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Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
one-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013-10-03 to 2012 -12-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: In accordancewith OECD test guideline and GLP compliant
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Department of Safety Assessment Advinus Therapeutics Limited Bangalore 560 058, India
- Age at study initiation: (P) x 10 weeks;
- Weight at study initiation:
- Control : Males 287.50 ± 16.62 Females 203.59 ± 16.14
- Low dose : Males 281.70 ± 17.04 Females 202.25 ± 15.49
- Mid dose : Males 283.55 ± 18.79 Females 200.73 ± 17.70
- High Dose : Males 286.38 ± 18.76 Females 203.97 ± 12.51

HOUSING
- Pre mating: Two rats of same sex were housed per cage in sterilized standard polysulfone cages (Size: L 425 x B 266 x H 175 mm), with stainless steel top grill having facilities to pelletted food and drinking water in polycarbonate bottles.
- Mating: During mating, two rats (one male and one female) were housed in standard polysulfone cages with stainless steel top grill having facilities for pelletted food and drinking water in polycarbonate bottles.
- Post-mating: After confirming presence of sperm in the vaginal smear or vaginal plugs (Day 0 of pregnancy), the mated pairs were separated. Males were housed with their former cage mates while females were housed individually in polysulfone cages. The sterilised nesting material (paper shreds) was provided near-term.
- Diet (e.g. ad libitum): Teklad Certified (2014C) Global 14 % Protein Rodent Maintenance Diet -Pellet (Certified) manufactured by Harlan Laboratories B.V. Maasheseweg 87c PO Box 553, 5800, AN Venray, The Netherlands, was provided to animals’ ad libitum.
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):19-24°C
- Humidity (%): relative humidity 59 - 67%,
- Air changes (per hr):2-15 air changes/hour
- Photoperiod (hrs dark / hrs light):12 hours light and 12 hours dark cycle

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The required amount of test item for each dose was weighed and carefully transferred into a mortar. A small aliquot of the vehicle was added to wet the test item and triturated well to obtain a pasty mash. An aliquot of vehicle was added to teh mass and triturated to obtain the test item solution/suspension and then transferred to a dry labelled breaker. The solution/suspension was brought to volume with the vehicle to obtain the derired concentration.

VEHICLE
- Concentration in vehicle: 20, 60 or 200 mg/mL
- Amount of vehicle (if gavage): 5mL/kg bw
- Vehicle: Corn oil
- Lot/batch no. (if required): Sigma Aldrich MKBF860V
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: not specified - continued until pregnancy confirmed or 2 weeks
- Proof of pregnancy: evidence of sperm in the vaginal smear and /or vaginal plug - identified as day 0 of pregnancy
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test item was found to be homogeneous in all the dose formulation samples received from Department of Safety Assessment which met the acceptance limits of variation (85 to 115%) from the theoretical concentrations and % RSD less than 10 %.
Duration of treatment / exposure:
Males: The dose formulation was administered orally by gavage to specific group of rats at approximately the same time (± 2 hours from the time of test item administration on day 1 except on day 17, wherein it exceeded ±2 hours) each day for 2 weeks prior to mating and the treatment was continued during the mating period and until approximately 80% of the females delivered.
Females: The dose formulation was administered orally by gavage to the specific group of rats at approximately the same time (± 2 hours from the time of test item administration on day 1 except on day 17, wherein it exceeded ±2 hours) each day for 2 weeks prior to the mating period and continued through mating, pregnancy and up to lactation day 3.
Frequency of treatment:
Daily
Details on study schedule:
The test item was administered in graduated doses to three groups of male and female rats. There was also a vehicle control group.

The males were dosed for a period of six weeks, up to and including the day before scheduled sacrifice (when approximately 80% of the females had delivered). This included a minimum of two weeks prior to mating, during the mating period and two weeks post mating.

Females were dosed throughout the treatment period. This included two weeks prior to mating (with the objective of covering at least two complete oestrous cycles), the variable time to conception, the duration of pregnancy and four days after delivery, up to and including the day before scheduled sacrifice.
Remarks:
Doses / Concentrations:
0, 100, 300 or 1000mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
20 males and 20 females per group. A total of 80 males and 80 females in the study
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Three dose levels of 100, 300 and 1000 mg/kg bw/day were selected as suggested by the sponsor. In addition to the test doses, a vehicle control group was included.
- Rationale for animal assignment (if not random): Grouping was done by body weight stratification and distributed as follows: rats were weighed and grouped into body weight ranges (Males: 241 – 310g, females: 161 – 240 g). Males in the range of 241 - 300 g and females in the range of 171 - 230 g were selected for the study. These body weight stratified rats were distributed to all the study groups, so that there were no statistically significant differences among group mean body weight within a sex. Grouping was done one day prior to initiation of treatment. The rats with extreme body weights were discarded.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: All rats were observed once daily for appearance, behaviour and clinical/toxic signs. All animals were observed for morbidity and mortality twice daily. As there were no clinical signs of concern, the observation were carried out once during holidays. Dams and offspring were observed for physical or gross behavioural abnormalities.

DETAILED CLINICAL OBSERVATIONS: Daily

BODY WEIGHT: Individual body weights were recorded at the beginning (on day 1) of the treatment and at least weekly thereafter and at termination. All dams were weighed on GD 0, 7, 14 and 20 and on Lactation Days (LD) 0 and 4 and weights were recorded

OTHER:
Cage wise food consumption was calculated by using the food consumed at weekly interval per cage and dividing by the number of rats per cage and the number of days in the intervening period to determine the food intake/rat/day. Food consumption was not measured during the cohabitation period. Food consumption of pregnant dams was recorded on days GD 7, 14 and 20 and for Day 4 of lactation period.

Food consumption of dams was calculated for the period GD 0-7, 7-14 and 14- 20 and for Day 0-4 of lactation period.

The duration of gestation (Gestation Length) was calculated from day '0' of pregnancy to day of parturition (i.e. Lactation Day 0). Females were observed for signs of difficult or prolonged parturition.
Oestrous cyclicity (parental animals):
The duration of gestation (Gestation Length) was calculated from day '0' of pregnancy to day of parturition (i.e. Lactation Day 0). Females were observed for signs of difficult or prolonged parturition. The number of implantation sites and corpora lutea were recorded for all the dams.
Sperm parameters (parental animals):
Parameters examined in male parental generations: testis weight, epididymis weight, Histopathological examination of the testes included a qualitative assessment of stages of spermatogenesis
Litter observations:
STANDARDISATION OF LITTERS: NO

PARAMETERS EXAMINED: At birth, all the pups (both dead and alive) in a litter from each dam were observed for any external deformities which were recorded. The number of pups born (litter size), sex and individual pup body weight of male and female pups on lactation days 0 and 4 were recorded.
The litters were observed daily in order to note the number of alive, dead and cannibalised pups.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.

.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals when approximately 80% of females had delivered

- Maternal animals: All surviving animals [describe when, e.g. after the last litter of each generation was weaned.]

GROSS NECROPSY

Male rats were sacrificed after approximately 80% of females had delivered.
Pups were sacrificed on Lactation Day 4 along with females (dams). One
female (Ro2222) in the low dose group did not deliver till Day 25 post-coitum
and was sacrificed on Day 26 post-coitum.

Rats to be sacrificed at term were anaesthetized with isoflurane, weighed
and exsanguinated. The parent rats were subjected for detailed necropsy by a
veterinary pathologist and findings were recorded. All the dead and sacrificed
pups were examined for malformations and subjected to gross pathological
examination. Dead pups were examined for possible defects and/or cause of
death.
The number of implantation sites and corpora lutea were recorded for all the
dams.
HISTOPATHOLOGY / ORGAN WEIGHTS
Testes and epididymides of all adult males were weighed. The organ weights as percentage of body weights were determined and presented in the report.

..

Microscopic examination took place of epididymes, ovaries, and testes

In addition all gross lesions, ovidcuts, prostate, seminal vesicles and coagulating glands, and uterus with cervix and vagina were collected and preserved

Tissues collected from all animals in the control and high dose groups were examined microscopically for histopathological changes. . As there were no test item related histopathological changes observed in the high dose group, the reproductive organs were not
examined in the lower dose groups. The ovaries of the one non-pregnant female rat were examined microscopically.
Postmortem examinations (offspring):
SACRIFICE
The rats to be sacrificed at term were anaesthetised with isoflurane, weighed and exsanguinated. The parent rats were subjected for detailed necropsy by a veterinary pathologist and findings were recorded. All the dead sacrificed pups were examined for malformations and subjected to gross pathological examination. Dead pups were examined for possible defects and/or cause of death.

GROSS NECROPSY
Male rats were sacrificed after approximately 80% of females had delivered. Pups were sacrificed on lactation day 4 along with females (dams). One female in the low dose group did not deliver till day 25 post-coitum and was sacrificed on day 26 post-coitum.

HISTOPATHOLOGY
On completion of the gross pathology examination, the tissues listed in the table below were collected and preserved in 10% neutral buffered formalin for histopathological examined. Tissues were examined microscopically for histopathological changes. Histopathological examination of the testes included a qualitative assessment of spermatogenesis. The tissues were processed for routine paraffin embedding and 4-5 micron sections were stained with Mayer’s Haematoxylin Eosin stain. In addition, testes were sectioned at 3-4 µm and stained with PAS reagent and haematoxylin to aid in qualitative assessment of spermatogenesis.
Statistics:
The statistical analysis of the experimental data was carried out using the validated package in Excel and also using licensed copies of SYSTAT Statistical package ver.12.0. All quantitative variables like body weight, food intake, organ weights and organ weight ratios were tested for normality (Shapiro-Wilk test) and homogeneity of variances (Levene’s test) within the group before performing a one-factor ANOVA modelling by treatment groups.

Non-optimal (non-normal or heteroschedastic) data was transformed, before ANOVA was performed. Comparison of means between treatment groups and control group was done using Dunnett’s test for the overall treatment when ‘F’ test was found significant. Pre-implantation loss (%), post implantation loss (%), no. of corpora lutea, implantations, pre-coital interval and gestation length (days) were analysed after suitable transformation (√ x + ½) of the data. One-way analysis of variance (ANOVA) was carried out for the transformed data. Dunnett’s pairwise comparison of the treated means with the control mean was done for the significant group differences. Z test was performed for testing the differences in proportions for mating and fertility indices.

All analyses and comparisons were evaluated at the 5% (P≤0.05) level. Statistically significant differences (P≤0.05), indicated by the aforementioned tests was designated by the superscripts throughout the report as stated below:

+/-: Significantly higher (+)/lower (-) than the vehicle control group
Reproductive indices:
REPRODUCTIVE INDICES (reproductive performance data of parents):
- Male mating index (%)
- Male fertility index (%)
- Female mating index (%)
- Fecundity index (%)
- Female fertility index (%)
- Mean number of corpora lutea (CL)/group
- Mean number of implantations/group
- Total number of implantations
- Implantation index
- Pre-implantation loss (%)
- Post implantation loss (%)
- Gestation index
Offspring viability indices:
- Mean litter size per group
- Live birth index (%)
- Day 4 survival index (%)
Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs or mortalities observed at any of the doses tested.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The mean body weights were not significantly affected at any of the doses tested, relative to controls. Food consumption was significantly lower during weeks 1, 2, 5 and 6.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The mean body weights were not significantly affected at any of the doses tested, relative to controls. Food consumption was significantly lower during weeks 1, 2, 5 and 6.
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
There were no clinical signs or mortalities observed at any of the doses tested.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Males: The mean body weights were not affected at any of the doses tested, when compared to vehicle control. Females: The mean body weights were not significantly different from the vehicle control prior to cohabitation period at all the doses tested, when compared to the vehicle control group. Mean maternal body weights and weight changes were unaffected by the treatment during different intervals of gestation period at all the doses tested, when compared to vehicle control. The maternal body weights and food consumption were unaffected by the treatment during lactation period at all the doses tested, when compared to vehicle control.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
There were no treatment-related effects on the mean pre-coital time, gestation length and in the fertility indices between controls and treated groups. The significantly lower male fertility, female fecundity and fertility indices at 100 mg/kg bw/day dose and gestation index at 300 mg/kg bw/day dose was considered toxicologically insignificant due to lack of dose relationship. No effect of test item administration was observed on the number of implantations, and percentage of pre and post implantation losses at any of the tested doses.

ORGAN WEIGHTS (PARENTAL ANIMALS)
There were no test item-related changes in terminal body weights in both the sexes. The organ weights of testes and epididymides remained unaffected by test item administration

GROSS PATHOLOGY (PARENTAL ANIMALS)
There were no test item-related changes in terminal body weights in both the sexes. The organ weights of testes and epididymides remained unaffected by test item administration. There were no test item related gross and microscopic changes in the reproductive organs of males and females at all the doses tested. In females at ≥ 300 mg/kg bw/day dose groups and in males at 1000 mg/kg bw/day dose group, test item-related thickening of non-glandular stomach was observed.

HISTOPATHOLOGY (PARENTAL ANIMALS)
There were no test item related gross and microscopic changes in the reproductive organs of males and females at all the doses tested.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
VIABILITY (OFFSPRING)
Test item had no effects on the mean litter size and mean viable litter size. There were no external abnormalities in live or dead pups in any of the groups. The significantly lower live birth index was observed at 100 and 300 mg/kg bw/day and was considered incidental because of lack of dose relationship. The Day 4 survival index was significantly lower at 100, 300 and 1000 mg/kg bw/day doses, when compared to vehicle control group. This finding was mainly contributed by the loss of an entire litter in each group (female No. Ro2229 of group G2, female No. Ro2244 of group G3 and female No. Ro2263 of group G4) during lactation period. As this isolated finding can be spontaneously observed (it was observed in a previous OECD 421 study performed in-house) the
lower Day 4 survival index observed at all the doses was considered incidental and thus not treatment-related.

BODY WEIGHT (OFFSPRING)
The mean number and weight of male, female and total pups per litter were unaffected by the treatment at all the doses tested, when compared to vehicle control group. However, the mean number of female pups per litter was significantly lower at 1000 mg/kg bw/day on LD 4 and was considered
incidental as the total mean number of pups per litter was unaffected.

SEXUAL MATURATION (OFFSPRING)
Not conducted

ORGAN WEIGHTS (OFFSPRING)
Not measured

GROSS PATHOLOGY (OFFSPRING)
No gross pathological changes were observed in dead pups and pups sacrificed on lactation Day 4 at all the doses tested

HISTOPATHOLOGY (OFFSPRING)
Not performed
Reproductive effects observed:
not specified
Conclusions:
On the basis of results obtained in the present study, No Observed Adverse Effect Level (NOAEL) for reproductive/developmental toxicity in Wistar rats is 1000 mg/kg bw/day.
Executive summary:

The oral administration of decan-1,2 -diol to male and female Wistar rats at the dose levels of 100, 300 and 1000 mg/kg bw/day produced no clinical signs or mortality. There were no toxicologically significant effects on body weights and food consumption in males and females during the entire dosing period.

 

The mean number and weight of male, female and total pups per litter were unaffected by the treatment. The test item had no effects on the mean litter size and mean viable litter size. There were no external abnormalities in live or dead pups in any of the groups. The Day 4 survival index was significantly lower at all the doses tested, when compared to vehicle control group. This finding was mainly contributed by the loss of an entire litter in each group during lactation period. As this isolated finding can be spontaneously observed (it was observed in a previous OECD 421 study performed in-house), the lower Day 4 survival index observed at all the doses was considered incidental and thus not treatment-related.

 

No toxicologically significant changes were observed in pre-coital time, gestation length, mating and fertility parameters. No treatment-related effects were observed on the number of implantations, percentage of pre and post implantation losses. There were no test item-related changes in terminal body weights in both the sexes. The organ weights of testes and epididymides remained unaffected by test item administration. There were no test item related gross and microscopic changes in the reproductive organs of males and females at all the doses tested. No gross pathological changes were observed in dead pups and pups sacrificed on lactation Day 4 at all the doses tested. In females at ≥ 300 mg/kg bw/day dose groups and in males at 1000 mg/kg bw/day dose group, test item-related thickening of non-glandular stomach was observed.

The No Observed Adverse Effect Level (NOAEL) for reproductive/developmental toxicity in Wistar rats is 1000 mg/kg bw/day.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
High
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Oral administration of decan-1,2-diol (100, 300 and 1000 mg/kg bw/day ) to Wistar rats produced no clinical signs or mortality. There were no toxicologically significant effects on body weights and food consumption in males and females during the entire dosing period.

 

The mean number and weight of male, female and total pups per litter were unaffected by the treatment. The test item had no effects on the mean litter size and mean viable litter size. There were no external abnormalities in live or dead pups in any of the groups. The Day 4 survival index was significantly lower at all the doses tested, when compared to vehicle control group. This finding was mainly contributed by the loss of an entire litter in each group during lactation period. As this isolated finding can be spontaneously observed (it was observed in a previous OECD 421 study performed in-house), the lower Day 4 survival index observed at all the doses was considered incidental and thus not treatment-related.

 

No toxicologically significant changes were observed in pre-coital time, gestation length, mating and fertility parameters. No treatment-related effects were observed on the number of implantations, percentage of pre and post implantation losses. There were no test item-related changes in terminal body weights in both the sexes. The organ weights of testes and epididymides remained unaffected by test item administration. There were no test item related gross and microscopic changes in the reproductive organs of males and females at all the doses tested. No gross pathological changes were observed in dead pups and pups sacrificed on lactation Day 4 at all the doses tested. In females at ≥ 300 mg/kg bw/day dose groups and in males at 1000 mg/kg bw/day dose group, test item-related thickening of non-glandular stomach was observed.

The No Observed Adverse Effect Level (NOAEL) for reproductive/developmental toxicity in Wistar rats is 1000 mg/kg bw/day.


Short description of key information:
The oral administration of decan-1,2 -diol to male and female Wistar rats at the dose levels of 100, 300 and 1000 mg/kg bw/day produced no clinical signs or mortality. There were no toxicologically significant effects on body weights and food consumption in males and females during the entire dosing period.

The mean number and weight of male, female and total pups per litter were unaffected by the treatment. The test item had no effects on the mean litter size and mean viable litter size. There were no external abnormalities in live or dead pups in any of the groups. The Day 4 survival index was significantly lower at all the doses tested, when compared to vehicle control group. This finding was mainly contributed by the loss of an entire litter in each group during lactation period. As this isolated finding can be spontaneously observed (it was observed in a previous OECD 421 study performed in-house), the lower Day 4 survival index observed at all the doses was considered incidental and thus not treatment-related.

No toxicologically significant changes were observed in pre-coital time, gestation length, mating and fertility parameters. No treatment-related effects were observed on the number of implantations, percentage of pre and post implantation losses. There were no test item-related changes in terminal body weights in both the sexes. The organ weights of testes and epididymides remained unaffected by test item administration. There were no test item related gross and microscopic changes in the reproductive organs of males and females at all the doses tested. No gross pathological changes were observed in dead pups and pups sacrificed on lactation Day 4 at all the doses tested. In females at ≥ 300 mg/kg bw/day dose groups and in males at 1000 mg/kg bw/day dose group, test item-related thickening of non-glandular stomach was observed.

Justification for selection of Effect on fertility via oral route:
Study conducted to OECD test guideline 421 and GLP compliant

Effects on developmental toxicity

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

No effects upon fertility, reproductive performance or adverse effect on pups were observed at doses upto 1000mg/kg bw/day. Decan-1,2-diol is not classifiable as reproductive toxicant.

Additional information