Details on exposure:

Formulations were prepared weekly.
The test item formulations were formulated as a suspension in 0.1% Myrj S-50-PA, 1.0% Klucel LF add 100% with 0.9% physiological saline solution. The formulations were stored at room temperature (15 to 25°C) in a sealed container, protected from light.

Duration of treatment / exposure:

Males: 42 days
Females: up to 64 days

Frequency of treatment:

once daily

Post exposure period:

24 hours

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Control

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

Low dose

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Remarks:

Intermediate dose

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

High dose

No. of animals per sex per dose:

males: 5
females: 8 (control group), 4 (low and intermediate dose), 5 (high dose)

Control animals:

yes, concurrent vehicle

Positive control(s):

The positive control animals were dosed with cyclophosphamide (20 mg/kg).

Examinations

Tissues and cell types examined:

bone marrow smears

Details of tissue and slide preparation:

DETAILS OF SLIDE PREPARATION: Bone marrow was flushed from the marrow cavity with foetal bovine serum. The collected samples were then filtered through cellulose columns. Once filtered, the bone marrow cells were pelleted by centrifugation and the
supernatant aspirated and discarded. Additional serum was added to the tubes followed by gentle resuspension of the cell pellet. The cells were pelleted again (by centrifugation) and the supernatant aspirated to leave one or two drops and the cell pellet. The pellet was mixed into this small volume of serum by using a Pasteur pipette, and from each tube one drop of suspension was placed on the end of each of three uniquely labelled slides. A smear was made from the drop by drawing the end of a clean slide along the labelled slide. Slides were air-dried prior fixing for 10 minutes in absolute methanol. Once fixed the slides were rinsed several times in distilled water and air-dried. Two slides per animal were stored at <-10°C with desiccant for subsequent staining. On the day of staining the slides were fixed and washed again (as described above) and immediately stained for 5 minutes in 12.5 μg/mL acridine orange made up in 0.1 M phosphate buffer pH 7.4. Stained slides were rinsed in phosphate buffer, then dried and stored protected from light at room temperature prior to analysis. The dried, unstained slides were initially stored at <-10°C with desiccant. Once final results were confirmed the reserve slides were transferred to storage at room temperature.

METHOD OF ANALYSIS: Scoring was carried out using fluorescence microscopy at an appropriate magnification. A sample of slides from the vehicle control animals and the positive control slides were checked for quality and/or response prior to analysis. All slides (including the positive control slides) were allocated a random code and analysed by an individual not connected with the dosing phase of the study. Initially the relative proportions of polychromatic erythrocytes (PCE), seen as bright orange enucleate cells, and normochromatic erythrocytes (NCE), seen as smaller dark green enucleate cells, were determined until a total of at least 500 cells (PCE plus NCE) had been analysed. Then at least 4000 PCE/animal (2000 PCE per positive control slide) were examined for the presence of MN (micronucleus).

The following criteria were applied during slide assessment:
1. Cells were to be of normal cell morphology
2. Areas where erythrocytes overlap were to be ignored
3. A MN was to be round or oval in shape
4. A cell containing more than one MN was scored as a single micronucleated cell
5. MN which were refractive, improperly stained or not in the focal plane of the cell were judged to be artefacts and were not scored.

Slide analysis was performed by a competent analyst trained in the applicable Covance Laboratories standard operating procedures.

DATA EVALUATION: After completion of microscopic analysis and decoding of the data the following were calculated:
1. %PCE for each animal and the mean for each group. The group mean %PCE values were examined to see if there was any decrease in groups of treated animals that could be taken as evidence of bone marrow toxicity
2. %MN PCE for each animal and the group mean %MN PCE (±standard deviation).

The numbers of MN PCE in the vehicle control animals were compared with the laboratory's standard micronucleus historical control data to determine whether the assay was acceptable.

Evaluation criteria:

For valid data, the test article was considered to induce clastogenic / aneugenic damage if:
1. A statistically significant increase in the frequency of MN PCE occurred at one or more dose levels
2. At points that were significant the group mean MN PCE value was outside the 95% historical standard micronucleus vehicle control range
3. At the same dose levels the distribution of MN PCE in the majority of animals exceeded the laboratory’s historical vehicle control data
4. A dose-response trend in the proportion of MN PCE (where more than two dose levels were analysed) was observed.
The test article was considered positive in this assay if all of the above criteria were met.
The test article was considered negative in this assay if none of the above criteria were met and bone marrow exposure was confirmed.
Results which only partially satisfied the above criteria were dealt with on a case-by-case basis. Evidence of a dose-related effect was considered useful but not essential in the evaluation of a positive result. Biological relevance was taken into account, for example consistency of response within and between dose levels.

Statistics:

For each group, inter-individual variation in the numbers of MN PCE was estimated by means of a heterogeneity chi-square calculation. For female data the numbers of MN PCE in each treated group were compared with the numbers in vehicle control groups by using a 2 x 2 contingency table to determine chi-square. For males the heterogeneity chi-squared calculation showed a significant difference and therefore the male data were evaluated using Wilcoxon Rank Sum test. The tests were interpreted with one-sided risk for increased frequency with increasing dose. Probability values of p≤0.05 were accepted as significant. A further statistical test (for linear trend) was used to evaluate possible dose-response relationships.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The data confirm that:
1. The vehicle control MN PCE data were comparable with the laboratory’s standard micronucleus historical vehicle control ranges
2. The positive control chemical (CPA) induced a clear increase in the frequency of MN PCE that was comparable with the laboratory’s historical positive control data
3. Adequate numbers of cells and doses were analysed.
The assay data were therefore considered valid.

Any other information on results incl. tables

Hydroxyprogesterone: Summary of Micronucleus Data:

							Heterogeneity		Statistics a)
Group/Sex/Treatment (mg/kg/day)	PCE Scored	Number of MN PCE	%PCE	MN / 4000 PCE	%MN PCE	SD	Chi²	Significance	P-value	Significance
1M / Vehicle (0)	20000	62	48.16	12.40	0.31	0.09	4.63	NS	-	-
2M / Hydroxyprogesterone (100)	20000	62	50.64	12.40	0.31	0.14	9.97	P≤0.05	0.5238	NS
3M / Hydroxyprogesterone (300)	20000	30	47.68	6.00	0.15	0.06	4.34	NS	0.9802	NS
4M / Hydroxyprogesterone (1000)	20000	31	50.16	6.20	0.16	0.05	2.39	NS	0.9881	NS
1F / Vehicle (0)	32000	67	51.63	8.38	0.21	0.10	12.67	NS	-	-
2F / Hydroxyprogesterone (100)	16000	27	49.90	6.75	0.17	0.07	3.38	NS	0.70	NS
3F / Hydroxyprogesterone (300)	16000	24	46.75	6.00	0.15	0.09	7.01	NS	1.69	NS
4F / Hydroxyprogesterone (1000)	20000	37	51.96	7.40	0.19	0.08	5.58	NS	0.25	NS
Positive control slides	4000	199	51.80	199.0	4.98	0.46	-	-	-	-

 

Linear Trend tests:

Males: P-value: 0.9945 NS

Females:P-value (z) -0.367 NS

a Wilcoxon Rank Sum Test for male data, Chi-squared 2 x 2 Contingency for female data

M Male

F Female

MN Micronucleus

NS Not significant

PCE Polychromatic erythrocyte

SD Standard deviation

Applicant's summary and conclusion

Conclusions:

negative

Executive summary:

The potential for Hydroxyprogesterone to induce micronuclei (MN) in the polychromatic erythrocytes (PCE) of the bone marrow was evaluated in male and female rats following daily oral (gavage) administration for at least 42 days. Bone marrow was sampled 1 day after the final dose administration (Day 43 for male rats or for females either Day 14 post-partum or Day 26 after mating for females that did not litter).

The vehicle control data for %PCE and MN PCE were comparable with the laboratory’s standard micronucleus historical vehicle control data. The positive control slides exhibited a clear increase in MN PCE over the concurrent vehicle control. The assay was therefore accepted as valid.

Male and female rats treated with Hydroxyprogesterone at all doses had group mean %PCE that were similar to the concurrent vehicle control group and which were comparable to the laboratory’s standard micronucleus historical vehicle  control data, thus confirming there was no evidence of test article related bone marrow toxicity.

Male and female rats treated with Hydroxyprogesterone at all doses had MN PCE frequencies that were similar to the concurrent vehicle control group and which were considered consistent with the laboratory's standard micronucleus historical vehicle control data. There were no statistically significant increases in micronucleus frequency for any of the groups receiving the test article, compared to the concurrent vehicle controls.

It is concluded that Hydroxyprogesterone did not induce micronuclei in the polychromatic erythrocytes of the bone marrow of male and female rats treated up to 1000 mg/kg/day, under the experimental conditions employed.