Etocrilene

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Toxicity to fertility: NOAEL = 1000 mg/kg bw/day (OECD 422, oral)

In a combined repeated dose toxicity study with the reproductive/developmental screening test in Wistar rats, the NOAEL (no observed adverse effect level) for general, systemic toxicity was 300 mg/kg bw/d based on decreased food consumption and decreased body weight parameters as well as clinical chemistry changes at 1000 mg/kg bw/d. The NOAEL for reproductive performance and fertility was 1000 mg/kg bw/d for the F0 parental rats. The NOAEL for developmental toxicity in the F1 offspring was 1000 mg/kg bw/d, the highest tested dose.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 Sep 2014 - 26 May 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Qualifier:

according to guideline

Guideline:

other: EPA, Health Effects Test Guidelines; OPPTS 870.3650

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 11 - 13 weeks
- Weight at study initiation: Male animals: 328.2 g - 368.9 g; Female animals: 190.2 g - 215.9 g
- Housing: individually in Polycarbonate cages type III; during overnight matings, male and female mating partners were housed together in Polycarbonate cages type III. Pregnant animals and their litters were housed together until PND 4.
- Diet: ground Kliba maintenance diet mouse/rat “GLP” meal, ad libitum
- Water: supplied from water bottles (ad libitum).
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24°C
- Humidity (%): 30-70
- Air changes (per hr): 115
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

1%

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
For the preparation of the administration suspensions the test substance was weighed in a calibrated beaker depending on the dose group, topped up with 1% Carboxymethylcellulose suspension in drinking water and intensely mixed with a homogenizer. During administration, the preparations were kept homogeneous with a magnetic stirrer.

VEHICLE
- Concentration in vehicle: 1.00, 3.00 and 10.00 g/100ml
- Amount of vehicle (if gavage): 10 ml/kg bw

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: maximum of 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical verifications of the stability of the test substance in 1% Carboxymethylcellulose suspension in drinking water for a period of 96 hours at room temperature were carried out prior to the start of the study. Samples of the test substance preparations were sent to the analytical laboratory once during the study period for verification of the concentration. The samples, which were taken for the concentration control analysis at the beginning of the administration period, were also used to verify the homogeneity. Three samples (one from the top, middle and bottom in each case) were taken from the beaker with a magnetic stirrer running. Of each sample, one additional reserve sample was retained. Details of the sampling schedule were recorded with the raw data.
The various analyses:
• Demonstrated the stability of the test substance in 1% Carboxymethylcellulose suspension in drinking water over a period of 96 hours at room temperature
• Confirmed the homogeneous distribution of the test substance in 1% Carboxymethylcellulose suspension in drinking water
• Verified correct concentrations of the test substance preparations.

Duration of treatment / exposure:

The duration of treatment covered a 2-week pre-mating and a mating period in both sexes, approximately 4 days post-mating in males, and the entire gestation period as well as approximately 4 weeks of the lactation period.

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
100, 300, 1000 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: A check for moribund or dead animals was made twice daily on working days or once daily (Saturday, Sunday or on public holidays). A cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations (DCO) were performed in all animals once prior to the first administration (day 0) and at weekly intervals during the administration period. The examinations started in the morning. The findings were ranked according to the degree of severity, if applicable.

BODY WEIGHT: Yes
In general, the body weight of the male and female parental animals was determined once a week at the same time of the day (in the morning) until sacrifice. The following exceptions are notable for the female animals:
• During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
• Females with litter were weighed on the day of parturition (PND 0) and on PND 4.
Females without positive evidence of sperm, without litter or waiting for necropsy, were weighed weekly. These body weight data were solely used for the calculations of the dose volume.

FOOD CONSUMPTION
Generally, food consumption was determined once a week for male and female parental animals, with the following exceptions:
• Food consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (male and female F0 animals).
• Food consumption of the F0 females with evidence of sperm was determined on gestation days (GD) 0 - 7, 7 - 14, and 14 - 20.
• Food consumption of F0 females which gave birth to a litter was determined on PND 1 - 4.
Food consumption was not determined in females without positive evidence of sperm during the mating and the gestation period and in females without litter during the lactation period.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: study day 32 (males) and 56 (females).
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: 5 male and 5 female animals (with litter) per group
- Parameters examined: Leukocyte count (WBC), Erythrocyte count (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume, (MCV),
Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (PLT), Differential blood count, Reticulocytes (RET), Prothrombin time (Hepato Quick’s test) (HQT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: study day 32 (males) and 56 (females).
- Animals fasted: Yes
- How many animals: 5 male and 5 female animals (with litter) per group
- Parameters examined: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), γ-Glutamyltransferase (GGT), Sodium (NA), Potassium (K), Chloride (CL), Inorganic phosphate (INP), Calcium (CA), Urea (UREA), Creatinine (CREA), Glucose (GLUC), Total bilirubin (TBIL), Total protein (TPROT), Albumin (ALB), Globulins (GLOB), Triglycerides (TRIG), Cholesterol (CHOL), Bile acids (TBA)

URINALYSIS: Yes
- Time schedule for collection of urine: Urinalysis was carried out on urine from 5 male and 5 female animals (with litter) per group on study day 30 (males) and 52 (females).
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: pH, Protein (PRO), Glucose (GLU), Ketones (KET), Urobilinogen (UBG), Bilirubin (BIL), Blood, Specific gravity, Sediment, Color, turbidity (COL, TURB), Volume (VOL)

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: On study day 28, a functional observational battery and motor activity measurement were carried out in five male animals per group. On study day 50, a functional observational battery and motor activity measurement were carried out in five female animals (with litter) per group.
- Battery of functions tested: Home cage observations, Open field observations, Sensory motor tests/Reflexes, Motor activity measurement

Sperm parameters (parental animals):

Stages of spermatogenesis in the testes

Litter observations:

All pups delivered from the F0 parents (F1 litter) were examined as soon as possible on the day of birth to determine the total number of pups, the sex and the number of liveborn and stillborn pups in each litter. At the same time, the pups were also being examined for macroscopically evident changes. Pups, which died before this initial examination, were defined as stillborn pups. In general, a check was made for any dead or moribund pups twice daily on workdays (once in the morning and once in the afternoon) or as a rule, only in the morning on Saturdays, Sundays or public holidays. The number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PND 1-4 (lactation period) were determined. The number of live pups/litter was calculated on the day after birth, and on lactation day 4. On the day of birth (PND 0) the sex of the pups was determined by observing the distance between the anus and the base of the genital tubercle; normally, the anogenital distance is considerably greater in male than in female pups. The sex of the pups was finally confirmed at necropsy. The live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams and documented for each pup. The pups were weighed on the day after birth (PND 1) and on PND 4. Pups' body weight change was calculated from these results.

GROSS EXAMINATION OF DEAD PUPS:
All stillborn pups and all pups that died before PND 4 were examined externally, eviscerated and their organs were assessed macroscopically.

Postmortem examinations (parental animals):

GROSS PATHOLOGY: Yes
All parental animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology, special attention being given to the reproductive organs.

ORGAN WEIGHTS
The following weights were determined in all animals sacrificed on schedule: Anesthetized animals, Epididymides, Testes
The following weights were determined in 5 animals/sex and test group (females with litters, same animals as used for clinical pathology examinations): Adrenal glands, Brain, Heart, Kidneys, Liver, Spleen, Thymus

HISTOPATHOLOGY: Yes
see table below for details. Special attention was given on the stages of spermatogenesis in the testes.

Postmortem examinations (offspring):

All pups with scheduled sacrifice on PND 4 were sacrificed under isoflurane anesthesia with CO2. All pups were examined externally and eviscerated; their organs were assessed macroscopically. All pups without notable findings or abnormalities were discarded after their macroscopic evaluation. Animals with notable findings or abnormalities were evaluated on a case-by-case basis, depending on the type of finding.

Statistics:

• DUNNETT-test (twosided): Food consumption (parental animals), body weight and body weight change (parental animals and pups; for the pup weights, the litter means were used), gestation days.
• FISHER'S EXACT test (one-sided): Male and female mating indices, male and female fertility indices, females mated, females delivering, gestation index (females with liveborn pups), females with stillborn pups, females with all stillborn pups.
• WILCOXON test (one-sided) with BONFERRONI-HOLM adjustment: Mating days until day 0 pc, %postimplantation loss, pups stillborn, %perinatal loss, Implantation sites, pups delivered, pups liveborn, live pups day x, viability Index, Urinalysis parameters (apart from pH, urine volume, specific gravity, color and turbidity.
• WILCOXON test (twosided): % live male day x, %live female day x
• KRUSKAL-WALLIS test (two-sided): Rearing, grip strength of forelimbs and hindlimbs, landing foot-splay test, motor activity, Blood parameters, Urine pH, volume, specific gravity, color and turbidity, Weight parameters (pathology)

Reproductive indices:

Male mating index, Male fertility index, Female mating index, Female fertility index, Gestation index, Live birth index, Postimplantation loss

Offspring viability indices:

Viability index, Sex ratio

CLINICAL SIGNS AND MORTALITY
There were no test substance-related or spontaneous mortalities in any of the groups. No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any male or female F0 generation parental animal during the entire study period.

BODY WEIGHT AND WEIGHT GAIN
While in the mean body weights of the high-dose parental males were without statistical significance nearly comparable to the concurrent control values, the body weight change of this group was statistically significantly below the concurrent control values during premating days 0 - 7 and 0 - 13 (about 27% and 29%, respectively).
The mean body weights of the high-dose parental females were statistically significantly below the concurrent control values on premating day 13 (about 4%), during GD 7 - 14 (up to 6%) and on PND 4 (about 6%). The body weight change of the high-dose parental females was statistically significantly below the concurrent control values during premating days 7 - 13 and 0 - 13 (about 69% and 51%, respectively).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Food consumption of the high-dose F0 females (1000 mg/kg bw/d) was statistically significantly below the concurrent control values during GD 7 - 14 and GD 0 - 20 (about 8%, respectively). This was assessed as treatment related. The food consumption of the high-dose F0 females (1000 mg/kg bw/d) during the premating and lactation period, as well as the food consumption of all test substance-treated F0 male animals (100, 300 and 1000 mg/kg bw/d) and the mid- and low-dose F0 females was comparable to the concurrent control values throughout the entire study period.

HAEMATOLOGY
At the end of the administration period in females of test groups 1 and 3 (100 and 1000 mg/kg bw/d), platelet counts were lower compared to controls. In test group 1 the mean was not dose-dependently altered and therefore the change was incidental and not treatmentrelated. In test group 3 the median platelet count was higher compared to test group 1, but the mean was lower. Mean and median platelet counts in females of test group 3 (1000 mg/kg b w/d) were below the historical control range. Therefore, this decrease has to be regarded as adverse.

CLINICAL CHEMISTRY
No treatment-related changes among clinical chemistry parameters were observed.
At the end of the administration period in males of test groups 1 and 2 (100 and 300 mg/kg bw/d), aspartate aminotransferase (AST) activities were lower compared to controls. In males of test groups 2 and 3 (300 and 1000 mg/kg bw/d) glucose levels were increased and in females of test group 2 (300 mg/kg bw/d) urea levels were decreased. All mentioned parameters were not dose-dependently changed and therefore the alterations were regarded as incidental and not treatment-related.

URINALYSIS
No treatment-related changes among urinalysis parameters were observed.
In females of test group 2 (300 mg/kg bw/d) urine volume was decreased and urine specific gravity was increased (not statistically significantly). These alterations were not dosedependent and therefore they were regarded as incidental and not treatment-related.

NEUROBEHAVIOUR
No test substance-related or spontaneous findings were observed in male and female animals of all test groups during the home cage observation. The open field observations did not reveal any test substance-related findings in male and female animals of all test groups. There were no test substance-related findings in male and female animals of all test groups. No test substance-related impaired parameters were observed in male and female animals of all test groups. Comparing single intervals of treated animals with the control group, no significant deviations were measured during motor activity merasurement with the exception of the decreased mean value at interval 2 in male animals of test group 3 (1000 mg/kg bw/d). As the overall motor activity measurement in male animals of test group 3 (1000 mg/kg bw/d) was not statistically significant impaired and in females in the comparable intervals increased values (without statistical significance) were seen, the change was assessed as being spontaneous and not related to treatment.

ORGAN WEIGHTS
When compared to control group 0 (set to 100%), the mean absolute kidney weights were significantly decreased in mid dose females. The significant kidneys weight decrease was regarded as incidental. All other mean absolute weight parameters did not show significant differences when compared to the control group 0.
When compared to control group 0 (set to 100%), the mean relative weights of the brain (mid dose females and high dose male), epididymides (high dose males) and kidneys (mid and high dose males and high dose females) were statistically significantly increased. The significant weight increase of the brain, epididymides and kidneys in males of test group 3 (1000 mg/kg bw/d) were secondary to the decreased terminal body weight. They were within the historical control values and showed no histopathological correlate. The significant increased weight of the kidneys in females was within the historical values and showed no histopathological correlate. The significant weight increases observed in males and females of test group 2 (300 mg/kg bw/d) showed no dose-dependency and were regarded as incidental. All other mean relative weight parameters did not show significant differences when
compared to the control group 0.

GROSS PATHOLOGY
All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

HISTOPATHOLOGY: NON-NEOPLASTIC
All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
For nearly all F0 parental males, which were placed with females to generate F1 pups, copulation was confirmed. Copulation was not confirmed for high-dose male No. 37 paired with high-dose female No. 137. Thus, the male mating index was 100% in the control, low- and mid-dose group and 90% in the high-dose group. Fertility was proven for most of the F0 parental males within the scheduled mating interval for F1 litter. One high-dose male (1000 mg/kg bw/d - No. 37) and one mid-dose male (300 mg/kg bw/d -No. 29) did not generate pregnancy. Thus, the male fertility index ranged between 90% and 100% without showing any relation to dosing. This reflects the normal range of biological variation inherent in the strain of rats used for this study. The apparently infertile male rats did not show relevant gross lesions.
The female mating index calculated after the mating period for F1 litter was 100% in test groups 0 - 2 and 90% in test group 3. The mean duration until sperm was detected (GD 0) varied between 2.2 and 3.6 days without any relation to dosing. All female rats delivered pups or had implants in utero with the following exceptions:
• High-dose female No. 137 (mated with male No. 37) did not become pregnant.
• Mid-dose female No. 129 (mated with male No. 29) did not become pregnant.
The fertility index varied between 90% in test group 2 and 100% in test groups 0 - 1 and 3. These values reflect the normal range of biological variation inherent in the strain of rats used for this study. None of the non-pregnant females had any relevant gross lesions. The mean duration of gestation was similar in all test groups (i.e. between 21.9 and 22.2 days). The gestation index was 100% in test groups 0 - 1 and 3 and 88.9% in test group 2. These
values reflect the normal range of biological variation inherent in the strain of rats used for this study. Implantation was not affected by the treatment since the mean number of implantation sites was comparable between all test substance-treated groups and the control, taking normal biological variation into account (11.9 / 11.7 / 10.3 and 12.6 implants/dam in test groups 0-3, respectively). Furthermore, there were no indications for any test substance-induced intrauterine embryo-/fetolethality since the post-implantation loss did not show any statistically significant differences between the groups, and the mean number of F1 pups delivered per dam remained unaffected (11.6 / 11.3 / 10.5 and 12.0 pups/dam in test groups 0 - 3, respectively).
One sperm negative mid-dose female (300 mg/kg bw/d - No. 130) did not deliver pups but show one implantation site at necropsy. Due to this single animal with 100% post implantation loss the mean value for post implantation loss in the mid dose (19,1%) was outside the historical control range (0,7 – 18,3 %). This deviation was assessed as incidental and not treatment-related. The rate of liveborn pups was also not affected by the test substance, as indicated by live birth indices of 100% / 99.1% / 100% and 99.1% in test groups 0 - 3, respectively. Moreover, the number of stillborn pups was not significantly different between the test groups. All values are well covered by the historical control range. Thus, the test substance Etocrilene did not adversely affect reproduction and delivery of the F0 generation parental females.

Dose descriptor:

NOAEL

Remarks:

general, systemic toxicity

Effect level:

300 mg/kg bw (total dose)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on decreased food consumption and decreased body weight parameters as well as clinical chemistry changes at 1000 mg/kg bw/d.

Dose descriptor:

NOAEL

Remarks:

reproductive performance and fertility

Effect level:

1 000 mg/kg bw (total dose)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no toxicity observed

VIABILITY (OFFSPRING)
The viability index indicating pup mortality during lactation (PND 0 - 4) varied between 99.3%/ 100% /100.0% and 99.2% in test groups 0 - 3, respectively, without showing any association to treatment.
The mean number of delivered F1 pups per dam and the rates of liveborn, stillborn, cannibalized and dead F1 pups were evenly distributed among the test groups. The respective values reflect the normal range of biological variation inherent in the strain used in this study, as they are in the historical control range.
The pup mortality based on total number of stillborn pups, dead pups, pups sacrificed moribund and cannibalized pups varied between 1 / 1 / 0 and 2 in test groups 0 - 3, respectively, without test substance-related influence.
The sex distribution and sex ratios of live F1 pups on the day of birth and PND 4 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature.

CLINICAL SIGNS (OFFSPRING)
There were no test substance-related adverse clinical signs observed in any of the F1 generation pups of the different test groups.

BODY WEIGHT (OFFSPRING)
No test substance-related influence on body weights and body weight change values of F1 pups were noted in in any test group (100, 300 and 1000 mg/kg bw/d). Five male and seven female runts were seen in test group 1, two female runts were seen in test group 2 and one male runt was seen in the test group 3.

GROSS PATHOLOGY (OFFSPRING)
There were no test substance-related adverse necropsy observations in any of the F1 generation pups of the different test groups.

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Generation:

F1

Effect level:

1 000 mg/kg bw (total dose)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no toxicity observed

Reproductive effects observed:

not specified

Conclusions:

Under the conditions of the present OECD 422 combined repeated dose toxicity study with the reproductive/developmental screening test in Wistar rats, the NOAEL (no observed adverse effect level) for general, systemic toxicity of Etocrilene was 300 mg/kg bw/d based on decreased food consumption and decreased body weight parameters as well as clinical chemistry changes at 1000 mg/kg bw/d. The NOAEL for reproductive performance and fertility was 1000 mg/kg bw/d for the F0 parental rats. The NOAEL for developmental toxicity in the F1 offspring was 1000 mg/kg bw/d, the highest tested dose.

Executive summary:

The test article was administered daily as an aqueous preparation to groups of 10 male and 10 female Wistar rats (F0 animals) by gavage at doses of 100, 300 and 1000 mg/kg body weight/day (mg/kg bw/d). Control animals (10 male and 10 female Wistar rats) were dosed daily with the vehicle only (1% Carboxymethylcellulose suspension in drinking water). The duration of treatment covered a 2-week pre-mating and a mating period in both sexes, approximately 4 days post-mating in males, and the entire gestation period as well as approximately 4 weeks of the lactation period. After 2 weeks of premating treatment the F0 animals were mated to produce F1 generation pups. Mating pairs were from the same test group. Mating was discontinued as soon as sperm were detected in the vaginal smear. F0 animals were examined for their reproductive performance including determination of the number of implantation sites and the calculation of postimplantation loss for all F0 females. A detailed clinical observation (DCO) was performed in all animals before initial test substance administration and, as a rule, thereafter at weekly intervals. Food consumption of the F0 parents was determined once weekly during premating. In dams food consumption was determined for gestation days 0 - 7, 7 - 14, 14 - 20 and lactation days 1 - 4. Body weights of F0 parents were determined once a week, in males throughout the study

and in females during premating. During gestation and lactation period, F0 females were weighed on gestation days (GD) 0, 7, 14 and 20, on the day of parturition (postnatal day [PND] 0) and on PND 4. The pups were sexed and examined for macroscopically evident changes on PND 0. They were weighed on PND 1 and on PND 4. Their viability was recorded. At necropsy on PND 4,

all pups were sacrificed with CO2, under isoflurane anesthesia, and examined macroscopically for external and visceral findings.

Clinico-chemical and hematological examinations as well as urinalyses were performed in 5 animals per sex and group towards the end of the administration period. At the end of the administration period a functional observational battery was performed and

motor activity was measured in 5 parental males and females per group. All F0 parental animals were sacrificed by decapitation, under isoflurane anesthesia, and were assessed by gross pathology. Weights of selected organs were recorded and a histopathological examination was performed. Parental animals of the high dose group (1000 mg/kg bw/d) showed slightly decreased food consumption in the females during gestation (up to about 8% below control). In addition, decreased body weights was recorded in the females on premating day 13 (about 4% below control), during GD 7 - 14 (up to 6% below control) and on PND 4 (about 6% below control). Decreased body weight change in the males and females during premating (up to 29% and 69% below control, respectively were also recorded. Furthermore, decreased platelet counts in females was evident and significantly decreased terminal body weight in males (about 6% below control). No test substance related findings were noted in parental animals of the mid and low dose group and in all F1 animals.

Under the conditions of the present OECD 422 combined repeated dose toxicity study with the reproductive/developmental screening test in Wistar rats, the NOAEL (no observed adverse effect level) for general, systemic toxicity of Etocrilene was 300 mg/kg bw/d based on decreased food consumption and decreased body weight parameters as well as clinical chemistry changes at 1000 mg/kg bw/d. The NOAEL for reproductive performance and fertility was 1000 mg/kg bw/d for the F0 parental rats. The NOAEL for developmental toxicity in the F1 offspring was 1000 mg/kg bw/d, the highest tested dose.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

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

In a GLP-compliant study following OECD guideline 422 study, the test compound was administered daily by gavage to groups of 10 male and 10 female Wistar rats to screen for potential reproductive and developmental toxicity. After a two-week premating period, these parental animals were mated and the females were allowed to give birth and bring up the offspring until sacrifice on PND 4. Analyses confirmed the overall accuracy of the prepared concentrations and the homogeneity of the test substance in the vehicle. The stability of these preparations was also demonstrated over a period of 7 days under ambient conditions. In the clinical examinations of the parental animals F0 no clinical symptoms were caused by the test compound up to the limit dose of 1000 mg/kg bw/d. In the subsequent investigations including the detailed clinical observation (DCO), the functional observational battery (FOB), and measurement of motor activity (MA) no treatment related differences to control were observed at any dose level. Food consumption was slightly reduced in the parental females of the high-dose group (1000 mg/kg bw/d) during gestation. This lower food consumption corresponded to slight, but not for a consistent statistically significant decrease of parental female body weights during premating, gestation and lactation. Additionally there was a statistically significant decrease in body weight gain in males and females during premating. In summary the affected body weight parameters of the rats of the high dose group point to systemic toxicity. This trend is only borderline; however, the total of minor changes is considered as an adverse effect of treatment. Regarding clinical pathology in females of test group 3 (1000 mg/kg bw/d) platelet counts were decreased which is an indication of a change in the coagulation homeostasis in these individuals. Regarding pathology, the decreased terminal body weight in males of test group 3 (1000 mg/kg bw/d) was regarded as treatment-related. Organ weight deviations in males of the high dose group were considered to be secondary to the terminal body weight decrease. All other weight deviations were incidental and not treatment-related. Macroscopic and microscopic findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Under the conditions of this study the test compound had no adverse effects on fertility and reproductive performance of the F0 parental animals of both sexes up to 1000 mg/kg bw/d. Most of the F0 parental animals proved to be fertile. Failure of pregnancy in two test substance-treated females could not be attributed to the treatment by gross and histopathological examinations of the respective animals of both genders. Mating behavior, conception, implantation and parturition were not influenced. No signs of developmental toxicity were noted in any of the treated groups, numbers of liveborn pups, pup survival and growth were not influenced by the test compound.

Under the conditions of the present OECD 422 combined repeated dose toxicity study with the reproductive/developmental screening test in Wistar rats, the NOAEL (no observed adverse effect level) for general, systemic toxicity was 300 mg/kg bw/d based on decreased food consumption and decreased body weight parameters as well as clinical chemistry changes at 1000 mg/kg bw/d. The NOAEL for reproductive performance and fertility was 1000 mg/kg bw/d for the F0 parental rats. The NOAEL for developmental toxicity in the F1 offspring was 1000 mg/kg bw/d, the highest tested dose.

Effects on developmental toxicity

Description of key information

Developmental toxicity/ teratogenicity: NOAEL = 1000 mg/kg bw/day (OECD 414, oral)

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

25 Jun 2018

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: ordered from Charles River Laboratories, Research Models and Services, Germany GmbH.
- Age at study initiation: 10-12 weeks (time-mated females)
- Body weight of pregnant animals on Day 0: 166.9 – 219.4 g

- Housing: individually in Polycarbonate cages type III supplied by TECNIPLAST, Hohenpeißenberg, Germany and Becker & Co., Castrop-Rauxel, Germany (floor area about 800 cm²). Dust-free wooden bedding was used in this study (the present supplier is documented in the raw data). As nesting material, compacted fibers of softwood (Typ SAFE® compact nesting large, supplied by J. Rettenmaier & Söhne GmbH + Co KG, Rosenberg, Germany) and cellulose wadding was offered toward the end of gestation in all pregnant females. For enrichment, wooden gnawing blocks (Typ SAFE® block large, supplied by J. Rettenmaier & Söhne GmbH + Co KG, Rosenberg, Germany) and play tunnel (Play tunnel large, Art. 14153, supplied by PLEXX B.V., Elst, The Netherlands) were offered.

- Diet (e.g. ad libitum): mouse and rat maintenance diet “GLP”, meal (Granovit AG, Kaiseraugst, Switzerland), ad libitum
- Water (e.g. ad libitum): drinking, ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 45-65
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5%

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance preparations were prepared at the beginning of the administration period
and thereafter at intervals, which took into account the period of established stability. The preparations were kept at room temperature. For the test substance preparations, the specific amount of test substance was weighed, topped up with 0.5% CMC suspension in deionized water in a calibrated beaker and intensely mixed with a homogenizer. Before and during administration, the preparations were kept homogeneous with a magnetic stirrer.

VEHICLE
- Concentration in vehicle: 0.5% CMC suspension in deionized water
- Amount of vehicle (if gavage): 10 ml/kg bw/day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test substance in 0.5% CMC suspension in deionized water over a period of 7 days at room temperature had been verified prior to the start of the study.
Samples of the test substance preparations were sent twice (at the beginning of administration and during the administration period) to the analytical laboratory for verification of the concentrations. The samples were also used to verify the homogeneity of the low- and high-concentrations (100 and 1000 mg/kg bw/d). Three samples (one from the top, middle and bottom in each case) were taken from the beaker with a magnetic stirrer running.

Details on mating procedure:

- The animals were paired by the breeder (“time-mated”)
- Proof of pregnancy: detection of vaginal plug/sperm referred to as Day 0 of gestation.

Duration of treatment / exposure:

GD 6-19

Frequency of treatment:

once a day

Duration of test:

on GD 20, all surviving females were sacrificed

Dose / conc.:

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

Remarks:

Test group 0 (vehicle)

Dose / conc.:

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

Remarks:

Test group 1

Dose / conc.:

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

Remarks:

Test group 2

Dose / conc.:

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

Remarks:

Test group 3

No. of animals per sex per dose:

25 females/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose level rationale: In a combined repeated dose toxicity study with the reproductive/developmental screening test (OECD 422) in Wistar rats, the NOAEL (no observed adverse effect level) for general, systemic toxicity was 300 mg/kg bw/d based on decreased food consumption and decreased body weight parameters as well as clinical chemistry changes at 1000 mg/kg bw/d. The NOAEL for reproductive performance and fertility was 1000 mg/kg bw/d for the F0 parental rats. The NOAEL for developmental toxicity in the F1 offspring was 1000 mg/kg bw/d, the highest tested dose. Based on these data the dose level for this study was set to 100, 300 and 1000 mg/kg bw/d.


- The oral route was selected since this has proven to be suitable for the detection of a toxicological hazard.
- The rat was the preferred animal species for developmental and reproductive toxicity studies according to the various test guidelines. The Crl:WI(Han) strain was selected since extensive historical control data is available from the test facility for Wistar rats. This specific strain has been proven to be sensitive to substances with a teratogenic potential.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Mortality was checked twice a day on working days or once a day on Saturdays, Sundays or on public holidays (GD 0-20).
A cage-side examination was conducted at least once daily before and after treatment period
(GD 0-5 and 20). During treatment period (GD 6-19) all rats were checked daily for any signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity before administration as well as within 2 hours and between 2 and 5 hours after administration.

BODY WEIGHT: Yes
- Time schedule for examinations: on GD 0, 1, 3, 6, 8, 10, 13, 15, 17, 18 and 20.
- Corrected body weight gain: calculated after terminal sacrifice (terminal body weight on GD 20 minus weight of the unopened uterus minus body weight on GD 6).

FOOD CONSUMPTION: Yes
- Time schedule for examinations: recorded for the intervals GD 0-1, 1-3, 3-6, 6-8, 8-10, 10-13,
13-15, 15-17, 17-19 and 19-20.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined (weight, fixation, histopathology): thyroid glands (all dams) (see also Table 1 in section "any other information on materials and methods incl. tables")

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
- Other: live fetuses, dead fetuses

Blood sampling:

- Blood: Yes
- Serum: Yes
- Volume collected: 1 mL

Fetal examinations:

- Weight of each fetus: Yes
- Sex: Yes
- Gross evaluation of placentae: Yes
- External examinations: Yes: all per litter
- Soft tissue examinations (fixation with Harrison's fluid): Yes: half of the fetuses
- Skeletal examinations (fixation in ethanol): Yes: half of the fetuses
- Anogenital distance (AGD): Yes: all liveborn fetuses

Statistics:

Please refer to section "any other information on materials and methods incl. tables"

Indices:

Conception rate % = no. of pregnant animals/ number of fertilized animals x 100

Pre-implantation loss % = no. ofcorpora lutea − no. of implantations/ no. of corpora lutea x 100

Post-implantation loss % = no. of implantations−no. of live foetuses/ no. of implantations x 100

Anogenital Index = anogenital distance (mm)/ cubic root of fetal weight (g)

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

- The mean body weights of test group 1-3 dams (100, 300 and 1000 mg/kg bw/d) were generally comparable to the concurrent control group throughout the entire study period.

- The body weight change of the dams in test groups 2 and 3 (300 and 1000 mg/kg bw/d) were
statistically significantly reduced during GD 6-8 (up to 32% and 42% below control) but recovered
afterwards, respectively, and was comparable to the concurrent control at the end of the treatment period. If calculated for the entire treatment period (GD 6-19), the mid- and highdose dams, each, gained 4% less body weight than the control (without attaining statistical significance). The impairments of body weights in the mid- and high-dose groups are considered to be treatment-related but not adverse. The mean body weights and the average body weight gain of the low-dose dams (100 mg/kg bw/d) were generally comparable to the concurrent control group throughout the entire study period.
- The corrected body weight gain of test groups 1, 2 and 3 (100, 300 and 1000 mg/kg bw/d) revealed no difference of any biological relevance to the corresponding control group. Moreover, mean carcass weights of all test groups remained unaffected by the treatment.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

- No necropsy findings which could be attributed to the test substance were seen in any dam. There occurred two spontaneous findings: a dilated renal pelvis in low-dose female No. 39 (both kidneys), in high-dose females No. 82 (both kidneys) and No. 84 (right kidney). Additionally, a cyst in the medulla of the left kidney in mid-dose female No. 54 was observed. These findings were detected in single females of different test groups and, therefore, were assessed as incidental.
- No macroscopic findings were noted in thyroid glands of control and all treatment groups.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

A 100% post-implantation loss was observed in one female of test group 3 (1000 mg/kg bw/d).

Early or late resorptions:

effects observed, non-treatment-related

Description (incidence and severity):

In high-dose female No. 87 only 10 early resorptions were recorded which were visible without staining (no viable fetuses). Since only one dam was affected, this isolated finding was
regarded as incidental and not treatment-related.

Dead fetuses:

no effects observed

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: Maternal toxicity

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

effects observed, non-treatment-related

Description (incidence and severity):

In test group 2, the mean% value of live fetuses (mean 89.8* [p ≤ 0.05 Dunnett-test]) was statistically significantly decreased. The mean number of live fetuses (10.8) in this test group was well within the historical control range (HCD: mean 10.6 [10.1 - 11.2]). The mean number of resorptions was very low in the concurrent control group (0.4), therefore, the mean% value of live fetuses (96.3) was exceptionally high. Furthermore, there was no dose-response relationship visible (mean% live fetuses: 96.3 / 92.5 / 89.8* / 91.4). Therefore, the statistically significantly decrease in test group 2 is assessed as incidental and biologically not relevant.

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

not specified

Anogenital distance of all rodent fetuses:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Skeletal malformations were detected in one fetus, each, of the control and the low-dose group (Table 6). All findings are assessed as not treatment-related since they occurred in single fetuses without a relation to dose.
The total incidences of skeletal malformations (Table 7) did not differ significantly from control and were comparable to the historical control data.

Details on Table 6 and 7 can be found in section "Any other information on results incl. tables".

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Three soft tissue variations were detected: short innominate in test groups 0 and 1, dilated renal pelvis in all test groups and dilated ureter in test group 3. The incidences of these variations did not differ significantly from the concurrent control group. All of them can be found in the historical control data at comparable incidences. Therefore, they are assessed as not treatment-related. The incidence of the finding ‘dilated ureter’ in two fetuses of two litters of test group 3 (affected fetuses/litter: 1.4%) was in the range of the historical control data (HCD: mean% 2.4 [0.8 - 4.0]) and, therefore, considered not biologically relevant.
For more details please refere to Table 5 in section "Any other information on results incl. tables".

Other effects:

effects observed, treatment-related

Description (incidence and severity):

FETAL SKELETAL VARIATIONS: EFFECTS OBSERVED, NOT TREATMENT-RELATED
For all test groups, skeletal variations of different bone structures were observed, with or without effects on corresponding cartilages. The observed skeletal variations were related to several parts of fetal skeletons and appeared in the majority of cases without a relation to dose (Table 8). The overall incidences of skeletal variations were comparable to the historical control data.
All skeletal variations with statistically significant differences between the control and any treated group were compiled in the table below (Table 9). All incidences were expressed on a fetus per litter basis and any statistically significant differences, which were outside the historical control range were marked in italicized bold types. Beside the treatment-related effect regarding the affected fetuses/litter rate of ‘unilateral ossification of sternebra (unchanged cartilage), the other increased incidences of skeletal variations were either not related to dose or they were clearly inside the historical control ranges. Therefore, these findings are assessed as not treatment-related.

FETAL SKELETAL VARIATIONS: EFFECTS OBSERVED, TREATMENT-RELATED
As can be seen from table 9, the affected fetuses/litter rate of ‘unilateral ossification of sternebra (unchanged cartilage)’ was statistically significantly increased in test group 3 (1000 mg/kg bw/d). The value was outside the historical control range and therefore a treatment-related effect cannot be ruled out.

FETAL SKELETAL UNCLASSIFIED CARTILAGE OBSERVATIONS: EFFECTS OBSERVED, NOT TREATMENT-RELATED
Additionally, some isolated cartilage findings without impact on the respective bony structures, which were designated as unclassified cartilage observations, occurred in all test groups (Tab. 10). The observed unclassified cartilage findings were related to the skull, the ribs and the sternum and did not show any relation to dosing.

For more details on Table 8, 9, 10 refer to section "Any other information on results incl. tables".

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: developmental toxicity

Abnormalities:

no effects observed

Developmental effects observed:

no

Table 5 Total soft tissue variations

		Test group 0 0mg/kg bw/day	Test group 1 100mg/kg bw/day	Test group 2 300mg/kg bw/day	Test group 3 1000mg/kg bw/day
Litter Fetuses	N N	25 126	25 127	25 130	24 126
Fetal incidence	N(%)	2(2.4)	3(2.4)	4(3.1)	5(4.0)
Litter incidence	N(%)	2(8.0)	3(12)	3(12)	3(13)
Affected fetuses/litter	Mean%	2.4	2.6	3.7	3.5

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Table 6 Individual fetal skeletal malformations

Test group	Dam No.-Fetus No., Sex	Finding
0 (0 mg/kg bw/d)	19-01 M	cleft sternum
1 (100 mg/kg bw/d)	41-07 M	malpositioned and bipartite sternebra
2 (300 mg/kg bw/d)	none
3 (1000 mg/kg bw/d)	none

mg/kg bw/d = milligram per kilogram body weight per day; No. = number; M = male; F = female

Table 7 Total skeletal malformations

		Test group 0 0mg/kg bw/day	Test group 1 100mg/kg bw/day	Test group 2 300mg/kg bw/day	Test group 3 1000mg/kg bw/day
Litter Fetuses	N N	25 137	25 142	25 140	24 136
Fetal incidence	N(%)	1(0.7)	1(0.7)	0.0	0.0
Litter incidence	N(%)	1(4.0)	1(4.0)	0.0	0.0
Affected fetuses/litter	Mean%	0.8	0.7	0.0	0.0

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Table 8 Total fetal skeletal variations

		Test group 0 0mg/kg bw/day	Test group 1 100mg/kg bw/day	Test group 2 300mg/kg bw/day	Test group 3 1000mg/kg bw/day
Litter Fetuses	N N	25 137	25 142	25 140	24 136
Fetal incidence	N(%)	129 (94)	138 (97)	137 (98)	132 (97)
Litter incidence	N(%)	25 (100)	25 (100)	25 (100)	24 (100)
Affected fetuses/litter	Mean%	94.9	97.1	97.2	96.6

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Table 9 Occurence of statistically significantly increased fetal skeletal variations (expressed as mean percentage of affected fetuses/litter)

Finding	Test group 0 0mg/kg bw/day	Test group 1 100mg/kg bw/day	Test group 2 300mg/kg bw/day	Test group 3 1000mg/kg bw/day	HCD Mean % (range)
Incomplete ossification of interparietal; unchanged cartilage	13.8	27.6**	15.1	24.7*	18.0 (11.8-26.6)
Dumbbell ossification of thoracic centrum; unchanged cartilage	0.8	1.4	3.7*	3.1	1.9 (0.0-5.5)
Bipartite ossification of thoracic centrum; dumbbell-shaped cartilage of centrum	0.0	2.2*	0.0	0.0	0.1 (0.0-1.3)
Incomplete ossification of sternebra; unchanged cartilage	74.6	82.5	80.9*	75.5	77.1 (67.6-84.2)
Unilateral ossification of sternebra; unchanged cartilage	0.0	0.0	0.0	3.1*	0.8 (0.0-2.2)

mg/kg bw/d = milligram per kilogram body weight per day; HCD = Historical control data; % = per cent
* = p =< 0.05 (Wilcoxon-test [one-sided])
** = p =< 0.01 (Wilcoxon-test [one-sided])

Table 10 Total unclassified cartilage observations

		Test group 0 0mg/kg bw/day	Test group 1 100mg/kg bw/day	Test group 2 300mg/kg bw/day	Test group 3 1000mg/kg bw/day
Litter Fetuses	N N	25 137	25 142	25 140	24 136
Fetal incidence	N(%)	94 (69)	91 (64)	106 (76)	92 (68)
Litter incidence	N(%)	24 (96)	24 (96)	25 (100)	24 (100)
Affected fetuses/litter	Mean%	67.9	63.9	73.0	67.3

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Conclusions:

The no observed adverse effect level (NOAEL) for maternal and prenatal developmental toxicity is the highest tested dose of 1000 mg/kg bw/d.

Executive summary:

The test substance was tested for its prenatal developmental toxicity in Wistar rats. The test substance was administered as an aqueous preparation to groups of 25 time-mated female Wistar rats by gavage at dose levels of 100, 300 and 1000 mg/kg body weight/day (mg/kg bw/d) on gestation days (GD) 6 through 19. The control group, consisting of 25 females, was dosed with the vehicle (0.5% Sodium carboxymethyl cellulose [CMC] suspension in deionized water) in parallel. A standard dose volume of 10 mL/kg body weight was used for each test group. At terminal sacrifice on GD 20, 25 females per group had implantation sites.

The following test substance-related adverse effects/findings were noted:
Test group 3 (1000 mg/kg bw/d):
• No test substance-related adverse effects on dams, gestational parameters or fetuses
Test group 2 (300 mg/kg bw/d):
• No test substance-related adverse effects on dams, gestational parameters or fetuses
Test group 1 (100 mg/kg bw/d):
• No test substance-related adverse effects on dams, gestational parameters or fetuses

Effect on developmental toxicity: 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 developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Toxicity to reproduction: other studies

Additional information

In a GLP-compliant prenatal developmental toxicity study (OECD 414), the test item (in 0.5% CMC) was administered as an aqueous preparation to groups of 25 time-mated female Wistar rats by gavage at dose levels of 100, 300 and 1000 mg/kg body weight/day (mg/kg bw/d) on gestation days (GD) 6 through 19. The control group, consisting of 25 females, was dosed with the vehicle (0.5% Sodium carboxymethyl cellulose [CMC] suspension in deionized water) in parallel. A standard dose volume of 10 mL/kg body weight was used for each test group. At terminal sacrifice on GD 20, 25 females per group had implantation sites.

No test substance-related adverse effects on dams, gestational parameters or fetuses occured in any dose tested.

Short description of key information:

A Prenatal Developmental Toxicity Test (OECD guideline 414) revealed no potential for maternal and developmental toxicity up to the highest dose level tested (1000 mg/kg bw).

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for the purpose of classification under Regulation (EC) No.1272/2008. Based on the data, classification for reproductive toxicity is not warranted under Regulation (EC) No.1272/2008.