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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
OECD 421
Type of information:
experimental study
Adequacy of study:
key study
Study period:
December 2017 - Date of study director signature; end of experimental phase 06 March 2018 (last day of necropsy)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The Sprague Dawley rat was the species and strain of choice because it is accepted by many regulatory authorities and there are ample experience and background data on this species and strain. The oral route was selected as it is a possible route of exposure of the test item in man.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Italia, S.p.A., Calco (Lecco), Italy.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 7-8 weeks
- Weight at study initiation: Males: 200-225 g; Females: 175-200 g
- Fasting period before study: no
- Housing: up to 5 of one sex to a cage, in polysulphone solid bottomed cages measuring 59.5×38×20 cm (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese).
- Diet: commercially available laboratory rodent diet (4 RF 21, Mucedola S.r.l., Via G. Galilei, 4, 20019, Settimo Milanese (MI), Italy) was offered ad libitum throughout the study.
- Water: Drinking water was supplied ad libitum to each cage via water bottles.
- Acclimation period: 5 weeks was allowed before the start of treatment.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): 15-20
- Photoperiod: artificial light for 12 hours each day

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
softened by reverse osmosis
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The required amount of test item was dissolved in the vehicle. During the formulation procedure, particular attention was paid to avoid foaming production as much as possible. The formulations were prepared daily (concentrations of 10, 30 and 50mg/mL). Concentrations were calculated and expressed in terms of test item corrected for purity.
Details on mating procedure:
- M/F ratio per cage: 1
- Length of cohabitation: the female was paired with the same male until positive identification of copulation occurred
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): The females were transferred to individual solid bottomed cages measuring 42.5×26.6×18 cm (Tecniplast Gazzada S.a.r.l.) for the gestation period, birth and lactation. Suitable nesting material was provided and changed as necessary.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis was performed in a separate study in order to validate the analytical method and the formulation procedure and to verify the stability of the formulations (RTC Study No. A2478). Samples of the formulations prepared during the current study (the first and the last week of treatment) were analysed to check the concentration. Chemical analysis was carried out by the Analytical Chemistry Department at RTC. The software used for this activity was Empower® 2 Build No. 2154.
Duration of treatment / exposure:
Main groups
Males
Animals of the main groups were dosed once a day, 7 days a week, for 2 consecutive weeks prior to pairing, through the mating period and thereafter through the day before necropsy (Day 15 or 16 of the mating phase), for a total of 30 or 31 days. Dose volumes were adjusted once per week for each animal according to the last recorded body weight.
Females
Animals were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing and thereafter during pairing, post coitum and post partum periods until Day 13 post partum or the day before sacrifice. Female no. X0780053 which had total litter loss was dosed up to Day 2 post partum (the day before sacrifice). Dose volumes were adjusted once per week for each animal according to the last recorded body weight up to mating. During the gestation and lactation periods, dose volumes were calculated according to the last recorded body weight.
Satellite groups
Animals were dosed once a day, 7 days a week, for 4 consecutive weeks until the day of necropsy (Day 29 of the premating phase). Animals were dosed for a total of 28 days. Dose volumes were adjusted once per week for each animal according to the last recorded body weight.
Frequency of treatment:
Once daily
Details on study schedule:
- Age at mating of the mated animals in the study: 12-13 weeks
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Groups 1 (main) and 5 (satellite)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Group 2
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
Group 3
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Remarks:
Groups 4 (main) and 6 (satellite)
No. of animals per sex per dose:
Main groups: 10 per sex per dose
Satellite groups: 5 per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels have been selected based on information from a previous repeated dose toxicity study
- Fasting period before blood sampling for clinical biochemistry: yes
Positive control:
none

Examinations

Parental animals: Observations and examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before commencement of treatment and at least once daily during the study
BODY WEIGHT: Yes / No / No data
- Time schedule for examinations:
Main groups: Males were weighed weekly from allocation to termination and on the day of necropsy. Females were weighed weekly from allocation to positive identification of mating and on gestation Days 0, 7, 14 and 20. Dams were also weighed on Days 1, 4, 7 and 13 post partum and on the day of necropsy.
Satellite groups: Animals were weighed on the day of allocation to treatment groups, on the day that treatment commenced, weekly thereafter and on the day of necropsy.
FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Oestrous cyclicity (parental animals):
Vaginal smears and oestrous cycle (Main groups)
Stock females
Oestrous cycle was monitored by vaginal smears in all stock females for at least 2 weeks before dosing in order to exclude from the study females with irregular cycle.
Females allocated to groups
Vaginal smears were taken in the morning from Day 1 of dosing up to positive identification of mating. The vaginal smear data were examined to determine the following:
– anomalies of the oestrous cycle;
– pre-coital interval (i.e., the number of nights paired prior to the detection of mating).
Vaginal smears were also taken from all females, before despatch to necropsy.
Mating (Main groups)
Matings were monogamous (one male to one female). Vaginal smears were taken from the day after the start of pairing until positive identification of copulation (sperm identification, vaginal plug in situ or copulation plug found on the cage tray). Each cage tray was checked each morning for the presence of copulation plugs. The female was paired with the same male until positive identification of copulation occurred.

Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no
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, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups.
GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals males were killed after the mating of all females.
- Maternal animals:
Females with live pups were killed on Day 14 post partum.
Female no X0780053 with total litter loss on Day 0 post partum was killed on Day 3 post partum.
Female no. X0780019 which did not give birth was killed on Day 26 post coitum.
GROSS NECROPSY
- Gross necropsy consisted of a detailed post mortem examination including examination of the external surface and orifices. Changes were noted, the requisite organs weighed and the required tissue samples processed for histopathological examination.
All females were examined also for the following:
– external and internal abnormalities
– number of visible implantation sites (pregnant animals)
– number of corpora lutea (pregnant animals)
HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues which were prepared for microscopic examination and weighed, respectively, are summarized in section “any other details on results incl. tables”.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at Day 4 post partum (culled pups) and at Day 14 post partum
- These animals were subjected to postmortem examinations as follows:
GROSS NECROPSY
All pups found dead in the cage were examined for external and internal abnormalities. All culled pups sacrificed at Day 4 post partum were subjected to an external examination. Sex was determined by internal gonads inspection. All live pups sacrificed at Day 14 post partum were examined for external abnormalities. Sex was determined by internal gonads inspection.
HISTOPATHOLOGY / ORGAN WEIGTHS
Thyroid was weighed from one male and one female from each litter (pups bled for thyroid hormone determination) and preserved. The thyroid weight (expressed in mg) was determined after fixation.
Statistics:
Standard deviations were calculated as appropriate. For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data. The criteria for statistical significance was p<0.05 and p<0.001.
Statistical analysis of histopathological findings was carried out by means of the nonparametric Kolmogorov-Smirnov test if n was more than 5.
The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters. Intergroup differences between the control and treated groups were assessed by the nonparametric version of the Williams test.
Reproductive indices:
Reproductive indices
1) Males:
- Copulation Index (%) = no. of animals mated x 100/ no. of animals paired
- Fertility Index (%) = no. of males which induced pregnancy x 100/ no. of animals paired
2) Females:
- Copulatory Index (%) = no. of animals mated x 100/ no. of animals paired
- Fertility Index (%) = no. of pregnant females x 100/ no. of females paired was calculated as a percentage from the formula:
3) Males and females:
- Copulatory Interval = Mean number of days between pairing and mating

Offspring viability indices:
Pre-implantation loss was calculated as a percentage from the formula:
[(No. of corpora lutea – No. of implantation) / No. of corpora lutea] * 100
Pre-natal loss was calculated as a percentage from the formula:
[(No. of visible implantations – live litter size at birth) / No. of visible implantations] * 100
Pup loss at Day 0 post partum was calculated as a percentage from the formula:
[(Total litter size – live litter size) / total litter size] * 100
Post natal loss at Day 4 post partum (before culling) was calculated as a percentage from the formula:
[(live litter size at birth – live litter size at day 4 before culling) / live litter size at birth] * 100
Post natal loss at Day 13 post partum (after culling) was calculated as a percentage from the formula:
[(live litter size at Day 4 after culling – live litter size at day 13) / live litter size at Day 4 after culling] * 100
Sex ratios were calculated at birth, on Day 4 and on Day 14 post partum and were presented as the percentage of males per litter.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No relevance was attributed to the hairloss and/or staining observed in one lowdose female, one mid-dose female of the main group or in one high dose male and two high dose females of the satellite groups. These signs were considered incidental. Since the hairloss was observed at fore- and hindlimbs this was caused by licking.
Dermal irritation (if dermal study):
not examined
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:
not examined
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Parental males
Thyroxine showed a statistically significant increase in a number of males dosed at 500 mg/kg/day (mean group value was 12 % above controls). Due to the minimal severity and the absence of other related changes, this finding was considered of no toxicological relevance.
Offspring - Day 14 post partum
A decrease of Thyroid Stimulating Hormone was recorded in some male pups of all treated groups. Compared with controls, changes were 21 % to 31 %, with no dose-relation. Due to the absence of dose-relation and of other related changes, this finding was not considered to be treatment-related.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Parental animals
No treatment-related changes were noted, following histopathology evaluation. Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages. As regular layering in the germinal epithelium was noted, there was no treatment-related effect on the spermatogenic cycle. All reported changes were considered spontaneous and incidental, having a comparable incidence in control and treated groups, and/or are characteristically seen in untreated
Sprague Dawley SD rats of the same age.

Satellite groups
No treatment-related changeswere noted, following histopathology evaluation of bone marrow, spleen and thymus. All reported changes were considered spontaneous and incidental, having a comparable incidence in control and treated groups, and/or are characteristically seen in untreated Sprague Dawley SD rats of the same age.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

Parental females
No mortality occurred during the study. One control female (no. X0780015) was proved not pregnant at necropsy and the mid-dose female no. X0780053 had total litter loss on Day 0 post partum.
The number of females with live pups on Day 14 post partum was: 9 in the control, 10 in the low dose (100 mg/kg/day), 9 in the mid-dose (300 mg/kg/day) and 10 in the high dose group (500 mg/kg/day).
No signs were recorded in males of the main groups during the study. No relevance was attributed to the hairloss and/or staining observed in one low dose female, one mid-dose female of the main group or in one high dose male and two high dose females of the satellite groups. These signs were considered incidental.
No differences of toxicological relevance were observed in body weight or body weight gain between the control and treated animals.
The food consumption was comparable between groups.

Parental males
Thyroxine showed a statistically significant increase in a number of males dosed at 500 mg/kg/day (mean group value was 12% above controls). Due to the minimal severity and the absence of other related changes, this finding was considered of no toxicological relevance.
Pups - Day 14 post partum.
A decrease of Thyroid Stimulating Hormone was recorded in some male pups of all treated groups. Compared with controls, changes were 21 % to 31 %, with no dose-relation. Due to the absence of dose-relation and of other related changes, this finding was not considered to be treatment-related.
Oestrous cycle, reproductive parameters, pairing combination and mating performance
Oestrous cycle and reproductive parameters (pre-coital intervals, copulatory and fertility indices) were similar in treated and control groups. All females were pregnant except for one control female (animal no. X0780015).
Implantation sites, pre-implantation loss data, pre-natal loss data and gestation length of females
Gestation periods were similar between treated and control group females. All pregnant females gave birth on Day 22 post coitum (mean value). Corpora lutea, implantation sites, total litter size, pre-implantation loss and pre-natal loss (percentage) were similar in control and treated groups.
Litter data at birth, on Day 1 and on Day 4 post partum (before culling), on Day 13 (after culling) post partum and sex ratio: No differences in litter data were seen between control and treated groups.
Sex ratios (calculated as a percentage of males) at birth and on Days 4 and 14 post partum did not show differences between groups.
Anogenital distance
Anogenital distance (normalised for the cube root of the pup weight collected on Day 1 post partum) was unaffected by treatment.
Clinical signs of pups and nipple observations
Pup no. 12 of dam X0780031 of the low dose group was found moribund and with no milk in the stomach on Day 4 post partum. This pup was selected for culling. No abnormalities were found at necropsy. No signs related to treatment were seen in the remaining pups.
No nipples were present in any male pup, when observed on Day 13 post partum. Necropsy findings in decedent pups, culled pups and in pups sacrificed on Day 14 post partum.
No treatment-related findings were described. Pups thyroid weight on Day 14 post partum. Thyroid weight in treated pups was comparable to controls.
Terminal body weight of treated animals of the three main groups (Groups 2, 3 and 4), as well as the high dose satellite group (Group 6) was comparable to the concurrent control group (Group 1 or 5).
No relevant changes were observed in absolute and relative organ weight in all treatment groups of both sexes, when compared to the concurrent control data. All organ weight variations between control and treated animals were considered to be within the physiological range of Sprague Dawley SD rats of this age.
Macroscopic observations
No treatment-related changes were noted, following gross pathology examination in either main or satellite groups. All observed changes were considered spontaneous and incidental, having a comparable incidence in control and treated groups and/or are characteristically seen in untreated Sprague Dawley SD rats of the same age.
Microscopic observations
Parental animals
No treatment-related changes were noted, following histopathology evaluation. Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages. As regular layering in the germinal epithelium was noted, there was no treatment-related effect on the spermatogenic cycle.
All reported changes were considered spontaneous and incidental, having a comparable incidence in control and treated groups, and/or are characteristically seen in untreated Sprague Dawley SD rats of the same age.
Satellite groups
No treatment-related changes were noted, following histopathology evaluation of bone marrow, spleen and thymus. All reported changes were considered spontaneous and incidental, having a comparable incidence in control and treated groups, and/or are characteristically seen in untreated Sprague Dawley SD rats of the same age.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed at highest dose level

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
Pup no.12 of dam X0780031 of the low dose group was found moribund and with no milk in the stomach on Day 4 post partum. This pup was selected for culling. No abnormalities were found at necropsy.
No signs related to treatment were seen in the remaining pups.
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:
not examined
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
A decrease of Thyroid Stimulating Hormone was recorded in some male pups of all treated groups. Compared with controls, changes were 21 % to 31 %, with no dose-relation. Due to the absence of dose-relation and of other related histopathological changes, this finding was not considered to be treatment-related.
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Thyroid weight in treated pups was comparable to controls.
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
500 mg/kg bw/day
Based on:
other: Treatment of parental generation
Sex:
male/female
Basis for effect level:
other: no adverse effects observed at highest dose level

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

Tissues which were prepared for microscopic examination and weighed:

Organs / tissues

Weight

Fixation / Preservation

Microscopic examination

Abnormalities

-

Yes

Yes

Adrenal glands

-

Yes

Yes

Bone marrow (from sternum), only satellite groups

-

Yes

Yes

Brain (cerebrum, cerebellum,

medulla/pons)

-

Yes

-

Clitoral gland

-

Yes

-

Epididymides

Yes

Yes

Yes

Kidneys

-

Yes

Yes

Liver

-

Yes

Yes

Mammary gland - Females

-

Yes

Yes

Mammary gland - males

-

Yes

Yes

Ovaries with oviducts

Yes

Yes

Yes

Parathyroid glands, weighed and preserved with thyroid gland

Yes

Yes

-

Pituitary gland

-

Yes

-

Penis

-

Yes

-

Prostate gland (dorsolateral and ventral)

Yes

Yes

-

Sciatic nerve

-

Yes

Yes

Seminal vesicles with coagulating glands

Yes

Yes

Yes

Spleen, only satellite groups

Yes

Yes

Yes

Testes

Yes

Yes

Yes

Thymus, where present, only in satellite groups

Yes

Yes

Yes

Thyroid gland

Yes

Yes

Yes

Uterus – cervix

Yes

Yes

Yes

vagina

-

Yes

Yes

Applicant's summary and conclusion

Conclusions:
The toxicity, as well as any possible effects of the test item on male and female reproductive performance were investigated in this study (main groups). Two satellite groups (control and high dose) sacrificed after 4 weeks of treatment were included for specific histopathology investigations of spleen, thymus and bone marrow.
The dosages used were 100, 300 and 500 mg/kg/day. The study was conducted according to OECD Test Guideline No. 421 adopted on 29 July 2016.
For parental main groups animals, no adverse effects were found on oestrous cycle, copulation, fertility, delivery or lactation gestation length, number of corpora lutea or implantation sites.
No adverse effects were found in anogenital distance, pup sex ratios, pup weight, pup viability and no treatment related findings were noted at necropsy. Thyroid weights of the parental animals and of pups on Day 14 post partum were comparable to the control group.
Macroscopic observation and organ weight were unaffected by treatment. No treatment related changes were described following histopathology evaluation in main and satellite groups, including spermatogenic cycle.
Slight changes noted in thyroid hormone levels were considered not related to treatment.
The histopathological examination performed in satellite group animals did not show any changes related to treatment.
The No Observed Adverse Effects Level (NOAEL) for reproductive and developmental toxicity including satellite group animals was considered to be 500 mg/kg/day for both parental animals and offspring, this being the highest dose investigated.
Executive summary:

In this study according to OECD guideline 421, GLP, the toxicity as well as any possible effects of “Sophorolipids: fermentation products of glucose and fatty acids, C18-unsatd., esters with glycerol with yeast Candida bombicola, partially hydrolysed” on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of conceptus and parturition, were investigated (main groups). Two satellite groups (control and high dose) sacrificed after 4 weeks of treatment were included for specific histopathology investigations of spleen, thymus and bone marrow.

The dosages used were 100, 300 and 500 mg/kg/day. For parental main groups animals, no adverse effects were found on oestrous cycle, copulation, fertility, delivery or lactation gestation length, number of corpora lutea or implantation sites.

No adverse effects were found in anogenital distance, pup sex ratios, pup weight, pup viability and no treatment related findings were noted at necropsy. Thyroid weights of the parental animals and of pups on Day 14 post partum were comparable to the control group.

Macroscopic observation and organ weight were unaffected by treatment. No treatment related changes were described following histopathology evaluation in main and satellite groups, including spermatogenic cycle.

Slight changes noted in thyroid hormone levels were considered not related to treatment.

The histopathological examination performed in satellite group animals did not show any changes related to treatment.

The No Observed Adverse Effects Level (NOAEL) for reproductive and developmental toxicity including satellite group animals was considered to be 500 mg/kg/day for both parental animals and offspring, this being the highest dose investigated.