Rhamnolipids: fermentation products of glucose with Pseudomonas putida, Pseudomonadaceae

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

In a screening assay conducted with the test substance Rhamnolipid (OECD 421 study) rats were dosed with 100, 300 and 1000 mg/kg bw/day via gavage. No effects on fertility and reproduction organs were observed in parental animals. Also, no effects were seen with regard to the pre- and postnatal development of the pups. A reduced T4 level of the male animals and a reduced thyroid weight of the female animals was observed in the high dose group at the end of the study. The premature deaths and the observed signs of clinical toxicity (especially breathing sounds) are due to the physical properties of the test item and not considered to be signs of systemic toxicity.

In the 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.

 

The developmental and reproduction toxicity potential of jelly mushroom glycolipids from Dacryopinax spathularia was studied in Crl:CD (SD) rats by daily oral gavage administration at doses of 150, 500 or 1000 mg/kg/day. Pregnant female rats in the developmental study received the test article from Gestation Days 6–19. F₀ and F₁ parental animals in the 2-generation reproduction toxicity study were dosed for a minimum of 70 days prior to mating and throughout mating, gestation, and lactation, until the day prior to euthanasia (following weaning of litters on postnatal day 21). The offspring of the F₀ and F₁ generations were potentially exposed to the test article in utero and via the milk while nursing. In the developmental study, there were no adverse effects on intrauterine growth and survival, or fetal morphology. In the 2-generation reproduction toxicity study, there were no adverse effects on observed parameters including macroscopic or microscopic findings, or organ weights for F₀ or F₁ animals, no effects on reproductive performance, and no test article-related effects on F₁ and F₂ postnatal survival, development, or growth. Therefore, the no-observed-adverse-effect level (NOAEL) for parental systemic toxicity, parental reproductive toxicity, and developmental/neonatal toxicity, was considered to be 1000 mg/kg/day, the highest dosage tested.

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes

Limit test:

no

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 or test system 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

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

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

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.

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: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

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.

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

Key result

Critical effects observed:

no

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

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

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

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

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

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.

Reference 2

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September - October 2016

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Results derived from Read-across approach

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl: CD (SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

Sexually mature male and virgin female Crl: CD rats (110/sex), approximately 30 days of age, were obtained from Charles River laboratories, Inc. Raleigh, NC, and were acclimated for 14 days. All F0 and F1 parental animals were house 2-3 per cage, by sex, from receipt (F0) or selection (F1) in solid bottom caging with ground corn-cob material. During co-habitation, the rats paired (1 female to 1 male) in solid bottom caging with bedding material. Following breeding F0 and F1 parental animals were individually housed or housed with their litter (females that delivered) until euthanasia or weaning of the litter.
The study rooms were maintained at a daily average temperature of 22.2 °C-23.0°C with a daily average relative humidity of 31.8%-60.1% and a 12-h light/12-h dark cycle.
PMI Nutrition International, LLC Certified Rodent LabDiet 5002 and reverse osmosis-purified (on-site) drinking water were provided ad libitum throughout the acclimation periods and during the experimental phase of each study.

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

F0 animals were approximately 6 weeks of age at the initiation of test article administration. The test article was administered to offspring selected to become the F1 parental generation following weaning, i.e. begininng on postnatal day (PND) 21. The F0 and F1 males continued to receive the test articele throughout mating and through the day prior to euthanasia. The F0 and F1 females continued to received test article throughout mating, gestation, and lactation, and through the day before to euthanasia. Offspring (25/sex/group) from the pairing of the F0 females were selected by PND 21 to consitutite the F1 generation. Nonselected F1 pups and all surviving F2 pups were ethanized on PND 21. F0 males and females were dosed for 138-148 consecutive days.

Details on mating procedure:

Vaginal lavages for each F0 and F1 females were collected for 21 days prior to cohabitation and continuing until evidence of mating was observed, or until the end of mating period to determined estrous cycle length, on the day of necropsy to determine the stage of estrus

Duration of treatment / exposure:

Fo and F1 received test article.70 days before mating, throughout mating, gestation, and lactation, and through the day before to euthanasia. F0 males and females were dosed for 128-133 conscutive dayss anf F1 males werde dosed for 138-148 consecutive days.

Frequency of treatment:

daily

Dose / conc.:

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

Dose / conc.:

150 mg/kg bw/day

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: the dose levels for the reproduction toxicity study were selected based on the results of a dose-range finding study in which administration of AM-1 to F0 male and female rats via oral gavage (500 mg/Kg/day and 1000 mg/kg/day) or drinking water (1650 and 5000 ppm) resulted in no reproductive toxicity at any dosage/exposure level. At the initial high AM-1 dose level of 11000 ppm in drinking water, excessive body weight losses correlating to reduced food and water consumption were noted during the first seven days of test article exposure. Reduced consumption of the treated water was attributed to lower palatability likely dure to the surfactant properties of the test article at higher concentrations and increased viscosity of the test article solution. Animals in this group were given a 3-day dosing pause before dosing was restarted via oral gavage at 500 mg/kg/day for the remainder of the study. Due to the technical challenges and potential indirect effects of drinking water administration, the definitive 2-generation reproduction study used only oral gavage as the route of test article administration.
- Rationale for animal assignment (random): Animals were allocated to the test groups by means of a computer-generated randomization program based on body weight stratification.
- Fasting period before blood sampling for clinical biochemistry: yes
- Study design: Three groups of male and females Crl: CD (SD) rats (25/sex/group) were administered the test article, AM-1, daily by oral gavage for at least 70 consecutive days prior to mating. Dosage levels were 150, 500, and 1000 mg/kg bw/day. Dosage levels were 150, 500 and 1000 mg/kg/day for the F0 and F1 generations, administered at a dose volume of 10 mL/kg. A concurrent control group of 25 rats/sex/group received the vehicle (reverse osmosis-treated water) on a comparable regimen. F0 animals were approximately 6 weeks of age at the initiation of test article administration. The test article was administered to offspring selected to become the F1 parental generation following weaning, i.e. beginning of postnatal day (PND) 21. The F0 and F1 males continued to receive the test article throughout mating, gestation, and lactation, and through the day prior to euthanasia. Offspring (25/sex/group) from the pairing of the F0 animals were selected by OND 21 to constitute the F1 generation. Non-selected F1 pus and all surviving F2 pups where euthanized on PND 21, F0 males and females were dosed for 138-133 consecutive days, and F1 males and females were dosed for 138-148 days

Positive control:

none

Parental animals: Observations and examinations:

All animals were observed twice daily for mortality and moribundy.Individual clinical observation were recorded daily and detailed physical examinations were recorded weekly and for all parental animals. Body weights for F0 and F1 males were recorded weekly throughout the study. F0 and F1 female body weight were recorded weekly until evidence of copulation was observed and then again after weaning of their litter until scheduled necropsy. Once evidence of mating was observed, female body weights were recorded on GD 0, 4, 7, 11, 14,17, and 20 and on Lactation Days (LD) 0 (when possible), 1, 4, 7, 11,14,17, and 21.
F0 and F1 male and female food consumption was measured weekly, except during the mating period, gestation , and lactation. Following evidence of mating, F0 and F1 femaels foof consumptions was recorded on the same schedule as body weight collection throughout gestation and lactation.

Sperm parameters (parental animals):

Quantitative assessment of spermatogenesis were evaluated for each F0 and F1 adult male rat. The right cauda epididymis of each male was used for determination of sperm motility using the HT IVOS* instrument. Sperm morphology was evaluated from differential count of 200 sperm per male by light microscopy via a modification of the wet mount evaluation technique. In addition, the left testis and epididymis from all teated F0 and F1 males were homogenized and a samples stained with a DNA specific fluorescent dey was analyzed using HT IVOS for determination of homogenization- resistant spermatid count and calculation of sperm prodcution rate.

Litter observations:

Beginning on the day parturition was initiated (PND0), pups, were sexed and examined for gross malformations, and the numbers of stillborn and live pups were recorded., All pups were individually identified by tattoo applied to the digits. Each litter was examined twice daily for survival, and all deaths were recorded. Litters were examined daily for any adverse changes in appearance or behavior. For both generations (F1 and F2), 8 pups/litter (4 pups/sex, when possible) were selected on PND 4 to reduced the variability among litters. On PNDs 1, 4, 7, 14 and 21 clinical observations were performed on each pup. Any abnormalities in nursing behavior were recorded. The sex of each pup was determined on PNDs 0, 4, 14, and 21. The following litter parameters were defined and calculated for each generation: mean live litter size, postnatal survival between birth and PND 0 or birth and PND4, postnatal survival for all other postnatale intervalrs (e.g., PND 0-1, 1-4, 4-7, etc.). Developmental landmarks were evaluated for the F1 rats selved to constitute the F1 generation, Daily evaluations of balanopreputial seperation in males and vaginal patency in females were performed beginning on PND 35 and PND 25, respectively.

Postmortem examinations (parental animals):

A complete necropsy was conducted on all F0 and F1 parental animals found dead, euthanized in extremis, or at termination. For females that delivered, the number of former implantation sited were recorded. The number of corpora lutea was also recorded for females necropsied during gestation through PND4. Selected tissues and organs were collected and placed in 10% neutral-buffered formalin or appropriate fixative. Absolute weights and organ-to-final body weight and brain weight rations were recorded for the following: adrenal glands, brain, epididymes (total and cauda), heart, kidneys, liver, ovaries, pituitaty gland, prostate gland, seminal vesicles, and coaulating glands (with accessory fluids), spleen, testes thyroid gland, thymus gland, and uterus with ovidctues and cervix. Ovarian primordial follicle counts were recorded for all F1 females in the control and high dose groups and for all F1 females suspected of reduced fertility.
Microscopic evaluations were performed on selected tissues (brain, cervix, coagulating gland, epididymis, kidneys, liver, mammary gland (female only), ovaries, pituitary gland, prostate gland, seminal vescicles, testis, uterus, vagina, vas deferens, and all gross (internal lesions) for all F0 and F1 parental animals from the control and high-dose groups and for all adult animals found dead.

Postmortem examinations (offspring):

Gross necropsies were performed on F1 weanlings not selected as parental animals and all surving F2 weanlings on PND 21 with emphasis on developmental morphology and organs of the reproductive system. At the time of necropsy, the brain, spleen, and thymus were weighed and retained from 2 survivin pups/sex/litter from both the F1 and F2 generations.

Statistics:

Parentalmating, fertility, copulation, and conception indice were analyzed using the Chi-square tests with Yets´ correction factor. All paraetric endpoints were subjected to a parametric one-way ANOVA to determine intergroup differences. If the parametric ANOVA reveled significant (p< 0.05) intergroup variance, Dunnett´s test was used to compare the test article treated group to the control group. All nonparametric endpoints were subjected to the Kruskal-Wallis

Reproductive indices:

Male and female reproductive performance was evaluated by calculating the following indices: male and females mating index, male and females fertility index, male copulation index, and female conceptions index. All females were allowed to deliver naturally and rear their young to weaning (PND21).

Clinical signs:

no effects observed

Description (incidence and severity):

These findings were generally noted in a dose-related manner throughout the treatment period for both generations. The increased occurrence of rales 1–2 h following dose administration was attributed to the surfactant properties of the test article combined with oral gavage dosing and was not considered adverse. Red and clear material findings around the nose and/or mouth are common following oral gavage administration and although dose- responsive, these observations only occasionally persisted to the de- tailed physical examinations or daily examinations, and therefore were considered non-adverse in the absence of other signs of systemic toxicity.

Mortality:

no mortality observed

Description (incidence):

There were no test article.related effects on F0 or F1 parental survival at any dosage level. One F0 male in the 500 mg/kg bw/day group and 1 F0 female in the 150 mg/kg bw/day group were found dead on Study Day 5.No remarkable clinical observations were noted for either animal prior to death. Because there were no deaths in the 1000 mg/kg/day group F0 males and females, the mortality noted in single animals in the 150 and 500 mg/kg/day groups was not considered test article-related.
In the control group, 1 F0 male was found dead on Study Day 9; there were no clinical observations noted for this male prior to death.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no test article-related effects on body weights and body
weight gains in the F0 generation (including during gestation and lactation) at any dosage level.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no test article-related effects on food consumption, or food efficiency in the F0 generation (including during gestation and lactation) at any dosage level.

Food efficiency:

no effects observed

Description (incidence and severity):

see above

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

not examined

Ophthalmological findings:

not examined

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

A significantly (p < 0.05) higher mean estrous cycle length was noted in the 150 mg/kg/day group compared to the concurrent control group; however, this difference was not considered test article-related because it did not occur in a dose-related manner, and the mean estrous cycle length was within the Charles River Ashland historical control data range. No test article-related effects were noted on mean gestation lengths or the process of parturition at any dosage/exposure level in the F0 and F1 generations, and the numbers of former implantation sites, pups born, and unaccounted-for sites were similar to control group values (Tables 3 and 4)

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

No test-related effects were observed on F0 an F1 male spermatogenesis parameters (motility, progressive motility, testicular and epididymal sperm concentration, sperm production rate, and the percentage of morphologically normal sperm) at any dosage level.

Reproductive performance:

no effects observed

Description (incidence and severity):

No test article-related effects were observed on F0 and F1 male and female mating and fertility, male copulation and female conception indices, estrous cycle lengths or pre-coital intervals

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

These findings were generally noted in a dose-related manner throughout the treatment period for both generations. The increased occurrence of rales 1–2 h following dose administration was attributed to the surfactant properties of the test article combined with oral gavage dosing and was not considered adverse. Red and clear material findings around the nose and/or mouth are common following oral gavage administration and although dose- responsive, these observations only occasionally persisted to the de- tailed physical examinations or daily examinations, and therefore were considered non-adverse in the absence of other signs of systemic toxicity.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

One F1 male each in the 500 and 1000 mg/kg/day groups was found dead on PND 22 and 60, respectivly. One F1 female in the 500 mg/kg/day group was found dead on presumed GD 21; this female was determined to be nongravid at necropsy. Two F1 females in the 1000 mg/kg/day group were found dead on PND 68 and LD 0, respectively; the female found dead during lactation was in dystocia. Based on macroscopic findings noted at necropsy (including dark red discoloration and/or areas of the lungs, lungs that were not fully collapsed, and/or foamy tracheal contents) and microscopic findings of inflammation, hemorrhage, and/or edema of the lungs in multiple animals that may have been related to inadvertent pulmonary aspiration of the test article during or following gavage, the aforementioned deaths in the F1 generation were likely the result of the intubation error, and therefore were not considered test article-related. One F1 male in the 1000 mg/kg bw/day group was found dead due to a mechanical injury within the home cage on PND 48; internal findings noted for this male at necropsy included dark red cranial contents around the brain, pituitary gland, and cervical spinal cord. All other F0 and F1 adults survived to the scheduled necropsies

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Test article-related lower mean body weight gains were noted in the 150, 500, and 1000 mg/kg/day group F1 males during PND 21–28, the first week of gavage dosing for these animals (data not presented). Mean body weight gains in these groups were generally similar to the control group throughout the remainder of the study. As a result of the initial lower mean body weights gains, mean body weights for F1 males were between 4.0% and 7.1% lower than the control group throughout the study (Fig. 3; Supplemental Data Table S-1). These differences were not considered adverse because the mean body weights in these groups at termination (PND 161) were only 5.4%–6.1% lower than the control group, demonstrating that the initial effects were ameliorated over of the course of generation. No test-article related effects on mean body weights, body weight gains. food consumption, and food efficienty were noted in the 150, 500, and 1000 mg/kg bw/day group F1 females during the post-weaning period

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The were no test article-related effects on mean food consumption of food efficiency for F1 males at any dosage level.

Food efficiency:

no effects observed

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

no effects observed

Ophthalmological findings:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

A significantly (p < 0.05) higher mean estrous cycle length was noted in the 150 mg/kg/day group compared to the concurrent control group; however, this difference was not considered test article-related because it did not occur in a dose-related manner, and the mean estrous cycle length was within the Charles River Ashland historical control data range. No test article-related effects were noted on mean gestation lengths or the process of parturition at any dosage/exposure level in the F0 and F1 generations, and the numbers of former implantation sites, pups born, and unaccounted-for sites were similar to control group values (Tables 3 and 4).

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

No test-related effects were observed on F0 an F1 male spermatogenesis parameters (motility, progressive motility, testicular and epididymal sperm concentration, sperm production rate, and the percentage of morphologically normal sperm) at any dosage level.

Reproductive performance:

no effects observed

Description (incidence and severity):

No test article-related effects were observed on F0 and F1 male and female mating and fertility, male copulation and female conception indices, estrous cycle lengths or pre-coital intervals

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other:

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Other effects:

no effects observed

Description (incidence and severity):

There were no test related effects noted on F1 and F2 mean number of pups born, liver size on PND 0, percentage of males, or posnatal survival. Mean F1 and F2 male female pups body weight changes in the 150, 500 and 1000 mg/kg bw/day groups were unaffected by test article administration throughout of postnatal period.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Reproductive effects observed:

no

Reference 3

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Source: sponsor; Batch: 1851T1031

- Purity, including information on contaminants, isomers, etc.:
Total Rhamnolipid (acidic form): 88.5 [wt%]
Ashes (>800°C from d.w.): 6.0 [wt%]
Water (Karl Fischer): 1.65 [wt%]

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature, dry
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Stable until December 2020

Species:

rat

Strain:

Crj: CD(SD)

Details on species / strain selection:

The rat is a commonly used rodent species for such studies

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Source: Mature CD rats bred by Charles River Laboratories Germany GmbH
- Females (if applicable) nulliparous and non-pregnant: [yes]
- Age at study initiation: Males and females: 71 days (at first dosing)
- Weight at study initiation: (P) Males: 301.4 g - 406.0 g; Females: 202.7 g - 252.5 g
- Fasting period before study:
- Housing: With exception of the mating period, the male and female animals (F0-Generation) are kept singly in MAKROLON cages (type III plus) with a basal surface of approximately 39 cm x 23 cm and a height of approximately 18 cm
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 3°C (maximum range)
- Humidity (%): 55% ± 10% (maximum range).
- Photoperiod: 12 hrs dark/12 hrs
Granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt/ Arkeburg, Germany) was used as bedding material in the cages. The cages were changed and cleaned once a week. Periodic analysis of the bedding material for contaminants based on EPA/USA is conducted by LUFA-ITL.
Diet
A certified commercial diet (ssniff® R-Z V1324, ssniff Spezialdiäten GmbH, 59494 Soest, Germany served as food. Food residue was removed and weighed.
IN-LIFE DATES: From: 2020-06-24 To: 2020-12-18

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

TEST ANIMALS
- Source: Mature CD rats bred by Charles River Laboratories Germany GmbH
- Females (if applicable) nulliparous and non-pregnant: [yes]
- Age at study initiation: Males and females: 71 days (at first dosing)
- Weight at study initiation: (P) Males: 301.4 g - 406.0 g; Females: 202.7 g - 252.5 g
- Fasting period before study:
- Housing: With exception of the mating period, the male and female animals (F0-Generation) are kept singly in MAKROLON cages (type III plus) with a basal surface of approximately 39 cm x 23 cm and a height of approximately 18 cm
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 3°C (maximum range)
- Humidity (%): 55% ± 10% (maximum range).
- Photoperiod: 12 hrs dark/12 hrs
IN-LIFE DATES: From: 2020-06-24 To: 2020-12-18
PREPARATION OF DOSING SOLUTIONS:
The administration formulations were freshly prepared every day.
The test item was suspended in the vehicle to the appropriate concentrations and administered orally at a constant administration volume of 5 mL/kg b.w. once daily.
The test item formulation was continuously agitated by stirring throughout the entire administration procedure. Foaming was avoided during the whole test item
preparation and administration period. The amount of the test item was adjusted to the animal's current body weight daily.
For the analysis of the test item-vehicle formulations, two aliquots approximately 2 mL were taken at the following times and stored at -20°C ± 10% until analysis.
At start of the treatment period (first dosing day): Analysis of concentration: Immediately after preparation of the test item vehicle formulation as well as after storage at room temperature for 8 and 24 hours (3 samples / dose level group; groups 2 - 4). Number of samples (aliquots): 3 x 3 = 9 (18).
Analysis of homogeneity: At the start of administration, during (middle) administration and before administration to the last animal of the dose level group (3 samples / dose level group; groups 2 - 4). Number of samples (aliquots): 3 x 3 = 9 (18)
Towards the end of the treatment period (when the majority of animals was dosed): Analysis of concentration: During treatment always before administration to the last animal/dose level group (1 sample / dose level group; groups 2 - 4) Number of samples (aliquots): 3 x 1 = 3 (6).
Total number of samples (aliquots): 21 (42)

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle: The test item was suspended in the vehicle to the appropriate concentrations and administered orally at a constant administration volume of 5 mg/kg b.w. once daily.
The test item formulation was continuously agitated by stirring throughout the entire administration procedure. Foaming was avoided during the whole test item
preparation and administration period.
- Amount of vehicle (if gavage): The amount of the test item will be adjusted to the animal's current body weight daily.
- Lot/batch no. (if required): Batch no. 191448151, Braun Melsungen AG; Carl-Braun-Str. 1, 34212 Melsungen, Germany

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: The female was placed with the same male until evidence of mating was observed or 2 weeks had elapsed.
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no

Analytical verification of doses or concentrations:

yes

Remarks:

The results of the HPLC analysis, showed that the actual concentrations of the test item within the formulations ranged from 100.0% to 105.6% of the nominal test item concentrations.

Details on analytical verification of doses or concentrations:

The results were well within the admissible limits of 90% to 110% of the theoretical
concentrations. The results of the HPLC analysis demonstrated that the test item formulations were correctly prepared, homogeneous and stable for 24 hours after storage at room
temperature.

Duration of treatment / exposure:

- 78 test days

Frequency of treatment:

daily

Details on study schedule:

- F1 parental animals not mated until [...] weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were [...] days of age.
- Age at mating of the mated animals in the study: [...] weeks

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

10

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale:
- Rationale for animal assignment (if not random):
- Fasting period before blood sampling for clinical biochemistry:
- Other:

Positive control:

none

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily. In case signs of toxicity occurred the frequency of observations was increased. Each animal was observed before and after dosing at each time of dosing for any signs of behavioural changes, reaction to treatment or illness. Any signs of illness or reaction to treatment were recorded. In addition, the animals were checked regularly throughout the working day from 7:00 a.m. to 3:45 p.m. On Saturdays and Sundays, the animals were checked regularly from 7:00 a.m. to 11:00 a.m. with a final check performed at approximately 3:30 p.m.
- Cage side observations: Behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity were recorded. Cage side observations included skin/fur, eyes, mucous membranes, respiratory and circulatory systems, locomotor activity and behaviour patterns
Further checks were made early in the morning and again in the afternoon of each working day to look for dead or moribund animals. On Saturdays and Sundays, a similar procedure was followed with a final check at approximately 3.30 p.m. If necessary, these provisions allowed to record any premortal symptoms in detail and a post-mortem examination to be carried out during the working period of a day.

BODY WEIGHT: Yes
- Time schedule for examinations: The individual body weights of the animals were recorded on each day of dosing for dose adjustment and at sacrifice (body weight at autopsy).
The report includes weekly values for the male animals (starting on test day 16) and the body weight one day before sacrifice (on test day 48). The days of body weight recording for the female animals were reported during the different study periods as follows:
Pre-mating period: Test days 15, 22, 28
Gestation period: Gestation days 0, 7, 14, 20
Lactation period: Lactation days 1, 4, 8, 13


FOOD CONSUMPTION:
- Relative food consumption (expressed as g/kg b.w./day): Total food given (g) – total food left (g)/ Number of animal days x body weight (g)
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Drinking water consumption was monitored daily by visual appraisal throughout the
study.

The weights of the following organs of all adult male and female animals were determined before fixation. Paired organs were weighed individually and identified as left or right. The weights of the thyroid glands were determined after fixation.

Weighed organs: Epididymis (2), Ovary (2), Testis (2) Uterus including cervix, Thyroid (1) (including parathyroids), As a whole: Prostate, seminal vesicles with coagulating glands

Oestrous cyclicity (parental animals):

During the 14-day pre-exposure period (TD 1 to TD 14), the animals were not yet allocated to the test groups and were kept in the study pool. During these 14 days the estrous cycle of each animal was monitored to select 40 females with regular estrous cycles. Only those animals that exhibit typical 4- to 5-day estrous cycles were considered in the randomisation process for the allocation of the animals to the test groups 1 to 4. This randomisation procedure was based on the body weight of the animals.
For futher information see Attached background information.
Most of the female animals that were used for the main study revealed 2 or 3 typical 4 to 5 day cycles during the 14-day pre-exposure period.
After the allocation of the animals to the treatment groups and the start of administration, the estrous cycle was examined by daily monitoring of vaginal smears throughout the pre-mating and mating periods until one day before a positive mating sign was noted. Finally, a vaginal smear was taken in the morning of the day of scheduled necropsy.

Sperm parameters (parental animals):

Parameters examined in P male parental generations:
Detailed histopathologic examination with special emphasis on the qualitative stages of spermatogenesis and histopathology of interstitial testicular structure was performed on one testis and one epididymis of all males of groups 1 and 4.

Litter observations:

As soon as possible after delivery, each litter was examined to establish the number and sex of pups, stillbirths, live births, runts (pups were considered as runts if their weight was less than 70% of the mean litter weight) and the presence of gross abnormalities.
Abnormal behaviour or changes in the external appearance of the pups noted during
the daily cage side inspections were recorded.
The following examinations/observations were done for the offspring:

Counting, sexing and weighing: Live pups were counted, sexed and weighed on post-natal days (PND) 1, 4 and 13.

Ano-genital distance: On PND 4 before litter adjustment the ano-genital distance (AGD) of all pups was determined using a scale. The AGD was normalised to the cube root of body weight.

Litter adjustment: After counting on PND 4 (lactation day 4), the litters were adjusted to 10 pups per litter (5 pups/sex/per litter) by eliminating (culling) surplus pups using a randomization scheme generated by Provantis®.
Selective elimination of pups, e.g. based upon body weight was not appropriate.
Whenever the number of male or female pups prevents having 5 pups/sex/per litter, partial adjustment (for example, 6 male and 4 female pups) was acceptable.

Blood sampling for thyroid hormone (T4) determination

On PND 4 and on PND 13 blood samples for determination of T4 hormone levels were taken from 2 surplus pups per litter, if possible from one male and one female pup. On PND 4 the culled surplus pups were used for blood collection. On PND 13 the same pups that were used for T4 analysis were also used for the preservation of the thyroids.

Counting of male nipples/areolae
Nipples/areolae were counted in all male pups on PND 12 or PND 13.

Postmortem examinations (parental animals):

On the day of necropsy, vaginal smears were obtained and examined to determine
the stage of estrous cycle and allow correlation with the histopathology of the
female reproductive organs.
The adult animals were euthanized by carbon dioxide (CO2) inhalation, exsanguinated by cutting the aorta abdominalis, weighed, dissected and inspected macroscopically (gross necropsy)

At the following times the blood samples were collected fot T4 determination

Adult males: On test day 49
Dams: On lactation days 14 to 16

Dissection of adult animals
At the time of sacrifice or premature death during the study, the adult animals were examined macroscopically for any abnormalities or pathological changes. Special attention was paid to the organs of the reproductive system.
During necropsy the number of implantation sites was recorded in the female animals.

Organ preservation: The following organ(s) or parts thereof of all adult male and female animals were fixed as follows:
Fixative:
a) modified Davidson's solution -> Epididymis (2); Testis (2)
b) Fixative: 7% neutral buffered formalin-> Gross lesions observed; Thyroid (including parathyroids); Ovary and oviduct (2); Uterus (including cervix); Prostate Seminal vesicles with coagulating glands

Histopathological examination was performed on the following organs of all adult
male and female animals of the control and high dose group (groups 1 and 4): Epididymis, Ovary, Testis.

Detailed histopathologic examination with special emphasis on the qualitative stages of spermatogenesis and histopathology of interstitial testicular structure was performed on one testis and one epididymis of all males of groups 1 and 4.
The other preserved organs will be examined when necessary. In case of changes noted in the high dose level group, examinations will be extended to the animals of other dose level groups when changes are seen in the highest dose group.

Postmortem examinations (offspring):

At the following times the blood samples were collected fot T4 determination:
Pups: On lactation days 13 to 16

Examination of the pups: Dead pups and pups terminally sacrificed were carefully examined externally for
gross abnormalities. The external reproductive genitals were examined for signs of
altered development.
At day 13 post-partum, the thyroid of 1 male and 1 female pup from each litter (preferably from those animals used for T4 ELISA) was preserved in 7% formalin.
Thyroid preparation and blood withdrawal for T4 analyses were performed for
1 male pup and 1 female pup from each litter. These pups were always sacrificed on lactation day 13.

Statistics:

The statistical evaluation of the parametrical values was done by Provantis® Integrated preclinical software, Version 10.2.1, Instem LSS Ltd. using the following settings:
Homogeneity of variances and normality of distribution were tested using the BARTLETT’s and SHAPIRO-WILK’s test. In case of heterogeneity and/or nonnormality of distribution, stepwise transformation of the values into logarithmic or rank values was performed prior to ANOVA. If the ANOVA yielded a significant
effect (p ≤ 0.05), intergroup comparisons with the control group were made by the
DUNNETT’s test (p ≤ 0.01 and p ≤ 0.05).
For the statistical evaluation of the histopathological data FISHER's exact test was
used.
The mean values and standard deviations were calculated to the highest possible degree of accuracy and then rounded to the reported number of decimal places.
Hence, deviations to the last decimal place of up to ±1 may occur caused by rounding.

Reproductive indices:

The reproductive parameters and reproductive indices listed below were determined to evaluate the reproductive performance:

Reproductive parameters: stages of the estrous cycle; pre-coital time; gestation length calculated from day 0 of pregnancy

Implantation sites: number per dam; absolute number per group; mean per group.

Reproductive indices:
- Female Fertility Index [%]: (Number of pregnant rats / Number of rats paired with a male) x 100
The female fertility index reflects the total number of dams that had achieved pregnancy, including dams which delivered at term, aborted or had fully resorbed litters.

Gestation Index [%]: (Number of dams with live pups / Number of pregnant rats) x 100

Offspring viability indices:

For each litter and group the following indices were determined:
Birth Index [%] = [(Total number of pups born (alive + dead)) / Number of implantation sites] x 100

Live Birth Index [%]= ((Number of pups alive on day 0/1 of lactation / Total number of pups (alive + dead)) x 100

Post-implantation loss[%]#1 = (Implantations - number of pups born alive) / Implantation sites x 100

#1: Twins (shared placentae) are considered as additional implantation sites in the calculation of the birth index and the post-implantation loss, to avoid a birth index above 100% or a negative post-implantation loss.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Males
No changes in behaviour, the external appearance or the appearance of the faeces were noted for the male animals of the control group.
At the low dose level (100 mg /kg b.w./day) salivation and/or areduced motility were noted for several animals. One male (no. 27) animal showed breathing sounds on 1 test day.
At the intermediate dose level (300 mg/kg b.w./day) salivation was noted for all animals (except for animal no. 44 which died prematurely) and a reduced motility for several animals. In addition, one male animal (no. 46) showed breathing sounds on 4 consecutive test days.
At the high dose level (1000 mg/kg b.w./day) salivation was noted for all animals and a reduced motility or breathing sounds for several animals.
Piloerection, laboured breathing, a thickened abdomen or a reduced body tension were additionally noted for male animal nos. 61 and / or 67 (see attached back ground information.

Females
No changes in behaviour, the external appearance or the appearance of the faeces were noted for the male animals of the control group.
At the low dose level (100 mg/kg b.w./day) salivation was noted for 5 of 10 animals on one test day each during the pre-mating and the mating period.
During the gestation period salivation was only noted for 1 of 9 pregnant animals on one test day.
Furthermore, a reduced motility was noted for 1 of 10 females during the pre-mating
and the mating period.
No observations were made during the lactation period for the females with litter.
At the intermediate dose level (300 mg/kg b.w./day) salivation was noted for several animals during the premating / mating period, the gestation period and the lactation period.
Breathing sounds were noted for a few animals during the pre-mating / mating and the gestation period and a reduced motility was noted for 2 females during the premating / mating period.
At the high dose level (1000 mg/kg b.w./day) salivation was noted for all animals during the pre-mating / mating period (10 of 10), the gestation period (8 of 8 pregnant animals) and the lactation period (7 of 7 animals with litter).
Breathing sounds were noted for several animals during the premating / mating period, the gestation period and the lactation period and a reduced motility for 2 females during the pre-mating / mating period and for one animal during the gestation period.
Further observations (discharge from vagina, piloerection, a decreased respiratory rate, laboured breathing) were noted during the pre-mating period for animal nos. 74 and / or 77, which were prematurely sacrificed during the gestation period due to poor health. During the lactation period piloerection, laboured breathing, a haemorrhagic canthus and / or a reduced body tension were noted for animal nos. 73 and / or 76 (see attached background information).

Start and duration of observations

Males and females

The symptoms started immediately after dosing. Observations of salivation and a reduced motility were short lasting post-dosing symptoms in nearly all cases. In contrast, breathing sounds were in nearly all cases long lasting observations which lasted for several hours or until the next dosing.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No systemic test item-related deaths were noted for the male and female animals treated orally with 100, 300 or 1000 mg/kg b.w./day of the test item.
The following not test item-related deaths occurred: One male animal (no. 44) of the intermediate dose level (300 mg/kg b.w./day) was found dead on test day 38. At the high dose level (1000 mg /kg b.w./day) three female animals (nos. 74, 77, 78) were prematurely sacrificed due to poor health (see attached background information). The premature death of male no. 44 and the poor health of female nos. 74, 77 and 78 were considered to be due to a reflux of the surface active test item into the lungs by regurgitation. Therefore, the deaths of these four animals are considered
to be test item-related and caused by the physical properties of the test item and not due to systemic toxicity.
Male animal no. 44 was found dead during test day 38. The external examination revealed haemorrhagic canthi and a haemorrhagic nose/snout. Necropsy revealed oedematous lungs. The left side of the lung was partly indurated and dark-red discoloured. This was most probably due to the regurgitation and subsequentinspiration of blood into the (naso-)pharynx and lungs following oral administration of the test item.
Female animal nos. 74 and 77 displayed slight salivation and breathing sounds on the days before premature sacrifice. Animal no. 74 additionally showed a body weight loss during the premating period of 25.2%. Due to the poor health, it was decided to prematurely sacrifice animal nos. 74 and 77. Inflated intestines with only little content were noted for animal no. 74 during necropsy and animal no. 77 revealed multiple brown foci in its lungs. little content were noted for animal no. 74 during necropsy and animal no. 77 revealed multiple brown foci in its lungs.
Female animal no. 78 was noted with severe discharge of red saliva before delivery
and with piloerection, a thickened abdomen and gasping on lactation day 1. Due to
the poor health and because it was doubted, animal no. 78 was able to take care
for its litter, animal no. 78 and also its litter were prematurely sacrificed. External examination revealed a markedly increased abdomen. During necropsy, inflated intestines were noted with only little content.
A microscopic examination was not performed for the affected organs of the prematurely deceased animals, as the microscopic examination was restricted to the reproductive organs of the control animals and the high dosed animals (see attached background information).

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No test item-related differences between the control group and the treatment groups
(100, 300 or 1000 mg HH-2016-680/kg b.w./day) were noted during the lactation period.
Slightly reduced pup body weights (statistically not significant) were noted at the intermediate and the high dose level on lactation days 1, 4 and 13 (between 4.9% and 7.9% below the control value, see Text table 7-26). However, these slight differences were still in the range of test laboratory background data and are considered to be spontaneous (see attached background information for a comparison with the background data on lactation day 13).
One runt (no. 54-05) was noted in the intermediate dose group.

Litter weight

No test item-related differences were noted for the litter weights between the control group and the low dose group (100 mg/kg b.w./day).
Reduced litter weights were noted at the intermediate and the high dose level on lactation days 1, 4 and 13, more pronounced at the high dose level than at the intermediate dose level as listed below. This was due to slightly reduced pup body weights at the intermediate and the high dose level (considered to be spontaneous; see attached background informtion 'Body weight of pups') and a reduced number of live pups at the high dose level on lactation day 1.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Males: Pre-mating period
No test item-related difference in food consumption was noted between the control group and the treatment groups (100, 300 or 1000 mg/kg b.w./day of the test item) during the pre-mating period after the start of treatment on test day 16 until mating on test day 30.

Females: Pre-mating, gestation and lactation period
No test item-related differences in food consumption were noted between the control group and the treatment groups (100, 300 or 1000 mg /kg b.w./day of the test item) during the pre-mating, the gestation and the lactation period.

No food intake was recorded during pairing when female animals were housed with males.

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

no effects observed

Description (incidence and severity):

No test item-related changes in drinking water consumption was noted for the male and female rats treated with 100, 300 or 1000 mg/kg b.w./day by visual assessment.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Males and females

Histological evaluation was performed on testes, epididymides and ovaries from all animals of the control group and the high dose group (1000 mg/kg b.w./day).
Ovaries of group 4 females did not show any effects related to the test item at microscopy. In addition, no test item-related microscopic changes were observed in the male reproductive organs (testes, epididymides) of group 4 animals.
Additionally, detailed histopathological examination of one testicle and one epididymis with special emphasis on the qualitative stages of spermatogenesis (proliferative, meiotic and spermiogenic phases) and histopathology of the interstitial testicular structure did not reveal any test item-related effects.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Monitoring of estrous cycle from start of dosing until mating

After allocation of the animals to the test groups and the start of treatment on test day 16, the estrous cycles werre furthre monitored during pre-mating and mating period until one day beofre mating sign was noted .

No test item-related differences were noted for the mean length and the mean number of estrous cycles per dam during the pre-mating period between the female animals of the control group and the female animals of the treatment groups (100, 300 or 1000 mg/kg b.w./day) (See Attached background information).

No complete estrous cycle were noted for famale no. 38 of the low dose group and no. 74, 80 of the high dose group. However, only no.74 did not became pregnat whereas nos 38 and 80 both littered live pups. No complete estrous was noted during the mating period.

Reproductive performance:

no effects observed

Description (incidence and severity):

Fertility index:

No test-item related influence on the fertility index of the female rats was noted for any treatment groups.

Two non-pregnant animals (nos. 74, 77) were noted at the high dose level.
However, in contrast to the non-pregnant animals of the control group and the low dose group, the 2 non-pregnant animals of the high dose group showed no evidence copulation during their mating period of 14 days. The reason may be due to the poor health of female nos. 74, 77, which were both prematurely sacrificed on day 7 of their pseudo-gestation period (for details see Attached background information). Hence, the reduced fertility index at the high dose level (80% in
comparison to 90% in the control group) could be considered as a secondary effect caused by the poor general condition of the animals.

Gestation index
No test item-related influence on the gestation index was noted for the female rats of the treatment groups (100, 300 or 1000 mg HH-2016-680/kg b.w./day).
All pregnant females of the control group, the intermediate dose group and the high dose group delivered live pups. Only one pregnant female of the low dose group (no. 34) delivered no live pups due to resorption of all implants. One female with resorption of all implants is in the range of normal variability (see Attached back ground information).

Gestation index: No test item-related influence on the gestation index was noted for the female rats of the treatment groups (100, 300 or 1000 mg/kg b.w./day).
All pregnant females of the control group, the intermediate dose group and the high dose group delivered live pups. Only one pregnant female of the low dose group (no. 34) delivered no live pups due to resorption of all implants. One female with resorption of all implants is in the range of normal variability.

Pre-coital time: No test item-related differences were noted in the length of the pre-coital time between the control group and the low and the intermediate dose group (100 or 300 mg /kg b.w./day).
A prolonged pre-coital time was noted at the high dose level of 1000 mg/kg b.w./day (mean duration of pre-coital time at the high dose level was 4.9 test days in comparison to 1.6 test days in the control group; p ≤ 0.5). This was due to animal nos. 74 and 77 with no positive mating sign during the whole mating period, resulting in a pre-coital time for both animals of 14 test days. As this could be related to the poor general health of both animals, the resulting prolongation of the mean pre-coital time at the high dose level could be considered as a secondary effect.

Gestation index: No test item-related differences were noted for the length of the gestation period between the rats of the control group and the treatment groups (100, 300 or 1000 mg/kg b.w./day).

Pups - Pre- and postnatal development - Birth indices and post-implantation loss: No test item-related differences were noted for the mean number of implantation sites per dam, the mean number of pups born (alive and dead) per dam and the mean number of live born pups per dam between the control group and the treatment groups (100, 300 or 1000 mg/kg b.w./day).
Also the reproductive indices as the birth index, the live birth index and the percentage of post implantation loss revealed no test item-related differences between the control group and the treatment groups (100, 300 or 1000 mg/kg b.w./day).
Increased numbers of resorptions and stillbirths were noted at the low dose level. In detail, 29 resorptions and 2 stillborns (sum = 31) were noted at the low dose level in comparison to 15 resorptions in the control group, 19 resorptions in the intermediate dose group and 16 resorptions in the high dose group. The mean values per dam were 1.7 resorptions/stillborns per dam in the control group, 3.4 in the low dose group, 1.9 in the intermediate dose group and 2.3 in the high dose group. As no dose response-relationship was noted, the increased number of resorptions that was noted at the low dose level is considered to be spontaneous

Males

No test item-related changes were noted between the control group and the low and the intermediate dose group (100 or 300 mg/kg b.w./day) for the T4 levels of the parental males.
A decreased T4 serum level was noted for the male animals of the high dose group (1000 mg/kg b.w./day) (29.3% below the control value; p ≤ 0.05).
The mean T4 serum concentration at the high dose level (36.6 ± 16.3 nmol/L) was below the background range (see Attached background information). This was mostly due to a markedly reduced T4 level that was noted for the male animal nos. 61 and 67 of the high dose group (12.1 nmol/L or 10.3 nmol(L). The reduced T4 level in the high dose group is considered to be test item-related.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

Key result

Critical effects observed:

no

Mortality / viability:

no mortality observed

Description (incidence and severity):

Viability index: Pre-cull period: No test item-related differences between the control group and the treatment groups (100, 300 or 1000 mg/kg b.w./day) were noted for the viability index of the pre-cull period.

Post-cull period: A reduced viability index was noted at the high dose level (1000 mg/kg b.w./day).
The reduced viability index at the high dose level was due to dam no. 73 (see Text table 7 24). All remaining 10 pups of dam no. 73 were cannibalized or found dead (without milk in their stomach) on lactation day 5. One day before, on lactation day 4, all 13 pups (before culling) of dam no. 73 were noted as cold to touch and without milk in their stomach, indicating an insufficient maternal care as the reason of death for the pups (see Table 19-3 in Section 9 'Pups - Unscheduled Deaths and Pup Observations'). The insufficient maternal care could be considered as a result of the poor general health of female no. 73 on the respective days. Gasping, piloerection, a haemorrhagic canthus, breathing sounds and a reduced body tension were noted for female no. 73 on lactation day 4. Hence, the reduced post-cull viability index is considered to be a secondary effect caused by poor general health.

Number of live pups: No test item-related differences were noted for the mean number of live pups between the control group and the treatment groups (100, 300 or 1000 mg/kg b.w./day) on lactation days 1 and 4.
A slightly reduced mean number of live pups was noted at the high dose level on lactation days 1 and 4. However, the mean number of live pups was still in the range of the test labor background and is considered to be spontaneous (see Table 7.29 for the mean number of live pups on lactation days 1, 4 and 13 and Table 7.30 for a comparison with the LPTtest labor background data). A reduced number of live pups was noted at the high dose level (1000 mg/kg b.w./day) on lactation day 13 (7.7 pups per dam in comparison to 10.0 pups per dam in the control group; see Table 7.29 below). This was mostly due to dam no. 73. All remaining pups of dam no. 73 (10 pups) died on lactation day 5, leading to zero pups for dam no. 73 on lactation day 13. Hence, the reduced number of live pups on lactation day 13 could be considered as a secondary effect to maternal toxicity (poor health of dam no. 73 that led to the death of its pups). 5 dams of the remaining 6 high dosed dams with live pups still had the maximum number of 10 live pups on lactation day 13 (dam no. 72 delivered only 4 live pups and hence, only 4 live pups were available on lactation day 13).

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No test item-related differences between the control group and the treatment groups (100, 300 or 1000 mg/day) were noted during the lactation period.
Slightly reduced pup body weights (statistically not significant) were noted at the intermediate and the high dose level on lactation days 1, 4 and 13 (between 4.9% and 7.9% below the control value, see Text table 7 26). However, these slight differences were still in the range of test laboratory background data and are considered to be spontaneous (see Table 7 27 for a comparison with the background data on lactation day 13).
One runt (no. 54-05) was noted in the intermediate dose group.

Litter weight: No test item-related differences were noted for the litter weights between the control group and the low dose group (100 mg/kg b.w./day).
Reduced litter weights were noted at the intermediate and the high dose level on lactation days 1, 4 and 13, more pronounced at the high dose level than at the intermediate dose level as listed below. This was due to slightly reduced pup body weights at the intermediate and the high dose level (considered to be spontaneous) and a reduced number of live pups at the high dose level on lactation day 13.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

No test item-related difference in the absolute and the relative ano-genital distance (value normalized to cube root of pup body weight) of the male and the female pups was noted between the control group and the treatment groups (100, 300 or 1000 mg/kg b.w./day of the test item).

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

No test item-related difference in the number of nipples was noted between the male pups of the control group and in the male pups of the treatment groups (100, 300 or 1000 mg/kg b.w./day).

Gross pathological findings:

no effects observed

Description (incidence and severity):

No gross abnormalities (e.g. malformations or variations) were noted during the external and internal macroscopic examination of the control pups and the pups from the dams treated with 100, 300 or 1000 mgkg b.w./day at necropsy.

Male to female ratio of the pups: No test item-related influence on the male to female ratio was noted for all treatment groups (100, 300 or 1000 mg /kg b.w./day).

Pups - T4 serum level: No test item-related differences between the control group and the treatment groups (100, 300 or 1000 mg/kg b.w./day) were noted for the T4 serum levels of the male and female pups on lactation day 13 (for futher details in attache background information).

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 1 000 mg/kg bw/day

Based on:

act. ingr.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Lowest effective dose / conc.:

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

Treatment related:

no

Table 1.1 Clinical Signs _Parental animals (males)

Observations noted for the male animals
Symptom		Group
		2	3^#	4
Salivation		7/10	9/9	10/10
Motility reduced		4/10	4/9	6/10
Breathing sounds		1/10	1/9	3/10
Piloerection		-	-	1/10
Laboured breathing		-	-	1/10
Thickened abdomen		-	-	2/10
Reduced body tension			-	1/10
#	prematurely deceased no. 44 excluded
…/…	no. of affected / no. of examined animals
-	not present

Table 1‑2:  Clinical signs - females -pre-mating / mating period

Observations noted for the female animals during the pre-mating / mating period
Symptom		Group
		2	3	4
Salivation		5/10	7/10	10/10
Motility reduced		1/10	2/10	2/10
Breathing sounds		-	3/10	5/10
Discharge from vagina		-	-	2/10^#
Piloerection		-	-	1/10^#
Respiratory rate decreased		-	-	1/10^#
Laboured breathing		-	-	1/10^#
#	nos. 74 and 77 (non-pregnant, prematurely sacrificed during pseudo-gestation period)
…/…	no. of affected / no. of examined animals
-	not present

Table 1‑3:   Clinical signs - females - gestation period

Observations noted for the female animals during the gestation period
Symptom		Group
		2^#	3	4^#
Salivation		1/9	7/10	8/8
Motility reduced		-	-	1/8
Breathing sounds		-	5/10	5/8
#	non-pregnant and/or prematurely deceased animals excluded
…/…	no. of affected / no. of examined animals
-	not present

Table 1‑4:     Clinical signs - females - lactation period

Observations noted for the female animals during the lactation period
Symptom		Group
		2	3	4
Salivation		-	6/10	7/7^#
Breathing sounds		-	-	3/7
Piloerection		-	-	1/7
Laboured breathing		-	-	1/7
Haemorhagic cantus		-	-	1/7
Reduced body tension		-	-	1/7
Gasping		-	-	1/7
#	Only females with live litters were considered.
…/…	no. of affected / no. of examined animals

Table 1‑5:  Body weight gain - males

Body weight gain of the male animals [%]
Test days 16 to 48	Group
	1	2	3	4
	+26.2	+22.3	+20.5	+13.5

Table 7‑1:     Reproductive Outcome of the female animals

HH-2016-680		Group 1 Control	Group 2 100 mg/kg	Group 3 300 mg/kg	Group 4 1000 mg/kg
Females started dosing		10	10	10	10
Females used for mating at the start of the mating period		10	10	10	10
Females without a positive mating sign (i.e. sperm detection) after a mating period of 14 days:		0	1 (34)^#1	0	2 (74, 77)
	- thereof pregnant   (mating sign was overlooked)	0	1 (34)^#1	0	0
	- thereof not pregnant	0	0	0	2 (74, 77)
Females pregnant		9	9	10	8
Females not pregnant		1 (14)	1 (33)	0	2 (74, 77)
Females died during the gestation or pseudo-gestation period		0	0	0	2 (74, 77)^#2
Females with total resorption of all implants		0	1 (34)^#1	0	0
Females with live born pups		9	8	10	8
Females died during lactation period		0	0	0	1 (78)^#3
Females with surviving pups until lactation day 13		9	8	10	7

(…)	No. of female animal(s)
#1	No. 34: No positive mating sign was noted during the mating period. After 14 days of mating a pseudo-gestation period was started for no. 34, which lasted until laparotomy on gestation day 23. Laparotomy revealed pregnancy of no. 34 but with resorption of all implants.
#2	Nos. 74 and 77 were prematurely sacrificed on gestation day 8 of their pseudo-gestation period. Their non-pregnancy status was confirmed by Salewski Staining.
#3	Animal no. 78 was prematurely sacrificed on lactation day 1 together with its litter.

Table 7‑2:     Table of prematurely deceased animals

Prematurely deceased animals
Group		No.	Sex	Test day	Reason for death
3		44	M	38	Found dead during the working day
4		74	F	52 (Pseudo-gestation day 8^#)	Prematurely sacrificed due to poor health
		77		52 (Pseudo-gestation day 8^#)
		78		56 (Lactation day 1)
	M; F: Male; Female
#	No positive mating sign was noted for nos. 74 and 77 after 14 days of mating. Their non-pregnancy status was confirmed by Salewski Staining at laparotomy.

Table 7‑3:     Macroscopic findings noted for the prematurely deceased animals at necropsy

Prematurely deceased animals
Group	No.	Sex	Macroscopic findings at necropsy
			Affected Organ	Finding
3	44	M	Nose/snout	haemorrhagic
			Canthus (both)	haemorrhagic
			Lungs	oedematous, left side partly indurated and dark-red discoloured
4	74^#	F	Intestines	Inflated, less content
	77^#	F	Lungs	multiple brown foci
	78	F	Abdomen	markedly increased, fluctuating, painful
			Intestines	Inflated, less content

M; F: Male; Female

No positive mating sign was noted for nos. 74 and 77 after 14 days of mating. Their non-pregnancy status was confirmed by Salewski Staining at laparotomy.

Table 7‑4:     Observations noted for the prematurely deceased animals during the daily cage side inspection on the days before death (common observations as salivation are not considered).

Prematurely deceased animals
Group		No.	Sex	Observations during the daily cage side inspections
				Symptom	Observed on test day (day of death)
3		44	M	Breathing sounds	35 (38)
4		74^#	F	Breathing sounds	GD 0 to 8 (8)
		77^#	F	Breathing sounds	GD 0 to 8 (8)
				Increased respiratory rate	GD 7 (8)
				Thickened Abdomen	GD 0 (8)
		78	F	Red salivation (extreme)	GD 21 (LD1)
				Gasping	LD 1 (LD1)
				Piloerection	LD 1 (LD1)
				Thickened abdomen	LD 1 (LD1)
	M; F: Male; Female
#	No positive mating sign was noted for nos. 74 and 77 after 14 days of mating. Their non-pregnancy status was confirmed by Salewski Staining at laparotomy.

Table 7‑5:   Observations noted during the daily cage side observations for the males

Observation (males)		Affected animals	First to last seen (test days)	Range of days observed^#1
Observations in group 2 (100 mg/kg b.w./day)
Salivation (slight to extreme)		7 of 10	16 - 34	1 - 2
Motility reduced (slight)		4 of 10	16 - 19	1 - 4
Breathing sounds		1 of 10	35	1 (27)
Observations in group 3 (300 mg/kg b.w./day)
Salivation (slight to moderate)		9 of 9^#2	22 - 48	1 - 5
Motility reduced (slight to moderate)		4 of 9	16 - 23	1 - 5
Breathing sounds		1 of 9	20 - 23	4 (46)
Observations in group 4 (1000 mg/kg b.w./day)
Salivation (slight to extreme)		10 of 10	19 - 48	3 - 15
Motility reduced (slight to moderate)		6 of 10	16 - 43	1 - 4
Breathing sounds		3 of 10	23 - 49	1 - 24
Piloerection		1 of 10	37	1 (61)
Laboured breathing		1 of 10	43 - 44	2 (61)
Thickened abdomen		2 of 10	43 - 49	1 - 4 (61, 67)
Body tension reduced		1 of 10	20	1 (67)
#1	If only 1 or 2 animals were affected, the animal numbers are listed in brackets.
#2	The prematurely deceased male no. 44 was excluded from this summary.

Table 7‑6:   Observations noted during the daily cage side observations for the females

Observation (females)		Affected females per period			Range of days observed per period^#1
		Pre-mating/ Mating	Gestation	Lactation	Pre-mating/ Mating	Gestation	Lactation
Observations in group 2 (100 mg/kg b.w./day)
Salivation (slight to moderate)		5 of 10	1 of 9^#2	n.d.	1	1 (39)	-
Motility reduced (slight)		1 of 10	n.d.	n.d.	1 (36)	-	-
Observations in group 3 (300 mg/kg b.w./day)
Salivation (slight to extreme)		7 of 10	7 of 10	6 of 10	1 - 7	1 - 5	1 - 2
Motility reduced (slight)		2 of 10	n.d.	n.d.	2 - 5 (51, 52)	-	-
Breathing sounds		3 of 10	5 of 10	n.d.	1 - 5	2 - 5	-
Observations in group 4 (1000 mg/kg b.w./day)
Salivation (slight to extreme)		10 of 10	8 of 8^#3	7 of 7^#4	1 - 15	3 - 13	6 - 12
Motility reduced (slight)		2 of 10	1 of 8	n.d.	1 - 2 (74, 77)	1 (72)	-
Breathing sounds		5 of 10	5 of 8	3 of 7	1 - 16	2 - 3	1 - 7
Discharge from vagina		2 of 10	n.d.	n.d.	2 (74, 77)	-	-
Piloerection		1 of 10	n.d.	2 of 7	8 (74)	-	3 - 7 (73, 76)
Respiratory rate decreased		1 of 10	n.d.	n.d.	8 (74)	-	-
Laboured breathing		1 of 10	n.d.	1 of 7	1 (77)	-	2 (76)
Haemorrhagic canthus		n.d.	n.d.	1 of 7	-	-	1 (73)
Body tension reduced		n.d.	n.d.	1 of 7	-	-	2 (73)
Gasping		n.d.	n.d.	1 of 7	-	-	1 (73)
n.d.	not detected
-	not present
#1	If only 1 or 2 animals were affected, the animal numbers are listed in brackets.
#2	The non-pregnant animal no. 33 was excluded from the gestation period of this summary.
#3	The non-pregnant and prematurely deceased (gestation day 8) animal nos. 74, 77) were excluded from the gestation period of this summary.
#4	The prematurely deceased animal no. 78 was excluded from the lactation period of this summary.

Table 7‑7:     Start and duration of observations noted for the male animals

Start and duration of symptoms - Males
Symptom	Time frames in relation to application		Frequency observed (in all groups)
	Appearing of the symptom	Disappearing of the symptom
Salivation	0 min to 5 min	5 min to 20 min	59
	0 min to 5 min	20 min to 60 min	50
	0 min to 5 min	1 h to 2 h	3
Reduced motility	0 min to 5 min	5 min to 20 min	1
	0 min to 5 min	20 min to 60 min	16
	5 min to 20 min	20 min to 60 min	14
	consistently during the day		1
Breathing sounds	0 min to 5 min	20 min to 60 min	1
	0 min to 5 min	2 h to 6 h	1
	0 min to 5 min	6 h to 24 h	16
	consistently during the day		26
Laboured breathing	consistently during the day		2

Table 7‑8:     Start and duration of observations noted for the female animals

Start and duration of symptoms - Females
Symptom	Time frames in relation to application		Frequency observed (in all groups and periods)
	Appearing of the symptom	Disappearing of the symptom
Salivation	0 min to 5 min	5 min to 20 min	121
	0 min to 5 min	20 min to 60 min	134
Reduced motility	0 min to 5 min	20 min to 60 min	6
	5 min to 20 min	20 min to 60 min	5
	consistently during the day		1
Breathing sounds	0 min to 5 min	2 h to 6 h	1
	0 min to 5 min	6 h to 24 h	32
	consistently during the day		32
Laboured breathing	consistently during the day		3
Decreased respiratory rate	consistently during the day		8

Table 7‑9:     Body weight gain of the male animals during the treatment period from test days 16 to 48

Males	Group 1 Control	Group 2 100 mg/kg	Group 3 300 mg/kg	Group 4 1000 mg/kg
Body weight gain (test day 16 to test day 48)	+26.2%	+22.3%	+20.5%	+13.5%

Table 7‑10:   Body weight gain of the female animals during the pre-mating period, the gestation period and the lactation period.

Body weight gain [%] (pre-mating period) (test days 16 to 30)		+5.7	+4.3	+5.7	+4.0
Body weight gain [%] (gestation period)		+63.5	+58.8	+57.6	+55.3
Body weight gain [%] (lactation period)		+12.8	+16.6	+14.8	+16.1

Table 7‑11:   Statistically significant differences in food consumption which are considered to be not of toxicological relevance but spontaneous

Parameter	Ref. table no.	Difference to control [%]	Group / Sex	Test days	Statistical significance	Reason
Food consumption (g/kg b.w./day)	6-1	+6.2	4 m	23 - 30	p £ 0.05	A

Table 7‑12:   Macroscopic findings noted for the male animals

Macroscopic findings - Terminal sacrificed animals - Males
Group		No.	Sex	Affected organ	Finding
1		4	M	Testis (left + right)	small
				Epididymis (left + right)	small
2		22	M	Testis (right)	small
				Epididymis (right)	small
4		61	M	Prostate	small
				Seminal vesicle	small
				Intestines	dilated and inflated
		67	M	Cecum	dilated and inflated
				Thymus	small
	M: Male

Table 7‑13:   Macroscopic findings noted for the female animals

Stage of estrous cycle at necropsy^#		Group 1 Control	Group 2 100 mg/kg	Group 3 300 mg/kg	Group 4 1000 mg/kg
Proestrus		-	-	-	-
Estrus		-	-	-	1 of 7
Metestrus		-	-	-	-
Diestrus		9 of 9	8 of 8	10 of 10	6 of 7
-	not detected
#	Non pregnant animals, animals with total resorption of all implants and prematurely deceased animals are not considered.

table 7‑14:   Stage of the estrous cycle at necropsy. The stage of the estrous cycle was evaluated from vaginal lavages that were taken at necropsy.

Stage of estrous cycle at necropsy^#		Group 1 Control	Group 2 100 mg/kg	Group 3 300 mg/kg	Group 4 1000 mg/kg
Proestrus		-	-	-	-
Estrus		-	-	-	1 of 7
Metestrus		-	-	-	-
Diestrus		9 of 9	8 of 8	10 of 10	6 of 7
-	not detected
#	Non pregnant animals, animals with total resorption of all implants and prematurely deceased animals are not considered.

Table 7‑15:   Changes of the thyroid weights in comparison to the control group

Organ		Sex	Changes in comparison to control [%]			Assess- ment
			Group 2	Group 3	Group 4
Thyroid with parathyroid (left, relative)		Male	- 8.3	- 2.3	- 8.1	A
Thyroid with parathyroid (left, absolute)		Male	- 10.0	- 6.9	- 18.9	A
Thyroid with parathyroid (left, relative)		Female	- 4.7	- 4.5	- 26.8*	B
Thyroid with parathyroid (left, absolute)		Female	- 9.7	- 8.3	- 26.4	B
*	Statistically significant from control at p ≤ 0.05 (DUNNETT's test)
#	Values taken from Table 11-1 'Absolute Organ Weights - Summary - Males' in Section 9
A	The differences that were noted for the male animals are most probably not considered to be test item-related (not statistically significant, no dose-response relationship). However, it cannot be completely ruled out.
B	The differences that were noted for the female animals are considered to be test item-related.

Table 7‑16:   Comparison of the T4 levels of the male animals with the LPT background data.

Parameter		Values from this study - males Group mean values ± SD [nmol/L]		LPT Background Data obtained from the control groups of 16 OECD 422 / 421 studies performed at LPT from 2016 - 2020
T4 serum level		Group 1	51.7 ± 10.0	Mean value from 160 control animals from 16 studies 63.6 ± 11.6 Range of the mean values from the control groups from 16 studies: 42.3 - 91.2
		Group 2	50.7 ± 6.8
		Group 3	52.0 ± 7.9
		Group 4	36.6 ± 16.3*
*	Statistically significant from control at p ≤ 0.05 (DUNNETT's test)

Table 7‑17:   Mean length and mean number of estrous cycles

Parameter		Group 1 Control	Group 2 100 mg/kg	Group 3 300 mg/kg	Group 4 1000 mg/kg
Pre-mating: Test day 16 (start of treatment) until pairing (test day 30)
Mean cycle length (days)		4.18	4.51	4.27	4.67
Number of cycles		2.5	2.3	2.2	2.2
Mating period
Mean cycle length (days)		-	-	-	-
Number of cycles		0.0	0.0	0.0	0.0
#2	During the mating period a complete estrous cycle was only noted for female no. 33.
-	not present

Table 7‑18:   Fertility indices per group

Group / Dose level		Fertility index%	Pregnant females / Females paired with a male partner^#1
Group 1 (Control)		90	9 / 10
Group 2 (100 mg/kg)		90	9^#2 / 10
Group 3 (300 mg/kg)		100	10 / 10
Group 4 (1000 mg/kg)		80	8^#3 / 10
#2	No. 34 with no live pups due to resorption of all implants is considered as pregnant.
#3	The 2 non-pregnant animals of group 4 (nos. 74, 77) showed no evidence of copulation (no positive mating sign in the form sperm detection) during their mating period of 14 days. Their non-pregnancy status was confirmed by laparotomy after they had been prematurely sacrificed on day 7 of their pseudo-gestation period.

Table 7‑19:   Gestation indices per group

Group / Dose level		Gestation index %	Dams with live pups / pregnant rats^#1
Group 1 (Control)		100	9 / 9
Group 2 (100 mg/kg)		89	8^#2 / 9
Group 3 (300 mg/kg)		100	10 / 10
Group 4 (1000 mg/kg)		100	8 / 8
#2	The pregnant animal no. 34 delivered no live pups due to resorption of all implants.

table 7‑20:Overview of the reproductive parameters

Parameter			Group 1 (control)	Group 2 (100 mg/kg)	Group 3 (300 mg/kg)	Group 4 (1000 mg/kg)
Parametrical values
Implantation sites (mean per dam)			16.0	16.1	15.9	15.3
Pups born alive and dead (mean per dam)^#1			14.4	12.9	14.0	13.0
Pups born alive (mean per dam)^#1			14.4	12.7	14.0	13.0
Indices [%]
Birth Index		mean per dam group	89.61 89.66	72.76 80.00	88.02 88.05	84.00 85.05
Live birth Index		mean per dam group	100.00 100.00	98.41 98.28	100.00 100.00	100.00 100.00
Post-implantation loss		mean per dam^#1 group	10.39 10.34	28.56 21.38	11.98 11.95	16.00 14.95
Resorptions and stillbirths
Difference between no. of implantation sites and no. of pups born alive		mean per dam       sum per group	1.7       15	3.4       31	1.9       19	2.3       16
Number of stillbirths			0	2	0	(3^#4)
#4	Three stillborns were noted for dam no. 78 (prematurely sacrificed due to poor health on lactation day 1). These stillborns were excluded from the summary.

Table 7‑21:   Viability indices and prematurely deceased pups during the pre-cull period

Pre-cull period
Parameter	Group 1 (control)	Group 2 (100 mg/kg)	Group 3 (300 mg/kg)	Group 4 (1000 mg/kg)
Prematurely deceased pups between lactation days 0/1 to 4 / Total number of live born pups^#	0 / 130	2 / 114	1 / 140	0 / 91
Viability index (group values)^#	100.00	98.25	99.29	100.00

Table 7‑22:   Dams with prematurely deceased pups during the pre-cull period

Number of prematurely deceased pups per dam (pre-cull period)
Group 1 (control)		Group 2 (100 mg/kg)		Group 3 (300 mg/kg)		Group 4 (1000 mg/kg)
dam no.	deceased pups	dam no.	deceased pups	dam no.	deceased pups	dam no.	deceased pups
		35	1	54	1	78	(14)
		36	1

Table 7‑23:   Viability indices and prematurely deceased pups during the post-cull period

Post-cull period

Parameter

Prematurely deceased pups between lactation days 5 and 13 / Number of pups alive on lactation day 4 after culling^#

Table 7‑24:   Dams with prematurely deceased pups during the post-cull period

Number of prematurely deceased pups per dam (post-cull period)^#1
Group 1 (control)		Group 2 (100 mg/kg)		Group 3 (300 mg/kg)		Group 4 (1000 mg/kg)
dam no.	deceased pups	dam no.	deceased pups	dam no.	deceased pups	dam no.	deceased pups
						73	10

Table 7‑25:   Male to female ratios in the test groups on lactation days 1 and 4

Male / Female ratio of the pups
Time point	Group 1 (control)	Group 2 (100 mg/kg)	Group 3 (300 mg/kg)	Group 4 (1000 mg/kg)
Lactation day	1.17	0.84	0.75	1.12
Lactation day	1.17	0.87	0.74	1.12

Table 7‑26:   Changes of pup body weight in comparison to the control group for the male and female pups combined

Pup body weight		Changes in comparison to the control group [%]
		Group 2 (100 mg/kg)	Group 3 (300 mg/kg)	Group 4 (1000 mg/kg)
Lactation day 1	males + females combined	- 3.3	- 6.3	- 7.9
Lactation day 4	males + females combined	+3.2	- 5.6	- 7.0
Lactation day 13	males + females combined	+4.4	- 4.9	- 5.6

Table 7‑27: Comparison of the pup body weights (group mean values) on lactation day 13 with the test labor background data

Parameter	Values from this study - male and female pups combined Group mean values ± SD [g]		Tes labor Background Data obtained from the control groups of 15 OECD 422 / 421 studies performed at test labor from 2016 - 2019
Pup body weight on lactation day 13	Group 1	29.8 ± 2.7	Mean value from the group mean values of 11 control groups: 28.9 ± 1.2 Range of group mean values from 11 control groups: 26.3 - 30.6

Table 7‑28:   Changes of litter weight in comparison to the control group for the male and female pups combined

Litter weight			Changes in comparison to the control group [%]
			Group 2 (100 mg/kg)	Group 3 (300 mg/kg)	Group 4 (1000 mg/kg)
Lactation day 1		males + females combined	- 4.6	- 8.5	- 19.3
Lactation day 4		males + females combined	+0.2	- 8.5	- 18.8
Lactation day 13		males + females combined	+4.4	- 6.7	- 17.7*
*	Statistically significant from control at p ≤ 0.05 (DUNNETT's test)

Table 7‑29:   Mean number of live pups per dam during the lactation period

Mean number of live pups per dam (males + females combined)		Group 1 (control)	Group 2 (100 mg/kg)	Group 3 (300 mg/kg)	Group 4 (1000 mg/kg)
Lactation day 1		14.4	12.7	14.0	13.0
Lactation day 4	before culling	14.4	12.4	13.9	13.0
	after culling	10.0	8.9	9.8	9.1
Lactation day 13		10.0	8.9	9.8	7.7

Table 7‑30:   Comparison of the number of live pups per dam (group mean values) on lactation day 1 with the test labor background data

Parameter	Values from this study - male and female pups combined Group mean values ± SD		LPT Background Data^#1 obtained from the control groups of 27 OECD 422 / 421 studies performed at test laborfrom 2016 - 2019
Number of live pups per dam on lactation day 1	Group 1	14.4 ± 2.5	Mean value from the group mean values of 27 control groups: 13.9 ± 0.9 Range of group mean values from 27 control groups: 12.0 - 15.1

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Experimental exposure time per week (hours/week):

168

Species:

rat

Quality of whole database:

The available study was conducted according to OECD guideline 416 and is of high quality.

Effects on developmental toxicity

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2020-11-08 to 2021-06-01 (draft final study report)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
[Describe why the read-across can be performed (e.g. common functional group(s), common precursor(s)/breakdown product(s) or common mechanism(s) of action]

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)

- Target substance:
EC No. 943-175-7;
EC Name: Rhamnolipids: fermentation products of glucose with Pseudomonas bactéria
Molecular formulas: C30H54O13; C32H58O13; C34H60O13; C34H62O13
Molecular weight [g/mol]: main component of the UVCB: 650.8; Range: 622.74 - 678.42

- Source substance 1:
Sophorolipids EC 941-809-7:
used for: Developmental toxicity (OECD 414)

- Source substance 2:
AM-1 natural mixture of congeneric glycolipids:
Used for: 90-day study in rat and dogs; Toxicity to reproduction (OECD 416)
Developmental toxicity (OECD 414)

3. ANALOGUE APPROACH JUSTIFICATION
).” According to the general rules for grouping of substances and read-across approach laid down in Annex XI, Item 1.5, of Regulation (EC) No 1907/2006, substances may be considered as analogues provided that their physico-chemical, toxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity. The target and source substances as well as the constituents of the target (listed in Table 1) are considered to apply to these general rules and the similarity is justified on basis of scope of variability and overlapping of composition, representative molecular structure, physico-chemical properties, toxicological profiles and supported by various QSAR methods. There is convincing evidence that these chemicals lie in the overall common profile with respect to the present analogue approach. The key points that the members share are:
(i) Common precursor and break-down products: surfactants glycolipids have a common metabolic fate that involves hydrolysis of the α- and β-glycosidic bond to the fatty acids and glucose. Glucose and glucose oligomers enter the carbohydrate metabolic pathway and are catabolised into pyruvate and subsequently to the major extent into acetyl-CoA, which is introduced into the citric acid cycle with the aim to generate reduction equivalents for energy generation in the oxidative phosphorylation. In addition to its function in the generation of energy by catabolic processes acetyl-CoA can also be used in anabolic processes like lipid synthesis, which is important for the storage of energy in form of large high-energy macromolecules. Alkyl glucoside undergo metabolism via P-450.
(ii) Similar structural features: The source and the target substances are glycolipids that consist of a hydrophobic alkyl chain derived from fatty acids and a hydrophilic saccharide structure derived from glucose.
(iii) Similar physico-chemical properties: Considering the physical state source and test substances are solid (marketed in liquid form). All substances have in common a high-water solubility and low vapour pressure.
(iv) Similar metabolic pathways: as mentioned in point (i), target and source substances as of alkyl glucosides undergo metabolism via P-450.
(v) Common levels and mode of human health related effects: The available data indicate that the target and source substances have similar intrinsic properties leading to similar toxicological profiles. Thus, based on the available data, the target and source substances show no acute oral and dermal toxicity, no skin irritation sensitisation properties. With regard to eye irritation/corrosion properties, assumption made for the target and source substances is the following: since analogue chemicals differ from each other by the number of -CH2- units, this group of compounds is very homogenous and it is assumed that either each compound exhibits the same toxic mode of action or there will be an evident relationship between chain length and the toxicological properties (for Details see Tab.5). No hazard is expected after short or long-term exposure. Furthermore, the target and source substances of the analogue approach are not mutagenic or clastogenic and are not considered to be hazardous for fertility and intrauterine development.

4. DATA MATRIX

See the attachment

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

January 2001

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

August 1998

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Source: sponsor; Batch: 1851T1031

- Purity, including information on contaminants, isomers, etc.:
Total Rhamnolipid (acidic form): 88.5 [wt%]
Ashes (>800°C from d.w.): 6.0 [wt%]
Water (Karl Fischer): 1.65 [wt%]

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature, dry

Species:

rat

Strain:

Crj: CD(SD)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC, United States
- Age at study initiation: 11–13 weeks
- Weight at study initiation: between 219 and 301 g
- Housing: Single/Individual. Solid-bottom cages containing appropriate bedding material (Bed-O-Cobs or other suitable material). Housing set-up was as specified in the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011). For enrichment, animals were provided items such as treats, a gnawing device, and/or nesting material, except when interrupted by study procedures/activities.
- Diet: ad libitum. PMI Nutrition International, LLC Certified Rodent LabDiet® 5002. It was considered that there were no known contaminants in the feed that would interfere with the objectives of the study.
- Water: ad libitum. Municipal tap water, treated by reverse osmosis and ultraviolet irradiation. Automatic watering system. Water bottles were provided, if required. It was considered that there were no known contaminants in the water that could interfere with the outcome of the study.
- Acclimation period: yes, duration not reported
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 25
- Humidity (%): 30 to 70
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: Completion of In-life: 2020-11-25

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

deionized water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Dose formulations were divided into aliquots where required to allow them to be dispensed on each dosing occasion. Frequency of preparation: approximately weekly. Dosing formulations were prepared at appropriate concentrations to meet dose level requirements. The dose formulations were stirred continuously at room temperature during dosing.
VEHICLE
- Concentration in vehicle: 0, 14.5, 43.5, 145 mg/mL
- Amount of vehicle: 5 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose analysis results were verified prior to dose/diet administration at each sampling interval. If results were deemed unacceptable, the formulations were prepared again and analysed. All samples to be analyzed were transferred to the Analytical Chemistry Department at the Testing Facility for same day analysis, where possible or stored for analysis within known formulation stability period.
Analytical Method
Analyses described below were performed by high performance liquid chromatography with ultraviolet absorbance detection (HPLC-UV) using a validated analytical procedure.
Concentration Analysis
Storage Conditions: Temperature set to maintain a target of 5°C.
Acceptance Criteria: Mean sample concentration within 100% ± 10% of theoretical concentration. Individual sample concentration of ± 15%.
Stability Analysis
Test substance formulations have been previously shown to be stable over the range of concentrations used on this study for at least 8 days (refrigerated). Therefore, stability of test substance formulations was not assessed on this study.

Details on mating procedure:

Time-mated female Crl:CD(SD) rats were received from Charles River Laboratories, Inc., Raleigh, NC on Gestation Day 2, 3, or 4.

Duration of treatment / exposure:

The test substance and vehicle were administered as a single daily oral gavage dose during Gestation Days 6 through 20.

Frequency of treatment:

Daily, at approximately the same time each day.

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

vehicle control

Dose / conc.:

72.5 mg/kg bw/day (nominal)

Dose / conc.:

217.5 mg/kg bw/day (nominal)

Dose / conc.:

725 mg/kg bw/day (nominal)

No. of animals per sex per dose:

25 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on information provided by the Sponsor based on preceding GLP-compliant studies in Sprague Dawley CD rats, including a 4-week oral toxicity study with mortality at 1000 mg/kg. The NOAEL was set at 500 mg/kg. For further information on dose selection rationale, please see section "any other information on materials and methods".
The dose levels chosen in this study were expected to produce graded responses to the test substance. It was anticipated that the high-dose level would show drug-specific effects but not produce an incidence of fatalities that would prevent a meaningful evaluation. The lower dose levels were selected at intervals that were predicted to be narrow enough to reveal any dose related trends.

- Time of day for (rat) dam blood sampling: prior to noon on each day of collection, around the same time each day, and within a 2-hour window on each collection day.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily (morning and afternoon), beginning upon arrival through termination/release.
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once daily, beginning with the day of animal arrival and continuing through (and including) the day of euthanasia.
BODY WEIGHT: Yes
- Time schedule for examinations: Gestation Days 0 (by supplier) and 5–21 (daily).

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #21
- Organs examined: thyroid gland, kidney, liver, muscle, diaphragm, placenta, spleen, ovary, uterus

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Blood sampling:

- Plasma: No
- Serum: Yes
- Volume collected: approximately 150 µL

Fetal examinations:

- External examinations: Yes, all per litter
- Soft tissue examinations: Yes, half per litter
- Skeletal examinations: Yes, half per litter
- Head examinations: Yes, half per litter
- Anogenital distance of all live rodent pups: yes

Statistics:

Yes. For details please refer to the document in section "Attached background material"

Indices:

Pre-Implantation Loss = (No. of corpora lutea – no. of implants x 100) / No. of corpora lutea.

Post-Implantation Loss = (No. of implants – no. of live fetuses x 100) / No. of implants.

Sex Ratio (% males) = (No. male fetuses x 100) / Total no. of fetuses.

Litter % of Fetuses with Abnormalities = (No. of fetuses in litter with a given finding x 100) / No. of fetuses in litter examined.

Historical control data:

The test facility has provided historical data on the background incidence of fetal malformations and developmental variations in the Crl:CD(SD) rat.
- Embryo-Fetal Developmental Historical Control Data
- Modal Distribution of Fetal Body Weights
- Historical Control Summary of Clinical Pathology Values

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related increased incidences of abnormal breathing sounds were observed in 8 females at approximately 2 hours post-dose in the 725 mg/kg/day group and persisted to the daily examinations in 3 females only during Gestation Days 7–21. These findings were considered non-adverse due to the animals being in otherwise general good health throughout the treatment period. Other clinical observations noted in the test substance-treated groups occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean maternal body weights, body weight gains, adjusted body weights, adjusted body weight gains in the 72.5, 217.5, and 725 mg/kg/day groups were unaffected by test substance administration. Any statistically significant differences from the control group were transient, did not occur in a dose-responsive manner, and/or did not impact mean absolute body weights.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In the 725 mg/kg/day group, test substance-related statistically significantly lower mean food consumption was noted during Gestation Days 6–9 compared to the control group. Mean food consumption in this group was comparable to the control group for the remainder of the dosing period (Gestation Days 9–21) and when the entire dosing period (Gestation Days 6–21) was evaluated. There were no corresponding effects on mean body weights or body weight gains, and therefore, the effects on mean food consumption in the 750 mg/kg/day group were considered non-adverse.
Mean food consumption in the 72.5 and 217.5 mg/kg/day were unaffected by test substance administration throughout the study. Differences from the control group were slight and not statistically significant.

Endocrine findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were test substance-related effects on thyroid hormone values (TSH, T3, and T4) at any dose level. Differences from the control group were not statistically significant, observed in a manner that was not dose-related, and were within the ranges of the Historical Control Data.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Gravid uterine weights in the 72.5, 217.5, and 725 mg/kg/day groups were unaffected by test substance administration.

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

725 mg/kg bw/day (nominal)

Based on:

act. ingr.

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

changes in number of pregnant

changes in pregnancy duration

clinical signs

dead fetuses

early or late resorptions

effects on pregnancy duration

endocrine findings

food consumption and compound intake

gross pathology

histopathology: neoplastic

histopathology: non-neoplastic

maternal abnormalities

mortality

necropsy findings

number of abortions

organ weights and organ / body weight ratios

pre and post implantation loss

total litter losses by resorption

Key result

Abnormalities:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance-related external malformations were noted in fetuses in this study. A single fetus in the 217.5 mg/kg/day group (No. 3518-09) was observed with proboscis and absent eye bulges, mouth, and lower jaw. These findings were noted in a single mid-dose fetus, and therefore were not considered test substance related. No other external malformations or external developmental variations were observed in fetuses on this study.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Skeletal malformations were noted for 0(0), 1(1), 2(2), and 6(3) fetuses (litters) in the control, 72.5, 217.5, and 725 mg/kg/day groups, respectively; incidences in the test substance-treated groups were not statistically significantly higher than the concurrent control group. In the 725 mg/kg/day group, different types of vertebral malformations were noted in 3 individual litters: Fetus Nos. 4504 05, 4504-16, and 4504-17 were observed with absent lumbar vertebra, Fetus Nos. 4513-01 was observed with an absent rib and thoracic hemivertebra; this fetus was also noted with a lower body weight (3.87 g) compared to the group mean value (5.92 g), and Fetus Nos. 4521-04 and 4521-10 were observed with supernumerary lumbar vertebra. Because the skeletal malformations in the 725 mg/kg/day group were of specific types to fetuses in individual litters, they were considered familial and not related to the test substance administration. In the 217.5 mg/kg/day group, Fetus No. 3503 10 was observed with fused ribs, cervical centrum, cervical arches, and thoracic arches and absent cervical centrum and Fetus No. 3518 09 was noted with multiple malformations in the skull (absent mandible and jugal bones, misshapen nasal, premaxilla, maxilla, squamosal, frontal, parietal, and interparietal bones, small premaxilla, nasal, maxilla, squamosal, tympanic annulus, frontal, parietal, and interparietal bones, large supraoccipital bone, and unossified parietal bone), corresponding to the external malformations noted in this fetus. In the 72.5 mg/kg/day group, Fetus No. 2501-03 was observed with an absent lumbar vertebra. Skeletal malformations in the 50 and 150 mg/kg/day groups were limited to single fetuses, and therefore were not considered test substance related.
Higher incidences (statistically significant) of skeletal developmental variations were noted in the 72.5 and 725 mg/kg/day groups. However, the individual findings were not observed in a dose related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the historical control data (version 2017.03). Therefore, the skeletal developmental variations were not considered test substance-related.

Visceral malformations:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

725 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

changes in sex ratio

changes in litter size and weights

changes in postnatal survival

external malformations

skeletal malformations

visceral malformations

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Dose Formulation Analyses

The analyzed dosing formulations contained 92.4% to 101% of the test substance which was within the protocol-specified range of target concentrations for solutions (90% to 110%). The test substance was not detected in the analyzed vehicle formulation that was administered to the control group (Group 1). Results of the analyses of dosing formulations are summarized below.
Results of Concentration Analyses (mean concentration, mg/mL % of target))
- Date of preparation: 2020-11-05. Group 2 (10 mg/mL) 9.49 mg/mL, Group 3 (30 mg/mL) 28.9 mg/mL, Group 4 (100 mg/mL) 95.1 mg/mL.
- Date of preparation: 2020-11-19. Group 2 (10 mg/mL) 9.24 mg/mL, Group 3 (30 mg/mL) 30.4 mg/mL, Group 4 (100 mg/mL) 93.6 mg/mL.

Mortality and Observations
There were no test substance-related effects on survival. All animals survived to scheduled euthanasia and were gravid.
Test substance-related increased incidences of abnormal breathing sounds were observed in 8 females at approximately 2 hours postdose in the 725 mg/kg/day group and persisted to the daily examinations in 3 females only during Gestation Days 7–21. These findings were considered non-adverse due to the animals being in otherwise general good health throughout the treatment period. Other clinical observations noted in the test substance-treated groups occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.

Body Weights and Gravid Uterine Weights
Mean maternal body weights, body weight gains, adjusted body weights, adjusted body weight gains, and gravid uterine weights in the 72.5, 217.5, and 725 mg/kg/day groups were unaffected by test substance administration. Any statistically significant differences from the control group were transient, did not occur in a dose-responsive manner, and/or did not impact mean absolute body weights.

Food Consumption
In the 725 mg/kg/day group, test substance-related statistically significantly lower mean food consumption was noted during Gestation Days 6–9 compared to the control group. Mean food consumption in this group was comparable to the control group for the remainder of the dosing period (Gestation Days 9–21) and when the entire dosing period (Gestation Days 6–21) was evaluated. There were no corresponding effects on mean body weights or body weight gains, and therefore, the effects on mean food consumption in the 750 mg/kg/day group were considered non-adverse.
Mean food consumption in the 72.5 and 217.5 mg/kg/day were unaffected by test substance administration throughout the study. Differences from the control group were slight and not statistically significant.

Thyroid Hormone Analyses
There were test substance-related effects on thyroid hormone values (TSH, T3, and T4) at any dose level. Differences from the control group were not statistically significant, observed in a manner that was not dose-related, and were within the ranges of the Historical Control Data.

Macroscopic Pathology
No test substance-related gross findings were noted. The gross findings observed were considered incidental, of the nature commonly observed in this strain and age of rat, and/or were of similar incidence in control and treated animals and, therefore, were considered unrelated to test substance administration.

Organ Weights
No test substance-related organ weight changes were noted at any dose level.

Microscopic Evaluations
No test substance-related microscopic findings in the thyroid gland or liver were noted. The microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rats, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to test substance administration.

Ovarian and Uterine Examinations
Intrauterine growth and survival were unaffected by test substance administration at dosage levels of 72.5, 217.5, and 725 mg/kg/day. Parameters evaluated included mean litter proportions of post implantation loss, mean number of live fetuses, mean fetal body weights, mean anogenital distance (absolute and relative to cube root of body weight), and fetal sex ratios. Differences from the control group were slight, not statistically significant, and/or noted in a manner that was not dose-related.
Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre implantation loss were similar across all groups.

Fetal Morphological Data
The numbers of fetuses (litters) available for morphological evaluation were 317(25), 318(25), 301(25), and 333(25) in the control, 72.5, 217.5, and 725 mg/kg/day groups, respectively. Malformations were observed in 0(0), 1(1), 2(2), and 6(3) fetuses (litters) in these same respective dose groups.

External Malformations and Variations
No test substance-related external malformations were noted in fetuses in this study. A single fetus in the 217.5 mg/kg/day group (No. 3518-09) was observed with proboscis and absent eye bulges, mouth, and lower jaw. These findings were noted in a single mid-dose fetus, and therefore were not considered test substance related. No other external malformations or external developmental variations were observed in fetuses on this study.

Visceral Malformations and Variations
No visceral malformations were noted in fetuses in this study.
No test substance-related visceral developmental variations were noted. Findings observed in the test substance treated groups were noted similarly in the control group.

Skeletal Malformations and Variations
Skeletal malformations were noted for 0(0), 1(1), 2(2), and 6(3) fetuses (litters) in the control, 72.5, 217.5, and 725 mg/kg/day groups, respectively; incidences in the test substance-treated groups were not statistically significantly higher than the concurrent control group. In the 725 mg/kg/day group, different types of vertebral malformations were noted in 3 individual litters: Fetus Nos. 4504 05, 4504-16, and 4504-17 were observed with absent lumbar vertebra, Fetus Nos. 4513-01 was observed with an absent rib and thoracic hemivertebra; this fetus was also noted with a lower body weight (3.87 g) compared to the group mean value (5.92 g), and Fetus Nos. 4521-04 and 4521-10 were observed with supernumerary lumbar vertebra. Because the skeletal malformations in the 725 mg/kg/day group were of specific types to fetuses in individual litters, they were considered familial and not related to the test substance administration. In the 217.5 mg/kg/day group, Fetus No. 3503 10 was observed with fused ribs, cervical centrum, cervical arches, and thoracic arches and absent cervical centrum and Fetus No. 3518 09 was noted with multiple malformations in the skull (absent mandible and jugal bones, misshapen nasal, premaxilla, maxilla, squamosal, frontal, parietal, and interparietal bones, small premaxilla, nasal, maxilla, squamosal, tympanic annulus, frontal, parietal, and interparietal bones, large supraoccipital bone, and unossified parietal bone), corresponding to the external malformations noted in this fetus. In the 72.5 mg/kg/day group, Fetus No. 2501-03 was observed with an absent lumbar vertebra. Skeletal malformations in the 50 and 150 mg/kg/day groups were limited to single fetuses, and therefore were not considered test substance related.
Higher incidences (statistically significant) of skeletal developmental variations were noted in the 72.5 and 725 mg/kg/day groups. However, the individual findings were not observed in a dose related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the Charles River Ashland historical control data (version 2017.03). Therefore, the skeletal developmental variations were not considered test substance-related.

Summary of External, Visceral, and Skeletal Examinations
The numbers of fetuses (litters) available for morphological evaluation were 317(25), 318(25), 301(25), and 333(25) in the control, 72.5, 217.5, and 725 mg/kg/day groups, respectively. Malformations were observed in 0(0), 1(1), 2(2), and 6(3) fetuses (litters) in these same respective dose groups.
When the total malformations and developmental variations were evaluated on a proportional basis, no statistically significant differences from the control group were noted, with the following exceptions. The mean litter proportion of fetuses with any skeletal developmental variations was statistically significantly higher in the 72.5 and 725 mg/kg/day groups compared to the control group; when individual developmental variations were evaluated, only the mean litter proportion of fetuses with incomplete ossification of the thoracic centrum was statistically significant in the 72.5 mg/kg/day group. Fetal malformations and developmental variations, when observed in the test article treated groups, occurred infrequently or at a frequency similar to that in the control group, did not occur in a dose-related manner, and/or were within the historical control data ranges. Based on these data, no fetal malformations or developmental variations were attributed to the test article.

Conclusions:

Based on the absence of adverse maternal or fetal effects, a dosage level of 725 mg/kg/day (the highest dose level tested) was considered to be the no observed adverse effect level (NOAEL) for maternal toxicity and embryo/fetal development when the test substance "Sophorolipids: fermentation products of glucose and fatty acids, C18-unsatd., esters with glycerol with yeast Candida bombicola, partially hydrolysed" was administered orally by gavage to time-mated Crl:CD(SD) rats.

Executive summary:

In this developmental toxicity study according to OECD guideline 414 and OPPTS 870.3700, the test substance "Sophorolipids: fermentation products of glucose and fatty acids, C18-unsatd., esters with glycerol with yeast Candida bombicola, partially hydrolysed" was administered to 25 time-mated female Crl:CD(SD) rats/dose at dose levels of 0, 72.5, 217.5 and 725 mg/kg bw/day. Animals were dosed via oral gavage once daily during Gestation Days 6–20.

The following parameters and end points were evaluated in this study: clinical signs, body weights, body weight gains, gravid uterine weights, food consumption, thyroid hormone parameters (T3, T4, and TSH), organ weights, macroscopic and microscopic examinations, intrauterine growth and survival, and fetal morphology.

All females survived to the scheduled necropsy on Gestation Day 21. Test substance-related clinical observations noted at the daily examinations or approximately 2 hours postdosing were limited to abnormal breathing sounds in the 725 mg/kg/day group. The animals were otherwise in good health throughout the treatment period, so these effects were not considered adverse.

There were no test substance-related effects on mean maternal body weights, body weight gains, adjusted body weights, adjusted body weight gains, or gravid uterine weights at any dose level.

Test substance-related lower mean maternal food consumption was noted in the 725 mg/kg/day group during Gestation Days 6–9 but was comparable to the control group thereafter. Based on the transient nature of these effects and the lack of corresponding body weight effects, these effects on food consumption were considered non-adverse. No test substance-related effects were observed on food consumption in the 72.5 or 217.5 mg/kg/day groups.

There were no test substance-related effects on maternal thyroid hormone values at any dose level.

There were no test substance-related effects on maternal macroscopic pathology, organ weights, or microscopic evaluations at any dose level.

There were no test substance-related effects on intrauterine growth or survival at any dose level.

There were no test substance-related effects on external, visceral, or skeletal fetal morphology at any dose level.

Based on the absence of adverse maternal or fetal effects, a dosage level of 725 mg/kg/day (the highest dose level tested) was considered to be the no‑observed‑adverse‑effect level (NOAEL) for maternal toxicity and embryo/fetal development when the test substance "Sophorolipids: fermentation products of glucose and fatty acids, C18-unsatd., esters with glycerol with yeast Candida bombicola, partially hydrolysed" was administered orally by gavage to time-mated Crl:CD(SD) rats.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

September-October 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Jelly mushroom Glycolipids (AM-1) was supplied by IMD Natural Solutions, GmbH (INS) (Dortmund, Germany) as a beige poweder with 95% glycolpid content as determinded by q NMR (the remaining ca. 5% was cmprised of residual water, protein and lipids).

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

Sexually mature, virgin female Crl: CD rats (120), approximately 71-89 days of age, were obtained from Charles River laboratories, Inc. Raleigh, NC, and were acclimated for at least 7 days. Upon arrival, all female rats were housed 2-3 per cage, by sex, in solid bottom caging with ground corn-cob material. Resident males were untreated, sexually mature rats utilized exclusively for breedings and these rats were maintained under similar laboratory conditions as the females. The rats were paired for mating in the home cage of the male. Following positive evidence of mating, the females were individually housed in clean, solid-bottom cages with bedding material.
The study rooms were maintained at a daily average temperature of 22.2 °C-23.0°C with a daily average relative humidity of 31.8%-60.1% and a 12-h light/12-h dark cycle.
PMI Nutrition International, LLC Certified Rodent LabDiet 5002 and reverse osmosis-purified (on-site) drinking water were provided ad libitum throughout the acclimation periods and during the experimental phase of each study.

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Reverse osmosis-treated water

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

24 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dosage levels for the developmental toxicity study were selected based on the results of a previous range-finding study in which pregnant female rats were administered to the test article at dosage levels of 150, 500 and 1000 mg/lg bw from Gestation Day (GD) 6-19 -In the previous study, a single female in the 1000 mg/kg/day group was found dead on GD11 following dosing. In the surviving animals, there were no significant effects on body weights, food consumption, reproductive parameters, or fetal weights noted in any dosage group tested. As a result, the same dosage levels of 150, 500, and 1000 mg/kg bw/day were used for this current definitive developmental toxicity study.
Three groups of 24 bred femalea CRL:CD (SD) rats were administered the test article, AM-1, once daily from GD 6-19. Dosage levels were 150, 500 and 1000 mg/ bw/day administered at a dose volume of 10 mL/kg. A concurrent control group composed of 24 bred females received the vehicle (reverse osmosis-treated water) on a comparable regimen.

Maternal examinations:

All rats were observed twice daily: once in the morning and once in the afternoon, for morbbundy and mortality. Individual clinical observation were recorded dauly during GD 0-20 (prior to dose administration during th treatment period). Animals were also observed for signs of toxicity 1 - 2 h following dose administration.
Individual maternal body weights and food consumption were recorded on GD 0 and 6 - 20 (daily).

Ovaries and uterine content:

Gravid uterine weight was collected and net body weight (the GD 20 body weight exclusive of the weight of the uterus and contents) and net body weight change (the GD 0-20 body weight change exclusive of the weight of the uterus and contents) were calculated and presented for each gravid female at the scheduled laparohysteroctomy. All rats were euthanized by carbon dioxide inhalation on GD 20 prior to laparohysterectomies and macroscopic examinations. The uteri, placentae, and ovaries were examined, and the numbers of fetuses, early and late resoprtions, total implantations, and corpora lutea were recorded.

Fetal examinations:

The fetuses were weighed, sexes, and examined for external, visceral, and skeletal malformations and developmental variations.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Rales was noted in a dose-dependent manner 1-2 h after dose administration for 2, 10 and 15 females in the 150, 500, and 1000 mg/kg bw/day groups, respectively, sporadically throughout the treatment period (GD6-19). Red and/or clear material around the nose and /or mouth was noted 1-2h after dose administration for 5 and 7 females in the 500 and 1000 mg/kg bw groups, respectively, sporadically during GD 7-16- The rales and material findings were considered test article-related but not adverse because they generally did not perisst to the daily examination the following day and were likely related to the physical properies of the surfactant-type test articles and route of administration.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

All females survived to the scheduled necropsy on GD 20.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean maternal body weights, body weight gains, net body weights, net body weight gains in all treatment groups were unaffected by test article administration. Differences from the control group were slight and not statistically significant with the followings exception. Mean net body weight change in the 150 mg/kg bw/day group were signficantly (p> 0.05) lower than the control group; however, this difference was not considered test article-related because it did not occur in a dose-response manner.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Mean maternal food consumption in the 1000 mg/kg bw/day group was significantly (p < 0.05 or p < 0.01) lower compared to the control group during GD 6 - 9. Mean food consumption in the 1000 mg/kg bw/day group was similar to the control group during GD 9-12 and 12-15. Mean food consumption for this group was significantly (p < 0.05 or p< 0.01) lower comapred to the control group during GD 15-20. The lower mean food consumption at the beginning and the end of the treatment period in the 1000 mg/kg bw/day group was considered test article-related but not adverse because mean overall food consumption (GD 15-20) in this group. Mean food consumption in the 500 mg/kg bw/day group was similar to the control group during GD 6-9 , 9-12, and 12-15. Significanly (p< 0.05) lower mean food consumption was noted in the 500 kg bw/day group during GD 15-20and was considered test article-related but adverse because mean overall food consumption (GD 6-20) in this group, as well as mean absoluted body weights, were similar to the control group. The food consumption in the 150 mg/kg bw/day groups was unaffected by test artivccle administration. Differences from the control groups were slight and not statistically significant.

Food efficiency:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

At the scheduled necropsy on GD 20, no test-article realted intermal findings were observed in any treatment group. Macroscopic findings observed in the test article treated groups occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related. One female in the control group amd 2 females in the 150 mg/kg bw /day group non gravid. Intrauterine growth and survival were unaffected by test article a.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

Postimplantation loss and the mean litter proportions of pre-implantation loss were similar across all groups.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

Mean litter proportions of pre-implantation loss were similar across all groups

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

One female in the control group and 2 females in 150 mg/kg bw /day group were nongravid.

Other effects:

no effects observed

Description (incidence and severity):

Mean numbers of corpora lutea were similar across all groups. Gravid uterine weights in the 150, 500 and 1000 mg/kg bw/day were unaffected by test article administration.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Fetal body weight changes were similar across all groups

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Fetal sex ratios were similar across all groups

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

Live litter size were similar across all groups

External malformations:

no effects observed

Description (incidence and severity):

No external developmental malformations or variations were observed in fetuses in the study.

Skeletal malformations:

no effects observed

Description (incidence and severity):

Skeletal malforamtions (vertebral anomaly with or without associated rib anomaly) were noted for one fetus each in the control and 150 mg/kg bw/day groups. This finding in the 150 mg/kg bw/day group was not considered test article-related because it was noted for only a single fetus and was also noted in the control group. No test-article related skeletal developmental variations were noted. Findings observed in the test article treated groups were noted infrequently similarly in the control group, were not observed in a dose-related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and /or the values were within the ranges of the CRO historical control database.

Visceral malformations:

no effects observed

Description (incidence and severity):

No soft tissue malformations were observed in fetuses in the test article-treated grouos. Situs inversus (i.e. lateral transposition of major visceral oragans) was noted for single fetus in the control group. No test article-related visceral developmental variation were noted. Findings observed in the test article treated groups were noted infrequently, similarity in the control group, were not observed in a dose-related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the CRO historical database .

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Developmental effects observed:

no

Conclusions:

In conclusion, a dosage level of 1000 mg/kg bw/day was considered to be the NOAEL for maternal toxicity and embryo/fetal developmetal toxicity when AM-1 was administered orally by gavage to bred feamles Crl:CD (SD) rats.

Justification for classification or non-classification

The available data for toxicity to reproduction do not meet the classification criteria according to Regulation (EC) 1272/2008 and are therefore conclusive but not sufficient for classification.