9-Octadecenoic acid (Z)-, sulfonated, potassium salts

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From September 19, 2016 to February 17, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 408

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EU Method B.26

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3100

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

SPF-bred Wistar Han rats

Details on species / strain selection:

Rationale: Recognized by international guidelines as the recommended test system (e.g. EPA, FDA, OECD and EC).

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test system Rat: Crl:WI(Han) (outbred, SPF-Quality).
Source: Charles River Deutschland, Sulzfeld, Germany.
Total number of animals: 40 males, 40 females (females were nulliparous and nonpregnant).
Age at start of treatment: Approximately 6 weeks.
Identification: Earmark and tattoo.
Randomization: By computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean.
Acclimatization period: At least 5 days before the start of treatment under laboratory conditions.
Health inspection: Upon receipt of the animals.

Conditions: Environmental controls for the animal room were a mean daily temperature range of 21 to 22°C, a mean daily relative humidity range of 44 to 63%, at least 10 air changes/hour, and a 12-hour light/12-hour dark cycle. The light/dark cycle was interrupted for study related activities.
Accommodation: Group housing of 5 animals per sex in Macrolon cages (MIV type, height 18 cm) with sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG) and paper as cage-enrichment (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd)). During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp).
Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF Spezialdiäten). During motor activity measurements, animals did not have access to food for a maximum of 1 hour and 43 minutes. Water: Free access to tap water. During motor activity measurements, animals did not have access to water for a maximum of 1 hour and 43 minutes.
Diet, water, bedding and cage enrichment evaluation for contaminants and/or nutrients was performed according to facility standard procedures. There were no findings that could interfere with the study.

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Method: Oral gavage, using a plastic feeding tube. Formulations were placed on a magnetic stirrer during dosing. A dose control system (DCS) was used as additional check to verify the dosing procedure according to Standard Operating Procedures.
Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated weekly according to the latest body weight.
Frequency: Once daily, 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Sampling: Samples (0.5 mL) were taken using a pipette, and were weighed on an analytical balance at 4 decimals precision. During sampling, formulations were placed on a magnetic stirrer. Immediately after sampling (accuracy and homogeneity samples) or after 5 hours at room temperature under normal laboratory light conditions (stability samples), samples were stored on dry ice until receipt.

Analysis: Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 5 hours at room temperature under normal laboratory light conditions was also determined (highest and lowest concentration). The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.

Duration of treatment / exposure:

90 d

Frequency of treatment:

daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 males and 10 females

Control animals:

yes, concurrent vehicle

Details on study design:

Estrous cycle determination: All females had a daily lavage from Day 70 up to and including Day 90 to determine the stage of estrous (according to Standard Operating Procedures).

Parental animals: Observations and examinations:

The following parameters were evaluated: clinical signs daily; functional observation tests in Week 13; body weight and food consumption weekly; ophthalmoscopy at pretest and in Week 13; clinical pathology and macroscopy at termination; organ weights and histopathology on a selection of tissues.

Oestrous cyclicity (parental animals):

Estrous cycle determination All females had a daily lavage from Day 70 up to and including Day 90 to determine the stage of estrous (according to Standard Operating Procedures).

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In the surviving animals, diarrhoea was observed on single occasions in seven 1000 mg/kg animals towards the end of the treatment period. Swelling/gas distention of the abdomen was observed in three 1000 mg/kg and two 300 mg/kg animals, each for single short and transient bouts. Rales were also observed for single short and transient bouts in five 1000 mg/kg animals and two 100 mg/kg animals. No findings were noted during the arena observations in this study.
A dose-related increase in salivation seen after dosing among treated groups was not considered toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response related to taste of the test substance rather than a sign of systemic toxicity.
Any other clinical signs noted during the treatment period occurred within the range of background findings.

Mortality:

mortality observed, treatment-related

Description (incidence):

There were two death males treated at 1000 mg/kg.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Slight (not statistically significant) trends towards a lower body weight and body weight gain were seen in 1000 mg/kg treated animals during a large proportion of the study.

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:

effects observed, treatment-related

Description (incidence and severity):

A statistically significant increase in the incidence of focal corneal edema was observed in 100 mg/kg females at Week 13, however this finding was not considered treatment-related as it occurred in a non-dose related manner.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

The following (statistically significant) changes in haematology parameters distinguished treated from control animals:
- White blood cell count was increased in 1000 mg/kg females, lymphocyte levels were reduced in 1000 mg/kg males and 1000 mg/kg females, and neutrophil levels were increased in 300 mg/kg males and 1000 mg/kg males and females (males not statistically significant).
- Red blood cell distribution width (%RDW) was increased in 1000 mg/kg females, and haemoglobin levels, mean red blood cell volume (MCV), and mean cell haemoglobin concentration (MCH) were reduced in 1000 mg/kg males and females.
Any other statistically significant changes in haematology parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

The following (statistically significant) changes in clinical biochemistry parameters distinguished treated from control animals:
- Alanine aminotransferase (ALAT) levels were significantly increased in both male and female 1000 mg/kg animals, and aspartate transaminase (ASAT) levels were increased in 1000 mg/kg females.
- Total protein and albumin levels were increased in 1000 mg/kg, and 100/300/1000 mg/kg males, respectively. Total bilirubin levels were increased in 100, 300 and 1000 mg/kg males.
- Urea levels were increased in 1000 mg/kg males and females, and creatinine levels were increased in 100 and 1000 mg/kg males and 1000 mg/kg females.
- Glucose and cholesterol levels both reduced in 1000 mg/kg males and females.
- Inorganic phosphate levels were increased in 1000 mg/kg males and females. Sodium levels were increased in 100 and 300 mg/kg males and females, however these findings occurred in the absence of a dose-related trend and therefore considered not toxicologically relevant.
Any other statistically significant changes in clinical biochemistry parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Foregrip strength was reduced in 300 and 1000 mg/kg males and 1000 mg/kg females in a statistically significant manner. And a slight (not statistically significant) reduction in total movement and ambulatory behavior was observed in 1000 mg/kg females.
Further observations, hearing ability, pupillary reflex, static righting reflex and hindgrip strenght were normal in all examined animals. Motor activity was similar between treated and control males. And all groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related microscopic findings were noted, starting at 300 mg/kg in the kidneys and mesenteric lymph nodes of males and females, and at 1000 mg/kg in the adrenal glands of males and females, the pancreas of males, and the spleen of females.
Kidney findings included an increased incidence and severity of tubular vacuolation, with pigmented material in the cortex, which was present in males and females starting at a dose of 300 mg/kg. Tubular degeneration and an increased severity and/or incidence in tubular basophilia was observed at 1000 mg/kg.
In the mesenteric lymph node, an increased incidence and severity of macrophage foci was recorded, starting at 300 mg/kg in both sexes.
In the adrenal gland, vacuolation of the zona glomerulosa was recorded at an increased incidence and/or severity in both sexes at 1000 mg/kg.
In the pancreas, increased apoptosis of acinar cells was recorded in males treated at 1000 mg/kg. One female at 1000 mg/kg with minimal increased apoptosis was considered to be within background levels.
In the spleen, increased incidence and severity of extramedullary hematopoiesis and increased severity of pigmentation was recorded in females treated at 1000 mg/kg.
The remaining histological changes were considered to be incidental findings or within the range of background pathology encountered in rats of this age and strain. There were no test substance-related alteration in the prevalence, severity, or histological character of those incidental tissue alterations.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Increased macrophages in male and female mesenteric lymph nodes starting at 300 mg/kg/day, vacuolation of the zona glomerulosa of male and female adrenal glands at1000 mg/kg/day and increased apoptosis in male pancreas at 1000 mg/kg/day were also considered to be non-adverse since these findings were slight in nature.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

No treatment related changes in estrous cycle length were noted across the dose groups during the period in which estrous cycle length was determined (Day 77 up to and including Day 98).

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

not examined

NOAEL based on on reproductive organs and male and female reproductive system function (e cycle, spermatogenesis)

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: estrous cycle regularity, spermatogenesis staging and morphology of gonads and accessory reproductive organs

Key result

Remarks on result:

not measured/tested

Key result

Remarks on result:

not measured/tested

Key result

Reproductive effects observed:

no

Treatment related:

no

Conclusions:

Under the study conditions, no toxicologically significant changes were noted in any of the reproductive parameters assessed in this study (estrous cycle regularity, spermatogenesis staging and morphology of gonads and accessory reproductive organs) revealed no treatment-related changes.

Executive summary:

A study was conducted to determine repeated dose toxicity of the test substance when administered to rats according to OECD Guideline 408, EU Method B.26 and EPA OPPTS 870.3100. The test substance, formulated in water, was administered daily for at least 90 d by oral gavage to SPF-bred Wistar Han rats. One control group and three treated groups were tested, each consisting of 10 males and 10 females, at the dose levels of 0, 100, 300 and 1000 mg/kg bw/d. Formulation analyses confirmed that formulations of test substance in water were prepared accurately and homogenously. Under the study conditions, no toxicologically significant changes were noted in any of the reproductive parameters assessed in this study (estrous cycle regularity, spermatogenesis staging and morphology of gonads and accessory reproductive organs) revealed no treatment-related changes (Beerens-Heijnen, 2017).

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

A study was conducted to determine repeated dose toxicity of the test substance when administered to rats according to OECD Guideline 408, EU Method B.26 and EPA OPPTS 870.3100. The test substance, formulated in water, was administered daily for at least 90 d by oral gavage to SPF-bred Wistar Han rats. One control group and three treated groups were tested, each consisting of 10 males and 10 females, at the dose levels of 0, 100, 300 and 1000 mg/kg bw/d. Formulation analyses confirmed that formulations of test substance in water were prepared accurately and homogenously. Under the study conditions, no toxicologically significant changes were noted in any of the reproductive parameters assessed in this study (estrous cycle regularity, spermatogenesis staging and morphology of gonads and accessory reproductive organs) revealed no treatment-related changes (Beerens-Heijnen, 2017).

Effects on developmental toxicity

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From October 13, 2016 to December 08, 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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

Test system Rat: Crl:WI(Han) (outbred, SPF-Quality). Untreated females were mated at the Supplier, and were at Day 0 or 1 post-coitum on arrival at the test facility (Day 0 post-coitum was the day of successful mating; confirmed by vaginal plug).
Source: Charles River Deutschland, Sulzfeld, Germany.
Total number of animals: F0-generation: 88 females; F1-generation: 951 fetuses.
Age at delivery Females were approximately 10-14 weeks.
Identification: by indelible ink.

Randomization: within one day after receipt, the animals of each subgroup were randomized by computer-generated random algorithm according to body weight. In the subgroups, body weights varied between ±20% to ±35% of the mean per subgroup. The overall group-means on Day 2 post-coitum were similar and showed no statistically significant differences when compared to controls. Females which were mated on the same day were classified in the same subgroup.
Acclimatization period: At least 5 days before the start of treatment under laboratory conditions.
Health inspection: Upon receipt of the animals.

Conditions: Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, at least 10 air changes/hour, and a 12-hour light/12-hour dark cycle. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.
Accommodation: Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm). Sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG) and paper as cageenrichment/ nesting material (Enviro-dri, Wm. Lillico & Son) were supplied.
Diet: free access to pelleted rodent diet (SM R/M-Z from SSNIFF Spezialdiäten GmbH).
Water: free access to tap-water.

Diet, water, bedding and cage-enrichment/nesting material evaluation for contaminants and/or nutrients was performed according to facility standard procedures. There were no findings that could interfere with the study.

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Elix, Millipore S.A.S., Molsheim, France

Details on exposure:

Test substance preparation:
Vehicle: water (Elix, Millipore S.A.S., Molsheim, France).
Rationale for vehicle: based on trial formulations performed at the labolatory.
Method of formulation: Formulations (w/w) were prepared daily within 5 hours prior to dosing and were homogenized to a visually acceptable level. No correction was made for the purity/composition of the test substance.
Appearance of formulations: light yellow to brown solutions for the test substance treatment groups.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples taken from dose preparations were analyzed according to a validated method. After sampling, all samples were stored and shipped on dry ice to the test site for formulation analysis. Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%.

The concentrations analyzed in the formulations of Groups 2, 3 and 4 (100, 300 and 1000 mg/kg bw/d)were in agreement with the target concentrations (i.e. mean accuracies between 90% and 110%). The formulations of Group 2 and Group 4 prepared were homogeneous (i.e. coefficient of variation ≤ 10%).

Duration of treatment / exposure:

From Days 6 to 20 post-coitum, inclusive.

Frequency of treatment:

Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Group 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Group 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Group 4

No. of animals per sex per dose:

22

Control animals:

yes, concurrent vehicle

Details on study design:

Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.

Maternal examinations:

Mortality: at least twice daily. Animals showing pain, distress or discomfort, which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints.
Clinical signs: at least once daily from Day 2 post-coitum onwards up to the day prior to necropsy. The time of onset, grade and duration of any observed signs were recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. In the data tables, the scored grades were reported, as well as the percentage of animals affected in summary tables.
Body weights: Days 2, 6, 9, 12, 15, 18 and 21 post-coitum.
Food consumption: Days 2-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post-coitum.
Water consumption: subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

Pathology:

All animals surviving to the end of the observation period and all moribund animals were sacrificed using an oxygen/carbon dioxide procedure and subsequently subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs.

All macroscopic abnormalities were recorded, collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution).
Necropsy was conducted on the following days:
Condition Day of necropsy: females surviving to planned necropsy - Day 21 post-coitum.
Euthanized in extremis (female no. 67): when pain, distress or discomfort was considered not transient in nature or was likely to become more severe.
Each ovary and uterine horn of all animals were dissected and examined as quickly as possible to determine:
- The number of corpora lutea.
- The weight of the (gravid) uterus (not for animals sacrificed before planned necropsy).
- The number and distribution of live and dead fetuses.
- The number and distribution of embryo-fetal deaths (early and late resorptions).
- The weight of each fetus (not for animals sacrificed before planned necropsy).
- The sex of each fetus from the ano-genital distance (during necropsy) and also from
gonadal inspections (during further fetal examination).
- Externally visible macroscopic fetal abnormalities.
In case implantations were not macroscopically visible, the uterus was stained using the Salewski technique in order to determine any former implantation sites.

For female no.67, early sacrificed in extremis, the findings in ovary and uterine are presentedseparetely. The following tissues and organs with macroscopic findings for female no.67 at necropsy were collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution):
Caecum
Colon
Duodenum
Ileum
Jejunum
Rectum
Stomach

Fetal examinations:

External:
Each viable fetus was examined in detail, weighed and sexed. All live fetuses were euthanized by administration of approximately 0.05 mL (=10 mg) of sodium pentobarbital into the oral cavity using a small flexible plastic or metal feeding tube. The fetuses of female no.67 were euthanized by decapitation, because oral administration was considered not feasible in fetuses of this age. For late resorptions a gross external examination was performed. (Late) resorptions were discarded.

Visceral (Internal):
Approximately one-half of the fetuses (live and dead) in each litter (all groups) were examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities were opened and dissected using a technique described by Stuckhardt and Poppe. This examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development as described by Woo and Hoar. The sex of all fetuses was confirmed by internal examination. The heads were removed from approximately one-half of the fetuses in each litter and placed in Bouin's solution for soft-tissue examination of all groups using the Wilson sectioning technique. After examination, the tissues without variation or malformations were discarded. Any remaining tissues (from the fetuses used for fresh visceral examination) were discarded. The carcasses were processed and stained with Alizarin Red S (as described below), but not examined.

Skeletal:
From the other one-half of the fetuses (live and dead) in each litter (all groups), the sex was confirmed by internal examination. All fetuses were eviscerated, fixed in 96% aqueous ethanol, macerated in potassium hydroxide and stained with Alizarin Red S by a method similar to that described by Dawson. Skeletal examination was done for one-half of the fetuses (i.e. the fetuses with heads). The specimens were archived in glycerin with bronopol as preservative.

Statistics:

Statistical Analyses
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control group.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
-The Mann Whitney test was used to compare mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and post-implantation loss, and sex distribution.
- Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test was used to compare the compound-treated groups to the control group.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment related clinical signs were observed in females surviving to scheduled necropsy and treated up to 1000 mg/kg.
Single or temporary observations of piloerection and/or hunched posture were observed among a few test substance treated females at 100 and 300 mg/kg. A relation to treatment was not apparent and in the absence of any corroborative finding and low incidence, these symptoms were considered incidental findings and of no toxicological significance.
Salivation seen after dosing among females of the 1000 mg/kg dose group during treatment period was considered to be a physiological response rather than a sign of systemic toxicity.
Scabs on the flews and rales were temporary observed in a single female at 1000 mg/kg. This finding occurred within the range of background findings to be expected. At this single incidence, these findings were considered of no toxicological relevance.

Mortality:

mortality observed, treatment-related

Description (incidence):

One high dose female treated at 1000 mg/kg (no. 67) was sacrificed in extremis on Day 14 post-coitum. Signs of ill health, comprising piloerection, hunched posture, rales and diarrhea became apparent from Day 11 post-coitum onwards. Its health status gradually declined and marked body weight loss and reduced food consumption were noted over Days 9 to 12 postcoitum. After a further body weight loss was seen on Day 14 post-coitum (body weight of 175 grams on Day 14 post-coitum) and yellow discoloration of the urine and emaciation were additionally observed, it was decided to sacrifice the animal. No further mortality was observed.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean body weights and body weight gain of treated animals remained in the same range as controls until Day 18 post-coitum. A markedly decreased body weight gain was observed in females at 1000 mg/kg over Days 18-21 post-coitum, resulting in lower mean body weights in these females on Day 21, reaching levels of statistical significance for both parameters in comparison with controls.
Body weights and body weight gain in females at 100 and 300 mg/kg were similar to controls on Day 21 post-coitum.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

In females treated at 1000 mg/kg, a lower food consumption before and after correction for body weight was observed during the treatment period (from Day 6 post-coitum onwards), achieving levels of statistical significance over the first part (Days 6-9 and 9-12 post-coitum) and the last part (Days 18-21 post-coitum) when compared to controls. The largest decrease in food consumption was observed over Days 18-21 post-coitum, which was likely to be related to the decreased body weight gain in these animals over the same period. Mean food consumption before or after correction for body weight for females treated at 300 and 100 mg/kg remained similar to the control level over the treatment period.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No macroscopic abnormalities were observed at necropsy in females surviving to scheduled termination. In the female sacrificed in extremis (no.67) on Day 14 post-coitum, tan colored, pasty content in the ceacum and emaciation were observed. These findings confirmed the in-life observations of diarrhea and lean appearance in this animal.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

A single high dose female (no.81) with nine corpora lutea and only three implantations sites determined the relatively high mean value, and corresponding large standard deviation, for pre-implantation loss in this group. Since implantation took place prior to start of treatment, the high pre-implantation loss in this animal was a fortuitous finding.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

not specified

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not specified

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

Two females at 100 mg/kg and one female at 1000 mg/kg were not pregnant.

Other effects:

not specified

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

mortality

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Male, female and combined fetal body weights were statistically significantly lower at 1000 mg/kg when compared to controls. The mean fetal body weights at 300 and 100 mg/kg remained in the same range as controls. Mean male fetal body weights were 5.4, 5.5, 5.3 and 4.7 grams for the vehicle control, 100, 300 and 1000 mg/kg, respectively. Mean female fetal body weights were 5.2, 5.2, 5.1 and 4.4 grams for the vehicle control, 100, 300 and 1000 mg/kg, respectively. The mean gravid uterus weight of high dose females was slightly lower (approx. 15%), but not statistically significant different from controls. This finding was likely to be related to the lower fetal body weights at 1000 mg/kg.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The male:female ratio was considered to be unaffected by treatment up to 1000 mg/kg. Mean sex ratios (males:females) were 48:52, 49:51, 50:50 and 57:43 for the control, 100, 300 and 1000 mg/kg groups, respectively.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

There were no treatment-related effects on litter size of any group. Mean litter sizes were 11.6, 11.0, 11.5 and 11.1 viable fetuses/litter for the control, 100, 300 and 1000 mg/kg groups, respectively.

Changes in postnatal survival:

no effects observed

External malformations:

effects observed, treatment-related

Description (incidence and severity):

In Group 4, a fetus with malrotated limbs was observed at external examination, which was correlated to the skeletal observation of bent limb bones in this fetus. Since bent limb bones were also observed in all other fetuses in the high dose group that were skeletally examined, i.e. one half of the total number of fetuses, the single occurrence of malrotated limbs was considered to be related to treatment. The other malformations observed in single fetuses of Group 4, i.e. cleft palate and anasarca, are occasionally seen in fetuses of this rat strain used in this type of study and were therefore considered to be chance findings and of no toxicological significance. The fetus in the control group had a small lower jaw that was skeletally confirmed. External variations were not seen in any fetuses in all groups.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

A dose related increase was observed in the number of fetuses with bent limb bones, achieving levels of statistical significance in Groups 3 and 4 when compared to controls. All fetuses in the high dose Group 3 were affected with this skeletal malformation, whereas no such malformations were observed in any of the fetuses in Group 1 and 2. In three Group 4 fetuses bent pelvic girdle bones (iliaca) were also observed. A malformation of the iliaca is rarely observed and is not seen previously in historical controls at the Test Facility. The dose related increase in litter incidence for bent ribs coincided with that observed for bent limb bones. All fetuses with bent limb bones also had bent ribs. The mean litter proportions for the skeletal variation of bent ribs were 7.7%, 12.2%, 60.7% and 99.3% per litter in Group 1, 2, 3 and 4, respectively. The incidences in Groups 3 and 4 were statistically significantly increased when compared to controls. The litter incidences of 14th full ribs and caudal shift of pelvic girdle showed a dose-related increase in Groups 3 and 4. Mean litter proportions of these respective variations were 11.3%, 3.3%, 16.4%, 21.2% and 6.1%, 3.8%, 12.0%, 18.9% per litter in Groups 1, 2, 3 and 4, respectively. Both the Group 3 and 4 incidences of these two related findings were nearby or above their historical control maximum value (13.1% for 14th full ribs and 12.8% for caudal shift of pelvic girdle) and considered to be related to treatment. Mean litter proportions of slight to moderate malaligned sternebrae were 22.4%, 20.2%, 21.7%, 40.0% per litter in Groups 1, 2, 3 and 4, respectively, achieving a level of statistical significance in Group 4 when compared to controls. Other skeletal malformations noted in this study were a vertebral centra anomaly in Group 4 fetus and a rib anomaly, sternoschisis and costal cartilage anomaly in three of control fetuses. Due to single occurrence and/or occurrence in control fetuses only, these malformations were not considered to be treatment related. Other skeletal variations that were noted occurred in the absence of a dose-related incidence trend.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related effects on visceral morphology following treatment up to 1000 mg/kg. Visceral variations were observed in 14.0%, 10.2%, 10.0% and 8.2% of fetuses per litter in Groups 1, 2, 3 and 4, respectively. The individual variations all occurred in the absence of a dose-related trend, infrequently and/or at frequencies that were within the range of available historical control data. Two viscerally malformed fetuses were observed in this study; one in each of Group 2 and 3. The Group 2 fetus had an interrupted aortic arch that was accompanied with an atrial septum defect and the Group 3 fetus had a diaphragmatic hernia and malpositioned right subclavian. The single occurrence and group distribution of the above malformations did not indicate a treatment relationship and were therefore considered to be fortuitous findings and of no toxicological significance.

Other effects:

not specified

Key result

Dose descriptor:

LOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

yes

Results

(A) Body weight

Body weights (gms) summary - Females - F0 generation

		GROUP 1 CONTROL	GROUP 2 100 MG/KG	GROUP 3 300 MG/KG	GROUP 3 1000 MG/KG
POST COITUM DAY 2	MEAN	216	217	211	215
	ST.DEV.	15.7	14.1	17.5	20.7
	N	22	20	22	21
DAY 6	MEAN	233	234	228	233
	ST.DEV.	15.9	15.8	18.2	22.1
	N	22	20	22	21
DAY 9	MEAN	242	241	236	238
	ST.DEV.	16.4	15.9	21.6	21.2
	N	22	20	22	21
DAY 12	MEAN	255	256	253	250
	ST.DEV.	16.5	18.0	23.8	24.1
	N	22	20	22	21
DAY 15	MEAN	270	270	265	265
	ST.DEV.	18.8	18.9	23.6	22.2
	N	22	20	22	20
DAY 18	MEAN	302	301	295	294
	ST.DEV.	21.5	21.2	27.5	26.1
	N	22	20	22	20
DAY 21	MEAN	342	339	334	315 **
	ST.DEV.	25.0	26.5	31.1	29.9
	N	22	20	22	20

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Body weight gain (%) summary - Females - F0 generation

		GROUP 1 CONTROL	GROUP 2 100 MG/KG	GROUP 3 300 MG/KG	GROUP 3 1000 MG/KG
POST COITUM DAY 2	MEAN	-7	-7	-8	-7
	ST.DEV.	2.1	1.7	1.5	1.5
	N	22	20	22	21
DAY 6	MEAN	0	0	0	0
	ST.DEV.	0.0	0.0	0.0	0.0
	N	22	20	22	21
DAY 9	MEAN	4	3	3	3
	ST.DEV.	1.2	1.3	2.8	3.0
	N	22	20	22	21
DAY 12	MEAN	9	9	11	8
	ST.DEV.	1.9	2.5	3.8	7.4
	N	22	20	22	21
DAY 15	MEAN	16	16	16	15
	ST.DEV.	2.4	3.1	2.4	4.4
	N	22	20	22	20
DAY 18	MEAN	29	29	29	27
	ST.DEV.	3.1	4.4	3.4	5.5
	N	22	20	22	20
DAY 21	MEAN	47	45	46	36 **
	ST.DEV.	5.3	7.3	4.5	7.6
	N	22	20	22	20

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

(B) Food consumption

Food consumption (g/animal/day) summary - F0 generation

		GROUP 1 CONTROL	GROUP 2 100 MG/KG	GROUP 3 300 MG/KG	GROUP 3 1000 MG/KG
POST COITUM DAYS 2-6	MEAN	21	21	21	21
	ST.DEV.	1.7	2.4	1.9	2.2
	N	22	20	22	21
DAYS 6-9	MEAN	20	21	20	18 *
	ST.DEV.	1.8	2.2	2.8	2.0
	N	22	20	22	21
DAYS 9-12	MEAN	21	22	21	19 *
	ST.DEV.	1.8	2.3	2.6	4.8
	N	22	20	22	21
DAYS 12-15	MEAN	23	23	22	22
	ST.DEV.	1.9	2.4	2.5	2.4
	N	22	20	22	20
DAYS 15-18	MEAN	24	24	23	22
	ST.DEV.	2.3	3.6	2.4	2.1
	N	22	20	22	20
DAYS 18-21	MEAN	24	24	23	20 **
	ST.DEV.	2.2	3.1	2.8	3.1
	N	22	20	22	20
MEAN OF MEANS		22	22	22	20

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Relative food consumption summary (g/kg bw/day) - Females - F0 generation

		GROUP 1 CONTROL	GROUP 2 100 MG/KG	GROUP 3 300 MG/KG	GROUP 3 1000 MG/KG
POST COITUM DAYS 2-6	MEAN	89	89	91	88
	ST.DEV.	7.0	8.0	6.7	7.6
	N	22	20	22	21
DAYS 6-9	MEAN	84	87	86	78 *
	ST.DEV.	6.2	7.1	7.3	8.5
	N	22	20	22	21
DAYS 9-12	MEAN	83	84	84	75 *
	ST.DEV.	5.3	5.5	4.8	18.7
	N	22	20	22	21
DAYS 12-15	MEAN	84	84	83	81
	ST.DEV.	4.7	6.1	6.4	8.0
	N	22	20	22	20
DAYS 15-18	MEAN	78	80	79	75
	ST.DEV.	4.8	7.9	3.7	4.6
	N	22	20	22	20
DAYS 18-21	MEAN	70	70	70	64 **
	ST.DEV.	3.8	4.6	4.3	8.5
	N	22	20	22	20
MEAN OF MEANS		81	82	82	77

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

(C) Macroscopic findings

Macroscopic findings summary - Females - F0 generation

	GROUP 1 CONTROL	GROUP 2 100 MG/KG	GROUP 3 300 MG/KG	GROUP 4 1000 MG/KG
POST COITUM Animals examined	22	22	22	22
Animals without findings	22	22	22	21
Animals affected	0	0	0	1
General observations Emaciated	0	0	0	1
Caecum Contents	0	0	0	1

For more parameter and data tables, kindly refer the attached background material section of the IUCLID

Conclusions:

Under the study conditions following NOAELs for the test substance were derived: maternal NOAEL: 300 mg/kg bw/d (based on the unscheduled mortality of a female and on treatment related changes in body weight and food consumption at 1000 mg/kg bw/d). Developmental NOAEL: 100 mg/kg bw/d (based on retardation of male and female fetal body weight at 1000 mg/kg bw/d and dose related increases in the incidence of several skeletal malformations and variations at 300 and 1000 mg/kg bw/d).

Executive summary:

A study was conducted to determine prenatal developmental toxicity of the test substance according to OECD Guideline 414, EU Method B.31 and OPPTS 870.3700. Eighty-eight mated female Wistar Han rats were assigned to four dose groups (0, 100, 300 and 1000 mg/kg bw/d, Groups 1, 2, 3 and 4 respectively). The test substance was administered once daily by oral gavage from Days 6 to 20 post-coitum. The rats of the control group received the vehicle, Water (Elix), alone. Females were checked daily for the presence of clinical signs. Food consumption and body weight were determined at periodic intervals. Formulations prepared on one day during treatment were analyzed for accuracy and homogeneity. All animals surviving to Day 21 post-coitum were subjected to an examination post-mortem and external, thoracic and abdominal macroscopic findings were recorded. Gross lesions were collected and fixed from all animals at necropsy. A laparohysterectomy was performed on each surviving female of the groups. The uteri, placentae and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations and corpora lutea were recorded. Gravid uterine weights were recorded, and corrected body weights (changes) were calculated. The fetuses were weighed, sexed and examined for external, visceral and skeletal malformations and developmental variations. All live fetuses were euthanized. One half of the fetuses were decapitated and the heads were fixed in Bouin’s fixative, these fetuses were dissected and examined for visceral anomalies. The other one-half of the fetuses were processed and stained with Alizarin Red S for skeletal examinations. Accuracy and homogeneity of formulations were demonstrated by analyses. In pregnant females at 1000 mg/kg bw/d, reduced body weight and food consumption were observed in particular shortly before scheduled cesarean section on Day 21 post-coitum. At termination of the study, body weights corrected for uterus weights were clearly lower in these high dose females in comparison to those in the other groups. One high dose female was early sacrificed because of a bad health status on Day 14 post-coitum. The cause of its bad health status could not be established in this study. However, the yellow discoloration of the urine and tan discoloured pasty ceacum-contents may indicate that treatment with the test substance was involved. Therefore, a relation to treatment was considered. No maternal toxicity was observed in the 300 and 100 mg/kg bw/d groups. Male and female fetal body weights were lower at 1000 mg/kg, when compared to controls. The mean fetal body weights at 300 and 100 mg/kg bw/d remained in the same range as controls. External examination of the high dose fetuses revealed malrotated limbs in one fetus. This malformation correlated to the skeletal malformation of bent limb bones, which was observed in all fetuses that were skeletally examined, including the fetus with the malrotated limbs. In addition, bent ribs were also observed in all these fetuses and the incidences of bent pelvic girdle bones (iliaca), caudal shift of pelvic girdle, malaligned sternebra, 14th full ribs and unossified metacarpal and/or metatarsal were increased at 1000 mg/kg. Increased incidences of most of these skeletal malformations or variations were also increased in the mid dose group treated at 300 mg/kg. The incidences of malaligned sternebra and unossified metacarpal and/or metatarsal remained within the same range as controls in the mid dose group fetuses. The skeletal malformations and variations in fetuses at 100 mg/kg bw/d remained in the same range as controls. There were no treatment related effects on visceral morphology following treatment up to 1000 mg/kg. Under the study conditions following NOAELs for the test substance were derived: maternal NOAEL: 300 mg/kg bw/d (based on the unscheduled mortality of a female and on treatment related changes in body weight and food consumption at 1000 mg/kg bw/d). Developmental NOAEL: 100 mg/kg bw/d (based on retardation of male and female fetal body weight at 1000 mg/kg bw/d and dose related increases in the incidence of several skeletal malformations and variations at 300 and 1000 mg/kg bw/d) (de Raaf-Beekhuijzen, 2017).

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

chronic

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

A study was conducted to determine prenatal developmental toxicity of the test substance according to OECD Guideline 414, EU Method B.31 and OPPTS 870.3700. Eighty-eight mated female Wistar Han rats were assigned to four dose groups (0, 100, 300 and 1000 mg/kg bw/d, Groups 1, 2, 3 and 4 respectively). The test substance was administered once daily by oral gavage from Days 6 to 20 post-coitum. The rats of the control group received the vehicle, Water (Elix), alone. Females were checked daily for the presence of clinical signs. Food consumption and body weight were determined at periodic intervals. Formulations prepared on one day during treatment were analyzed for accuracy and homogeneity. All animals surviving to Day 21 post-coitum were subjected to an examination post-mortem and external, thoracic and abdominal macroscopic findings were recorded. Gross lesions were collected and fixed from all animals at necropsy. A laparohysterectomy was performed on each surviving female of the groups. The uteri, placentae and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations and corpora lutea were recorded. Gravid uterine weights were recorded, and corrected body weights (changes) were calculated. The fetuses were weighed, sexed and examined for external, visceral and skeletal malformations and developmental variations. All live fetuses were euthanized. One half of the fetuses were decapitated and the heads were fixed in Bouin’s fixative, these fetuses were dissected and examined for visceral anomalies. The other one-half of the fetuses were processed and stained with Alizarin Red S for skeletal examinations. Accuracy and homogeneity of formulations were demonstrated by analyses. In pregnant females at 1000 mg/kg bw/d, reduced body weight and food consumption were observed in particular shortly before scheduled cesarean section on Day 21 post-coitum. At termination of the study, body weights corrected for uterus weights were clearly lower in these high dose females in comparison to those in the other groups. One high dose female was early sacrificed because of a bad health status on Day 14 post-coitum. The cause of its bad health status could not be established in this study. However, the yellow discoloration of the urine and tan discoloured pasty ceacum-contents may indicate that treatment with the test substance was involved. Therefore, a relation to treatment was considered. No maternal toxicity was observed in the 300 and 100 mg/kg bw/d groups. Male and female fetal body weights were lower at 1000 mg/kg, when compared to controls. The mean fetal body weights at 300 and 100 mg/kg bw/d remained in the same range as controls. External examination of the high dose fetuses revealed malrotated limbs in one fetus. This malformation correlated to the skeletal malformation of bent limb bones, which was observed in all fetuses that were skeletally examined, including the fetus with the malrotated limbs. In addition, bent ribs were also observed in all these fetuses and the incidences of bent pelvic girdle bones (iliaca), caudal shift of pelvic girdle, malaligned sternebra, 14th full ribs and unossified metacarpal and/or metatarsal were increased at 1000 mg/kg. Increased incidences of most of these skeletal malformations or variations were also increased in the mid dose group treated at 300 mg/kg. The incidences of malaligned sternebra and unossified metacarpal and/or metatarsal remained within the same range as controls in the mid dose group fetuses. The skeletal malformations and variations in fetuses at 100 mg/kg bw/d remained in the same range as controls. There were no treatment related effects on visceral morphology following treatment up to 1000 mg/kg. Under the study conditions following NOAELs for the test substance were derived: maternal NOAEL: 300 mg/kg bw/d (based on the unscheduled mortality of a female and on treatment related changes in body weight and food consumption at 1000 mg/kg bw/d). Developmental NOAEL: 100 mg/kg bw/d (based on retardation of male and female fetal body weight at 1000 mg/kg bw/d and dose related increases in the incidence of several skeletal malformations and variations at 300 and 1000 mg/kg bw/d) (de Raaf-Beekhuijzen, 2017).

Justification for classification or non-classification

Results from the prenatal developmental toxicity study in Wistar Han rats with the test substance revealed bent limb bones and bent ribs in all fetuses at the highest tested dose of 1000 mg/kg/day. The incidences of bent pelvic girdle bones (iliaca), caudal shift of pelvic girdle, malaligned sternebra, 14th full ribs and unossified metacarpal and/or metatarsal were increased at 1000 mg/kg/day. Increased incidences of most of these skeletal malformations or variations were also increased at the mid dose of 300 mg/kg/day. The fetal effects at 1000 mg/kg/day were observed in presence of maternal toxicity (treatment related changes in body weight and food consumption at 1000 mg/kg/day). However, the fetal effects observed at the mid dose were observed in absence of maternal toxicity at this dose. Based on these results, the test substance warrants classification as Repr. Cat. 1B according to EU CLP regulation (EC/1272/2008) criteria.