Hydroxyprogesterone

Repeated dose toxicity: oral

Administrative data

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

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

screening for reproductive / developmental toxicity

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 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.

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: up to 10 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: individually
- Animals were paired on the day following 14 days of dosing. During the pairing phase, one male was housed for up to 10 days with one female from the same dose group. Three animals of Group 1; one animal of Group 2; one animal of group 3; and one animal of Group 4 were paired on the day following 19 days in the pre-pairing phase (Day 20 of the study).

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

Details on study schedule:

- Age at mating of the mated animals in the study: 14-15 weeks

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 / 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

Parental animals: Observations and examinations:

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

Oestrous cyclicity (parental animals):

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

Sperm parameters (parental animals):

Testis weight, epididymis weight, qualitative testis examination

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 10 pups/litter (5/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups

GROSS EXAMINATION OF DEAD PUPS:
- macroscopic examination and recording of all lesions

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals on day 43 of the study (Post-pairing day 15)
- Maternal animals: All surviving animals on lactation day (LD) 14 or day 26 post coitum

GROSS NECROPSY
- Macroscopic examinations were conducted and all lesions were recorded.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [1] were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

SACRIFICE
Surplus pups were culled on PND (post natal day) 4. Remaining pups were sacrificed at PND 13.
Blood was sampled for thyroid hormone analysis. After termination, macroscopic examinations were conducted, and all lesions were recorded.

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).

Male and female mating, fecundity, and fertility indices were analyzed using a one-sided lower tail Fisher’s exact test.

Mean numbers of estrous cycles and mean cycle length were analyzed using the Kruskal-Wallis and Wilcoxon rank sum test.

Percent pregnant, percent delivering, and gestation index were analyzed using a one-sided lower tail Fisher’s exact test.

Percent of females with stillborn pups was analyzed using a one-sided upper tail Fisher’s exact test.

The number of pups delivered, live-born pups, implantation sites and live-birth index, Day 4 viability index, duration of gestation, and percent post-implantation loss were analyzed using the Kruskal-Wallis and Wilcoxon rank sum test.

Pup weights (male, female, and combined) were analyzed using analysis of covariance (ANCOVA), with litter size as the covariate.

Live pups/litters with live pups were analyzed using the Kruskal-Wallis and Wilcoxon rank sum test.

Reproductive indices:

Males:
Mating index % = (Number of males mating with at least 1 female / Number of males cohabitated with at least 1 female) x 100
Fecundity index % = (Number of males impregnating at least 1 female / Number of males mating with at least 1 female) x 100
Fertility Index % = (Number of males impregnating at least 1 female / Number of males cohabitated with at least 1 female) x 100

Females:
Mating index % = Mated females/females cohabited (excluding females sacrificed during Cohabitation) x 100
Fecundity index % = Pregnant females/mated females (excluding females with an undetermined pregnancy status) x 100
Fertility index % = Pregnant females/females cohabited (excluding females sacrificed during Cohabitation or with an undetermined pregnancy status) x 100

Gestation index % = (Number of females with live pups / Number of pregnant females) x 100
% post-implantation loss = (Number of implantations - number of live embryos) / Number of implantations) x 100

Offspring viability indices:

% live birth index (litter) = (Number of live pups per litter at birth / Number of pups born per litter) X 100
% live birth index (mean) = Sum of live pups per group at birth / Total number of litters
% Viability index 1-4 (litter) = (Number of pups alive Day 4 before culling / Number of pups born alive) X 100
% Viability index 1-4 (mean) = Sum of % litters on Day 4 before culling in each group / Total number of litters

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

Organ weight findings including organ / body weight ratios:

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. (Table 2)

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 and no 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

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Description (incidence and severity):

Qualitative testis examination did not indicate any abnormalities in the integrity of the various cell types present within the different stages of the spermatogenic cycle.

Reproductive performance:

no effects observed

Description (incidence and severity):

No test substance-related effects on mating, fecundity, or fertility were noted.
The number of implantation sites evident for females administered 1000 mg/kg/day was slightly lower than controls, and percentage post-implantation loss was statistically higher in this group, compared with controls (p<0.01). Slightly higher percentage post-implantation losses was also evident for females administered 300 or 1000 mg/kg/day compared with controls (p<0.05 or p<0.01 respectively). This resulted in slightly lower mean number of pups per litter in 300 or 1000 mg/kg/day litters, compared with control (-12 and -20% respectively) [see table 3]. Furthermore, a slightly higher percentage of male pups were noted in 1000 mg/kg/day litters, compared with controls (+27%), although statistical significance was never achieved. In the absence of any adverse clinical observations or higher rates of litter mortality after birth, these slight differences, compared with controls, were considered not to have represented adverse effects on the offspring.

The female reproductive NOAEL was established at 1000 mg/kg bw/day as the number of implantation sites in females administered 1000 mg/kg was only slightly lower than controls, as well as the only slightly higher post-implantation losses in females administered 300 or 1000 mg/kg bw/day compared to controls. In addition, there was a high standard deviation within the groups [see table 3].

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 treatment related effects observed

Remarks on result:

other: systemic and reproductive toxicity

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: systemic toxicity

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

reproductive performance

Remarks on result:

other: reproductive toxicity

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

reproductive performance

Remarks on result:

other: reproductive toxicity

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

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

In the absence of a convincing dose-related response, the statistically significantly increased body weights of pups from 100 or 300 mg/kg/day litters following the cull on PND 4 were considered to have arisen incidentally and were unrelated to administration of Hydroxyprogesterone.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Other effects:

not specified

Description (incidence and severity):

A slightly higher percentage of male pups were noted in 1000 mg/kg/day litters, compared with controls (+27%), although statistical significance was never achieved.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

- No effects on ano-genital distance and nipple/areolae count.
- No statistical significant effect on TSH of pups on PND 4 or 13

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

not specified

Basis for effect level:

other: no adverse effects observed

Reproductive effects observed:

no

Table 2:

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.

Table 3:

Dose level  Control  Low dose Intermediate dose  High dose   Pups delivered  Mean  11.85  11.56  10.44  10.20    Standard deviation 1.99   2.01  3.05 2.62     Number of animals 13   9  9  10  Implantation sites Mean   12.08 12.33  11.44  11.40     Standard deviation  2.18  1.66  3.13  2.41  Postimplantation loss Mean  0.23   0.78  1.00 1.20     Standard deviation  0.60 0.83  0.87   0.63

 xxx

Conclusions:

In conclusion, 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 and reproductive 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. Due to the only slight changes observed in number of implantation sites at 1000 mg/kg/day and in post-implantation losses at 300 and 1000 mg/kg/day and the high standard deviations the NOAEL for females for reproductive toxicity was established at 300 mg/kg/day.
The NOAEL for offspring development was considered to be 1000 mg/kg/day.

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. For details on general toxicity please refer to section 7.5 (Repeated Dose Toxicity, Main study, OECD 422).

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.

The number and length of estrous cycles were unaffected by administration of Hydroxyprogesterone, and no effect on mating, fecundity, or fertility indices occurred.

A slightly lower number of implantation sites were observed for females administered 1000 mg/kg/day and a slightly higher percentage of post-implantation losses for females administered 1000 or 300 mg/kg/day, were noted, resulting in slightly lower mean pups/litter for these dose groups. A slightly higher percentage of male pups were noted in 1000 mg/kg/day litters. No effect on thyroid hormones was noted on PND 4, and no thyroid weight changes, macroscopic abnormalities, or microscopic changes were noted in the thyroids of pups.

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 and reproductive 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 and reproductive toxicity.

The NOAEL for offspring development was considered to be 1000 mg/kg/day, based on the slightly higher number of male offspring noted at this dose level, compared with controls.

 

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 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

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:

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

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: 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.

Examinations

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.

Results and discussion

Results of examinations

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

Effect levels

1

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

2

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

Target system / organ toxicity

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

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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