O,O,O-triphenyl phosphorothioate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

In an OECD 421 study, no effects on reproduction performance have been noted up to 1000 mg/kg body weight. The no observed adverse effect level (NOAEL) for general systemic toxicity was 1000 mg/kg bw/d in females and 300 mg/kg bw/d in males based on the adverse changes in the thyroid glands. The NOAEL for developmental toxicity in the offspring was 1000 mg/kg bw/d in females and 300 mg/kg bw/d in males due to retained areolae/nipples.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15.11.2016-9.11.2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Qualifier:

according to guideline

Guideline:

other: EPA, Health Effects Test Guidelines; OPPTS 870.3550: Reproduction/Developmental Toxicity Screening Test (Jul 2000)

GLP compliance:

yes (incl. QA statement)

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories
- Females nulliparous and non-pregnant: yes
- Age at study initiation: male: 14-16 weeks, females: 12 weeks
- Average weight at study initiation: Males: 348.7 g - 415.7 g; Females: 186.6 g - 224.0 g
- Housing: During the study period, the rats were housed individually in Polycarbonate cages type III supplied by TECNIPLAST, Hohenpeißenberg, Germany and Becker & Co., Castrop-Rauxel, Germany, with the following exceptions: 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 13 in Polycarbonate cages type III.
- Diet: ground Kliba maintenance diet mouse/rat “GLP” meal, ad libitum
- Water: supplied from water bottles, ad libitum
- Acclimation period: 3 weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24°C
- Humidity: 30-70%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5% Sodium carboxymethyl cellulose suspension in drinking water

Details on exposure:

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

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: maximum of 14 days
- Proof of pregnancy: vaginal sperm in vaginal smear referred to as day 0 of pregnancy

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 0.5% Sodium carboxymethyl cellulose suspension in drinking water over a period of 7 days 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 twice during the study period for verification of the concentration (reserve samples collected at the beginning of the administration period and further samples collected during gestation). The samples which were taken for the concentration control analysis were also used to verify the homogeneity of the samples of the low- and high-concentration. From these concentrations 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.

Duration of treatment / exposure:

The treatment lasted up to one day prior to sacrifice. The male and female animals were sacrificed 30 and 58 days, respectively, after the beginning of the administration, and examined.

Frequency of treatment:

daily

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Fasting period before blood sampling for clinical biochemistry: yes

Positive control:

not included

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
A check for moribund or dead animals was made twice daily on working days or once daily (Saturday, Sunday or on public holidays).

DETAILED CLINICAL OBSERVATIONS: Yes
A cageside examination was conducted at least daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity before the administration as well as within 2 hours and within 5 hours after the administration. Abnormalities and changes were documented daily for each animal. Individual data of daily observations can be found in the raw data. The parturition and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams. Only particular findings (e.g. inability to deliver) were documented on an individual dam basis. On weekdays (except Saturday, Sunday and public holidays) the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings. The day of parturition was considered the 24-hour period from about 15.00 h of one day until about 15.00 h of the following day.

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 body weight change of the animals was calculated from these results. 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), 4, 7, 10 and 13.
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; therefore, these values are not reported in the Summary but in the Individual Tables.

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, 4-7, 7-10 and 10-13.
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.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / No data
- Time schedule for examinations:

OTHER:
Blood samples from all dams at PND 14 and all males at termination were taken by puncturing the retrobulbar venous plexus under isoflurane anesthesia. The adults were fastened before the blood sampling. Blood samples were assessed for serum levels for thyroid hormones (T4 and TSH). The concentrations of TSH were determined by radioimmunoassay (RIA), using commercially available RIA test kits and a Gamma-Counter (LB 2111, Berthold, Germany). T4 Elisa was measured with a Sunrise MTP-reader, Tecan AG, Maennedorf, Switzerland, and evaluated with the Magellan-Software of the instrument producer.

Oestrous cyclicity (parental animals):

For all females of the pool estrous cycle normality was evaluated before the beginning of the administration period. Estrous cycle length was evaluated by daily analysis of vaginal smear for all F0 female parental
rats for a minimum of 2 weeks prior to mating. Determination was continued throughout the pairing period until the female exhibited evidence of copulation. At necropsy, an additional vaginal smear was examined to determine the stage of estrous cycle for each F0 female with scheduled sacrifice.

Litter observations:

STANDARDISATION OF LITTERS
On PND 4, as a result of standardization, the surplus pups were sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations. All pups were examined externally and eviscerated; their organs were assessed macroscopically.
On PND 13, one selected male and one female pup per litter was sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations. Thyroid glands/parathyroid glands were fixed in neutral buffered 4% formaldehyde solution and were transferred to the Pathology Laboratory for possible further processing. All pups were examined externally and eviscerated, and their organs were assessed macroscopically.

PARAMETERS EXAMINED
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.
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. Dead pups were evaluated. The number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PND 1-4, 5-7 and 8-13 were determined. Pups which died accidentally or had to be sacrificed due to maternal death were not included in these calculations. The number of live pups/litter was calculated on the day of birth (PND 0), and on lactation days 4, 7 and 13. Furthermore, viability and survival indices was calculated according to the following formulas
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. Later, during the course of lactation, this initial sex determination was followed up by surveying the external appearance of the anogenital region and the mammary line. The sex of the pups was finally confirmed at necropsy. The sex ratio was calculated at day 0 and day 13 after birth.
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 (before standardization), 7 and 13. Pups' body weight change was calculated from these results. The individual weights were always determined at about the same time of the day (in the morning) and on PND 4 immediately before standardization of the litters. In the summary tables pup body weights and pup body weight gains are listed for males, females and males + females. “Runts” were defined on the basis of the body weights on PND 1. "Runts" are pups that weigh less than 75% of the mean weight of the respective control pups.
Anogenital distance (AGD) was determined in all live male and female pups on PND 1. These measurements were performed in randomized order, using a measuring ocular. They were conducted by technicians unaware of treatment group in order to minimize bias.
All surviving male pups were examined for the presence of nipple/areola anlagen on PND 13. The number of nipple/areola anlagen was counted.
Blood samples were taken from all surplus pups per litter at PND 4 as well as one male and one female pup per litter at PND 13 by decapitation under isoflurane anesthesia. Blood samples the PND 13 pups were assessed for serum levels for thyroid hormones (T4 and TSH). The concentrations of TSH were determined by radioimmunoassay (RIA), using commercially available RIA test kits and a Gamma-Counter (LB 2111, Berthold, Germany). T4 Elisa was measured with a Sunrise MTP-reader, Tecan AG, Maennedorf, Switzerland, and evaluated with the Magellan-Software of the instrument producer.

Postmortem examinations (parental animals):

GROSS NECROPSY
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.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following weights were determined in all animals sacrificed on schedule:
Anesthetized animals, Epididymides, Ovaries, Prostate, Seminal vesicles with coagulating glands, Testes, Thyroid glands, Uterus (with cervix),
The following weights were determined in 5 animals per sex/test group sacrificed on schedule (females with litters only, same animals as used for clinical pathological examinations): Adrenal glands, Brain, Heart, Kidneys, Liver, Spleen, Thymus

The following organs or tissues of all parental animals were fixed in 4% neutral-buffered formaldehyde solution or in modified Davidson’s solution: All gross lesions, Adrenal glands, Aorta, Bone marrow (femur), Brain, Cecum, Cervix, Coagulating glands, Colon, Duodenum, Eyes with optic nerve, Esophagus, Extraorbital lacrimal glands, Epididymides (modified Davidson’s solution), Femur with knee joint, Heart, Ileum, Jejunum (with Peyer’s patches), Kidneys, Larynx, Liver, Lungs, Lymph nodes (axillary and mesenteric), Mammary gland (male and female), Nose (nasal cavity), Ovaries (modified Davidson’s solution), Oviducts, Pancreas, Parathyroid glands, Pharynx, Pituitary gland, Prostate gland, Rectum, Salivary glands (mandibular and sublingual), Sciatic nerve, Seminal vesicles, Skeletal muscle, Spinal cord (cervical, thoracic and lumbar cord), Spleen, Sternum with marrow, Stomach (forestomach and glandular stomach), Testes (modified Davidson’s solution), Thymus, Thyroid glands, Trachea, Urinary bladder, Uterus (uteri of all apparently nonpregnant animals or empty uterus horns were stained according to Salewski E, 1964), Vagina

The testes and epididymides were histotechnical processed and examination by light microscopy in all control and high dose animals and in low and mid dose mating pairs suspected of reduced fertility. Ovaries, Liver and Thyroid glands were examined in all animals of all dose groups.

Postmortem examinations (offspring):

GROSS NECROPSY
On PND 4, as a result of standardization, the surplus pups were sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations. After sacrifice, the pups were examined externally and eviscerated, and the organs were assessed macroscopically. On PND 13, one selected male and one female pup per litter was sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations. Thyroid glands/parathyroid glands were fixed in neutral buffered 4% formaldehyde solution and were transferred to the Pathology Laboratory for possible further processing. The remaining pups were sacrificed under isoflurane by decapitation. After sacrifice, all pups were examined externally and eviscerated, and their organs were assessed macroscopically. All stillborn pups and all pups that died before weaning were examined externally, eviscerated and 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 noted.

HISTOPATHOLOGY / ORGAN WEIGTHS
On PND 13, one selected male and one female pup per litter were sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations. Thyroid glands/parathyroid glands were fixed in neutral buffered 4% formaldehyde solution and were transferred to the Pathology Laboratory for possible further processing (see chapter “Histopathology”).

Statistics:

- Simultaneous comparison of all dose groups with the control group using the DUNNETT-test (twosided) for the hypothesis of equal means: 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, anogenital distance, anogenital index.
- Pair-wise comparison of each dose group with the control group using FISHER'S EXACT test (one-sided) for the hypothesis of equal proportions: 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.
- Pair-wise comparison of the dose group with the control group using the WILCOXON test (one-sided+) with BONFERRONI-HOLM adjustment for the hypothesis of equal medians: Mating days until day 0 pc, %postimplantation loss, pups stillborn, %perinatal loss, nipple development.
- Pair-wise comparison of the dose group with the control group using the WILCOXON test (one-sided-) with BONFERRONI-HOLM adjustment for the hypothesis of equal medians: Implantation sites, pups delivered, pups liveborn, live pups day x, viability index, survival index.
- Comparison of the dose group with the control group was performed using the WILCOXON test (twosided) for the hypothesis of equal medians: % live male day x, %live female day x
- Non-parametric oneway analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (twosided) for the equal medians: Number of cycles and cycle length, Blood parameters, Weight parameters

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, Survival index

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

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 animals at dose levels of 100 and 300 mg/kg bw/d during premating, gestation, lactation and post-mating periods. During lactation period, one high-dose female (No. 138 - 1000 mg/kg bw/d) showed salivation after treatment (within the 2-hour examination interval, PND 17-21). During the 5-hour examination interval (i.e. >2h<5h after treatment), no clinical signs or changes of general behavior were detected in this female. Therefore, this was assessed as spontaneous and not treatment related. No further clinical signs or findings were detected in any male or female F0 generation parental animals at dose levels of 1000 mg/kg bw/d during the different study periods. One sperm-positive mid-dose female (No. 123 - 300 mg/kg bw/d) did not deliver F1 pups.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were no test substance-related or spontaneous mortalities in any of the groups (0, 100, 300 or 1000 mg/kg bw/d).

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The mean body weights and mean body weight changes of all test substance treated F0 parental male and female animals (test groups 1-3) during the entire study period were comparable to the concurrent control values. The statistically significantly decreased mean body weight of the low-dose females (100 mg/kg bw/d) during GD 7 and 14 (up to 5% below control), as well as the significantly decreased mean
body weight change values of the low- and mid-dose females (100 and 300 mg/kg bw/d) during GD 0-7 (approx. 32% and 19% below control) were considered as spontaneous in nature and not treatment-related as no dose-response relationship occurred.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Food consumption of the test substance-treated F0 males (test groups 1-3) was generally comparable to the respective control group during the entire treatment period. Food consumption of the F0 females in test group 2 and 3 (300 or 1000 mg/kg bw/d) was generally comparable to the respective control group during the entire treatment period. Food consumption of the F0 females in test group 1 (100 mg/kg bw/d) was statistically significantly decreased during gestation days 7-20 (up to 9% below control) and, as a consequence, during gestation days 0-20 (approx. 8% below control). During the other study periods (premating and lactation periods) the food consumption of the low-dose females was comparable to the concurrent control group. Thus, this sporadic event during gestation was considered as incidental and not treatment-related, as there was no dose-response relationship visible.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

After four-weeks of administration in F0 males thyroid stimulating hormone (TSH) levels were significantly increased and T4 levels were decreased although being not statistically significant. The TSH mean was above and that of T4 below the historical control range (males, TSH 5.07- 8.01 μg/L, T4 44.87-88.29 nmol/L). The corresponding hormone values in F0 females were not changed.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

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):

Treatment-related findings were observed in the liver and thyroid glands of males and females with incidences and grading according to the table below.

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

Estrous cycle data, generated during the last 2 weeks prior to mating for the F1 litter, revealed regular cycles in the females of test groups 0-3. The mean estrous cycle duration was statistically significantly decreased in test group 2: 4.00 / 4.47 / 3.78* / 3.97 days (p ≤ 0.05) in test groups 0-3, respectively. Although the mean value of test group 2 animals was outside the historical control data (cycles length: range of 3.99 – 4.46 days), there was no dose-response-relationship and, therefore, the finding was not related to treatment.

Reproductive performance:

no effects observed

Description (incidence and severity):

For all F0 parental males, which were placed with females to generate F1 pups, copulation was confirmed. Thus, the male mating index was 100.0% in test groups 0-3. Fertility was proven for most of the F0 parental males within the scheduled mating interval for F1 litter. One mid-dose male (No. 23 - 300 mg/kg bw/d) did not generate pregnancy. Thus, the male fertility index ranged between 90.0% and 100.0% 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 rat did not show relevant gross lesions.

The female mating index calculated after the mating period for F1 litter was 100.0% in all test groups. The mean duration until sperm was detected (GD 0) varied between 1.5 and 2.1 days without any relation to dosing. All female rats delivered pups or had implants in utero with the following exception:
• Mid-dose female No. 123 (mated with male No. 23) did not become pregnant.
The fertility index varied between 100.0% in test groups 0, 1 and 3 and 90.0% in test group 2. These values reflect the normal range of biological variation inherent in the strain of rats used for this study. The non-pregnant female had no relevant gross lesions. The mean duration of gestation was similar in all test groups (i.e. between 21.9 and 22.4 days). The gestation index was 100.0% in all test groups. 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 (11.6 / 12.0 / 12.7 and 13.1 implants/dam in test groups 0-3, respectively). Furthermore, there were no indications for test substance-induced intrauterine embryo-/fetolethality since the postimplantation loss did not show any significant differences between the groups, and the mean number of F1 pups delivered per dam remained unaffected (10.7 / 11.8 / 12.3 and 12.7 pups/dam in test groups 0-3, respectively). On average, test group 2 and 3 dams delivered 2 pups/litter more compared to control dams. The rate of liveborn pups was also not affected by the test substance, as indicated by live birth indices of 95.3% / 100.0% / 100.0% and 100.0% in test groups 0-3. Moreover, the number of stillborn pups was not significantly different between the test groups. Thus, the test substance did not adversely affect reproduction and delivery of the F0 generation parental females.

Dose descriptor:

NOAEL

Remarks:

fertility and reproductive performance

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effect

Dose descriptor:

NOAEL

Remarks:

general systemic toxicity

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

clinical biochemistry

histopathology: non-neoplastic

Dose descriptor:

NOAEL

Remarks:

general systemic toxicity

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no adverse effects observed

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

immune system

Organ:

thyroid gland

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Clinical signs:

no effects observed

Description (incidence and severity):

There were no test substance-related adverse clinical signs observed in any of the F1 generation pups of the different test groups

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

The mean number of delivered F1 pups per dam and the rates of liveborn 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.
- Pup viability: The viability index indicating pup mortality during early lactation (PND 0-4) varied between 99.1% (test group 2), 99.3% (test groups 1 and 3) and 100.0% (control) without relation to dosing. The survival index indicating pup mortality during PND 4-13 varied between 100.0% / 97.5% / 100.0% / 100.0% in test groups 0-3, respectively.
- Pup mortality: The pup mortality shown in Figure 4.2.2.1.3.1 is based on total number of stillborn pups, dead pups, pups sacrificed moribund and cannibalized pups.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean body weights of the test substance-treated F1 male and female pups (and both sexes combined) were below the concurrent control group during the entire lactation period, attaining statistical significance on different time points:
On PND 1, the mean body weights of the high-dose male pups (1000 mg/kg bw/d) were statistically significantly decreased (about 9% below control).
On PND 4, a statistically significant decrease in body weight was also observable in male pups and both sexes combined in test groups 1-3 (100, 300 or 1000 mg/kg bw/d - up to 13% below control), in female pups in test groups 2 and 3 (about 12% below control).
All values were within the historical control range (PND 1: male: 5.4 – 7.4 g; PND 4: male: 8.1 – 11.8 g, female: 8.0 – 11.2 g, both sexes: 8.1 – 11.4 g).
The following statistically significant decrease of pup body weights was observed on the further days:
• On PND 7, the mean body weight of the female and male pups and both sexes combined in test groups 1 and 3 (up to 10% below control)
• On PND 13, the mean body weights of the male F1 pups in test group 3, in female pups and both sexes combined in test groups 1 and 3 (up to 8% below control, respectively).
The mean body weight change of all test substance-treated male and female F1 pups (and both sexes combined) was statistically significantly decreased on PND 1-4 (about 18% below control, both sexes combined), furthermore, in the low- and high-dose female pups and in both sexes combined, on PND 1-13 (about 8% below control).

In general, control dams had a smaller litter size compared to the test groups 1-3 dams. Consequently, control pups exhibited higher body weights based on the low pup numbers compared to the test group pups. The mean numbers of F1 pups delivered were 10.7 / 11.8 / 12.3 and 12.7 pups/dam in test groups 0-3, respectively. On average, test groups 2 and 3 dams delivered 2 pups/litter more compared to control dams. For the test groups, dams had to take care of a higher number of pups and, therefore, the individual weight of the pups were lower compared to control.
Overall, the decrease in pup body weight in the respective test groups was due to differences in litter size. In cases where historical control data are available, all values were within the range of historical control data. Furthermore, the decrease in pup body weights on PND 7 and 13 was not dose-dependent since body weights of test group 2 pups were not altered. Therefore, the decrease in pup body weights of test groups 1-3 were not considered as treatment-related and adverse.
One male runt was seen in the control, one female runt was seen in test group 3, one male and one female runt was seen in test groups 1 and 2. The findings were not assessed as treatment-related since they were observed only in single pups or were not related to dose.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

In F1 PND13 female pups of test group 13 (1000 mg/kg bw/d), T4 values were significantly decreased with no significant changes of TSH. However, the T4 mean was within the historical control range (females, PND13 T4 41.14-75.73 nmol/L). No hormone changes were observed in PND13 males.

Anogenital distance (AGD):

effects observed, non-treatment-related

Description (incidence and severity):

Neither on anogenital distance nor anogenital index test substance-related effects were noted in all treated F1 offspring (100, 300 and 1000 mg/kg bw/d). The statistically significantly decreased anogenital distance of male pups in test group 1 (100 mg/kg bw/d) is considered as spontaneous in nature due to the lack of dose-response relationship.

Nipple retention in male pups:

effects observed, treatment-related

Description (incidence and severity):

The percentage of male pups having nipple / areolae was statistically significantly increased in test group 3 (1000 mg/kg bw/d) when examined on PND 13 (44.7% / 67.3% / 78.7% / 90.0%*
[p ≤ 0.05] in test groups 0-3). The mean value was outside the historical control range (males with areolae/nipples: range of 0.0 – 81.7 %). The mean number of nipple / areolae in the respective male pups was also statistically significantly increased in test group 3 (1.2 / 1.8 / 2.1 / 2.5* [p ≤ 0.05] in test group 0-3). The mean value was on the border of the historical control data (nipple number, range of 0.0 – 2.5). Due to the dose-related increase of male pups having nipples/areolae and the statistically significant increase in both parameters (mean number of nipple / areolae and percentage of affected males), a relation to treatment cannot be excluded. Therefore, the findings concerning nipple development in males of test group 3 were assumed to be treatment-related.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

A few F1 pups showed spontaneous findings at gross necropsy, such as partly cannibalized, small testis and dilated renal pelvis. These findings occurred without any relation to dosing and/or some of them can be found in the historical control data at comparable incidences. None of the findings was considered to be related to the treatment.

Description (incidence and severity):

The sex distribution and sex ratios of live F1 pups on the day of birth and PND 13 did not show substantial differences between the control and the test substance-treated groups. The statistically significantly increased number of male pups in test group 3 and decreased number of female pups on PND 0 was considered to be spontaneous in nature. The mean values of the males and females were within the range of the historical control data (live males: 36.6 – 60.5 %; live females: 39.5 – 63.4 %). The concurrent control group showed, however, a rather low number of male pups which may have led to this statistical significance without having any biological relevance. Therefore, the finding was not assessed as treatment-related.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: development, retained areolae/nipples

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no adverse effect

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

endocrine system

Organ:

thyroid gland

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Reproductive effects observed:

no

Absolute and relative organ weights

Absolute organ weights

When compared to control group 0 (set to 100%), the mean absolute weights of following organs were significantly increased (statistically significant changes printed in bold):

Absolute weights	Males			Females
Test group (mg/kg bw/day)	1 (100)	2 (300)	3 (1000)	1 (100)	2 (300)	3 (1000)
Liver	107%	104%	119%**	94%	103%	117%**
Ovaries				96%	114%*	117%**
Heart				88%**	99%	98%

All other mean absolute weight parameters did not show significant differences when compared to the control group 0.

Relative organ weights

When compared to control group 0 (set to 100%), the mean relative weights of following organs were significantly increased (statistically significant changes printed in bold):

Relative weights	Males			Females
Test group (mg/kg bw/day)	1 (100)	2 (300)	3 (1000)	1 (100)	2 (300)	3 (1000)
Liver	105%*	104%	121%**	100%	107%	119%**
Ovaries				101%	117%*	120%**
Thyroid gland				96%	108%	123%*

The significant absolute and relative liver weight increase in males and females of test group 3 was regarded as treatment-related and correlated with centrilobular hepatocellular hypertrophy.

The significant absolute weight increase of the ovaries in females of test groups 2 and 3 (118.3 and 121.5 mg, respectively) was within the historical control range (101.2 – 125.0 mg). However, the relative weight increase in test group 3 (0.054 %) was minimally above the historical control range (0.042 – 0.052%), whereas in test group 2 the relative weight increase was weak significant and was within the historical range (0.052 %). Since no histopathological correlate was found in the ovaries to explain these weight deviations, the significant weight increase in test groups 2 and 3 was assumed to be incidental.

The significant relative weight increase of the thyroid glands (0.01%) in females of test group 3 was above the historical control range (0.007 – 0.009%) and correlated with histopathological findings.

The significant relative weight increase of the liver in test group 1 males and the significant absolute weight decrease of the heart in test group 1 females occurred both incidentally and were not considered treatment-related. All other mean relative weight parameters did not show significant differences when compared to the control group 0.

Histopathology

Treatment-related findings were observed in the liver and thyroid glands of males and females with incidences and grading according to the table below:

LIVER

	Male animals				Female animals
Test group (mg/kg bw/day)	0 (0)	1 (100)	2 (300)	3 (1000)	0 (0)	1 (100)	2 (300)	3 (1000)
No. of animals	10	10	10	10	10	10	10	10
Liver
Hypertrophy, centrilobular	0	0	2	8	0	0	1	10
·    Grade 1			2	7			1	7
·    Grade 2				1				3

The centrilobular hepatocellular hypertrophy in males and females of test group 2 and 3 was considered treatment-related but not adverse.

THYROID GLAND

	Male animals				Female animals
Test group (mg/kg bw/day)	0 (0)	1 (100)	2 (300)	3 (1000)	0 (0)	1 (100)	2 (300)	3 (1000)
No. of animals	10	10	10	10	10	10	10	10
Thyroid gland
Hypertrophy / hyperplasia, follicular	2	3	8	8	0	2	1	4
·    Grade 1	2	3	4	3		2	1	4
·    Grade 2			4	5
Altered colloid	3	4	6	5	2	3	2	4
·    Grade 1	3	3	2	2	1	2	2	3
·    Grade 2		1	4	3	1	1		1

The hypertrophy/hyperplasia of follicular cells and altered colloid in males of test group 2 and 3 was considered treatment-related although no dose-dependent relationship in incidence and/or grading occurred between both groups. In females of test group 3, a minimal increase in the incidence of the hypertrophy/hyperplasia of follicular cells and the altered colloid was assumed to be treatment-related.

All other 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.

Thyroid Hormones

Males:

		Test Group 0/ M 0 mg/kg bw/d	Test Group 1/ M 100 mg/kg bw/d	Test Group 2/ M 300 mg/kg bw/d	Test Group 3/ M 1000 mg/kg bw/d
T4 [nmol/L] day 30	Mean	50.10 k	43.80	45.03	40.92
	S.d.	8.22	5.78	7.54	5.36
	N	10	10	10	10
	Median	47.21	44.11	46.96	39.91
	Deviation Vs Control		-12.58	-10.12	-18.31
TSH [µg/L] day 30	Mean	8.87 v	8.69	13.79	12.44 *
	S.d.	2.50	2.19	7.43	3.36
	N	10	10	10	10
	Median	8.91	8.57	11.41	12.30
	Deviation Vs Control		-1.96	55.46	40.26

Statistic Profile = Kruskal-Wallis + Wilcoxon test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics k=KRUSKAL-WALLIS

Females:

		Test Group 0/ F 0 mg/kg bw/d	Test Group 1/ F 100 mg/kg bw/d	Test Group 2/ F 300 mg/kg bw/d	Test Group 3/ F 1000 mg/kg bw/d
T4 [nmol/L] day 49	Mean	50.81 k	48.11	51.23	49.22
	S.d.	7.79	5.18	6.41	9.63
	N	10	10	9	10
	Median	48.83	48.7	49.44	47.60
	Deviation Vs Control		-5.32	0.83	-3.13
TSH [µg/L] day 49	Mean	6.92 k	7.39	6.96	7.75
	S.d.	2.44	2.84	3.01	1.70
	N	10	10	9	10
	Median	6.46	7.04	6.46	7.63
	Deviation Vs Control		6.78	0.61	12.07

Statistic Profile = Kruskal-Wallis + Wilcoxon test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics k=KRUSKAL-WALLIS

Male Pups

		G 10 / M	G 11 / M	G 12 / M	G 13 / M
T4 [nmol/L] day 13	Mean	70.67 k	62.87	66.72	60.25
	S.d.	10.79	8.04	7.22	9.27
	N	10	10	9	10
	Median	70.11	63.64	66.96	59.42
	Deviation Vs Control		-11.04	-5.59	-14.75
TSH [µg/L] day 13	Mean	3.80 k	3.90	4.36	4.45
	S.d.	0.86	0.87	1.07	1.06
	N	10	10	9	10
	Median	3.76	3.98	4.02	4.28
	Deviation Vs Control		2.63	14.56	16.96

Statistic Profile = Kruskal-Wallis + Wilcoxon test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics k=KRUSKAL-WALLIS

Female Pups

		G 10 / F	G 11 / F	G 12 / F	G 13 / F
T4 [nmol/L] day 13	Mean	69.61 v	63.27	69.28	57.89 *
	S.d.	11.07	11.03	5.81	8.07
	N	10	10	9	10
	Median	71.72	61.38	69.68	57.14
	Deviation Vs Control		-9.11	-0.47	-16.84
TSH [µg/L] day 13	Mean	3.88 k	4.24	4.28	4.29
	S.d.	0.79	1.18	1.69	0.65
	N	10	10	9	10
	Median	3.77	3.93	3.74	4.48
	Deviation Vs Control		9.25	10.40	10.62

Statistic Profile = Kruskal-Wallis + Wilcoxon test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics v=KRUSKAL-WALLIS-WILCOX; k=KRUSKAL-WALLIS

Conclusions:

In conclusion, under the conditions of the present Reproductive/Developmental Toxicity Screening Test the oral administration of the test item to male and female Wistar rats revealed signs of systemic toxicity such as adverse changes in thyroid hormones and corresponding hypertrophy/hyperplasia of follicular cells and altered colloid in the thyroid glands of adult males at 1000 mg/kg bw/d. Females showed no signs of systemic toxicity. Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 1000 mg/kg bw/d in females and 300 mg/kg bw/d in males based on the adverse changes in the thyroid glands. The NOAEL for fertility and reproductive performance was 1000 mg/kg bw/d for the F0 parental rats. The NOAEL for developmental toxicity in the offspring was 1000 mg/kg bw/d in females and 300 mg/kg bw/d in males due to retained areolae/nipples.

Executive summary:

The test article was given 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 (0.5% Sodium carboxymethyl cellulose suspension in drinking water [0.5% CMC]). The duration of treatment covered a 2-week pre-mating and a mating period in both sexes, one day post-mating in males, and the entire gestation period as well as approximately 20 days of the lactation period in females. 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. Food consumption of the F0 parental animals was determined once weekly during premating. In dams food consumption was determined for gestation days (GD) 0-7, 7-14, 14-20 and lactation days (PND) 1-4, 4-7, 7-10 and 10-13. Body weights of F0 parental animals 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 GD 0, 7, 14 and 20, on the day of parturition (PND 0) and on PND 4, 7, 10 and 13. Estrous cycle data were evaluated for F0 generation females over a two-week period prior to mating until evidence of mating occurred. Moreover, the estrous stage of each female was determined on the day of scheduled sacrifice. The pups were sexed and examined for macroscopically evident changes on PND 0. They were weighed on PND 1, 4, 7 and 13. Their viability was recorded. Anogenital distance measurements were conducted in a blind randomized fashion, using a measuring ocular on all live male and female pups on PND 1. At necropsy on PND 4 and PND 13, the pups were sacrificed with CO2, under isoflurane anesthesia, and examined macroscopically for external and visceral findings. On PND 13 thyroid glands and parathyroid glands were fixed for possible further processing. All surviving male pups were examined for the presence or absence of nipple/areola anlagen on PND 13. The number of nipple/areola anlagen were counted. Blood samples for clinical pathological investigations were taken from all surplus pups at PND 4 as well as one male and one female pup per litter at PND 13 during necropsy of pups. Blood samples from all dams at PND 14 and all males at termination were taken by puncturing the retrobulbar venous plexus under isoflurane anesthesia for hormone measurement. 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.

The following test substance-related adverse effects/findings were noted: In the high dose group, increased TSH values and decreased T4 values were reported in adult males, correlating with hypertrophy/hyperplasia in follicular cells of the thyroid in 8 out of 10 males (minimal to slight) and altered colloid in 5 out of 10 males (minimal to slight). No test substance-related adverse findings in females. In male pups of the high dose group the percentage of male pups having nipple / areolae was statistically significantly increased (90.0 %, control: 44.7 %) and the mean number of nipples in males was increased (2.5, control: 1.2) on PND 13. No adverse findigs were reported for the mid and low dose groups.

In conclusion, under the conditions of the present Reproductive/Developmental Toxicity Screening Test the oral administration of the test item to male and female Wistar rats revealed signs of systemic toxicity such as adverse changes in thyroid hormones and corresponding hypertrophy/hyperplasia of follicular cells and altered colloid in the thyroid glands of adult males at 1000 mg/kg bw/d. Females showed no signs of systemic toxicity. Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 1000 mg/kg bw/d in females and 300 mg/kg bw/d in males based on the adverse changes in the thyroid glands. The NOAEL for fertility and reproductive performance was 1000 mg/kg bw/d for the F0 parental rats. The NOAEL for developmental toxicity in the offspring was 1000 mg/kg bw/d in females and 300 mg/kg bw/d in males due to retained areolae/nipples.

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

The test item was administered daily by gavage to groups of 10 male and 10 female Wistar rats (F0 animals) at doses of 100, 300 and 1000 mg/kg bw/d. The duration of treatment covered a 2-week premating and a mating period in both sexes, approximately 2 days post-mating in males, and the entire gestation period as well as 13 days of lactation in females.

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 at room temperature. No signs of general systemic toxicity (clinical findings, food consumption, body weight) were observed in male and female parental animals of test groups 1-3 (100, 300 and 1000 mg/kg bw/d). Fertility, reproductive performance and delivery were not impaired by test substance up to a dose of 1000 mg/kg bw/d, as was demonstrated by unchanged fertility, gestation and live birth indices of pups in all test groups. The viability and survival indices as indicator for pup mortality were also not significantly altered. Concerning nipple development in male pups, percentage of affected males and mean number of male pups having nipple / areolae was statistically significantly increased in test group 3 (1000 mg/kg bw/d) on PND 13. The sex ratio on day of birth showed no indication for an increase in female pups. The number of male pups was even increased but was within the historical control range. However, since both parameters of nipple development were dose-related, statistically significantly altered, a relation to treatment cannot be excluded and, therefore, this was considered to be treatment related and adverse. Regarding clinical pathology, a hypothyroid effect in adult males of test group 3 (1000 mg/kg bw/d) cannot be excluded because of changes in thyroid hormone values (increased TSH and decreased T4 values). No effect on thyroid hormone levels was observed in adult females as well as in F1 PND13 pups. Regarding pathology, significant absolute and relative liver weight increases in male (absolute: +19 %; relative: +21 %) and female animals (absolute +17 %; relative: +19 %) of test group 3 (1000 mg/kg bw/d) were consistent with centrilobular hepatocellular hypertrophy in both male (minimal to slight) and female (minimal to slight) animals. These findings were regarded as treatment-related but not adverse. In the thyroid gland of males of test group 3, hypertrophy/hyperplasia of follicular cells and altered colloid were clearly increased in incidence and grading, when compared to control group animals. Since these findings correlated with increased TSH and decreased T4 values, they were considered treatmentrelated and adverse. In females of test group 3, a statistically weak significant increase of the relative thyroid gland weight correlated with a minimal increase in the incidence of hypertrophy/hyperplasia of follicular cells and altered colloid. Since these findings occurred without hormonal changes, they were assumed to be treatment-related but not adverse. In test group 2 (300 mg/kg bw/d), the incidence of centrilobular hepatocellular hypertrophy in the liver was very low in both male (two out of 10) and female animals (one out of 10). This finding was regarded as possible treatment-related but not adverse. In the thyroid gland of males, hypertrophy/hyperplasia of follicular cells and altered colloid were increased and comparable to the changes seen in test group 3. However, since these changes were not associated with TSH and T4 alterations, they were regarded as possible treatment-related but not adverse. In test group 1 (100 mg/kg bw/d), no treatment-related findings were seen in the liver and thyroid gland of male and female animals. All other 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 the present Reproductive/Developmental Toxicity Screening Test the oral administration of the test item to male and female Wistar rats revealed signs of systemic toxicity such as adverse changes in thyroid hormones and corresponding hypertrophy/hyperplasia of follicular cells and altered colloid in the thyroid glands of adult males at 1000 mg/kg bw/d. Females showed no signs of systemic toxicity. Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 1000 mg/kg bw/d in females and 300 mg/kg bw/d in males based on the adverse changes in the thyroid glands. The NOAEL for fertility and reproductive performance was 1000 mg/kg bw/d for the F0 parental rats. The NOAEL for developmental toxicity in the offspring was 1000 mg/kg bw/d in females and 300 mg/kg bw/d in males due to retained areolae/nipples.

Effects on developmental toxicity

Description of key information

In an OECD 414 study with rats there was no evidence for toxicologically relevant adverse effects of the test substance to the dams or on fetal morphology at any dose. The no observed adverse effect level (NOAEL) for maternal toxicity and for prenatal developmental toxicity is the highest dose level of 1000 mg/kg bw/d.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Jan 2019 - 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

Jun 2018

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

Aug 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
For the test substance preparations, the specific amount of test substance was weighed, topped up with 0.5% CMC suspension in drinking water in a calibrated beaker and intensely mixed with a homogenizer.
- Final preparation of a solid:
Preparations prepared at the beginning and thereafter at intervals. Preparations kept at room temperature

FORM AS APPLIED IN THE TEST (if different from that of starting material)
- suspension

Species:

rat

Strain:

Wistar

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: 10-12 weeks
- Weight at study initiation: 162.6 - 233.6 g (pregnant animals)
- Fasting period before study: no
- Housing: individually
- Diet: mouse and rat maintenance diet “GLP”, meal, supplied by Granovit AG, Kaiseraugst, Switzerland, ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 45-65
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12 (illumination from 6 am to 6 pm)

IN-LIFE DATES: From: 29/30/31 Jan 2019 To: 18/19/20 Feb 2019

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5% CMC in drinking water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Volume administered: 10 ml/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of the test substance preparations were sent four times (once at the beginning of administration and thrice at the end of the study) to the analytical laboratory for verification of the concentrations. Reserve samples were described by the suffix “R” in the report. The concentrations of the test item in the samples were calculated by means of their phosphor content. The method chosen was: determination of phosphor by inductively coupled plasma optical emission spectrometry (ICP-OES) after digestion with concentrated mineral acids.
Overall, the results of the analysis of the test substance preparations confirmed the correctness of the prepared concentrations. Almost all values of the measured concentrations corresponded to the expected values within the limits of the analytical method, i.e. were above 90% and below 110% of the nominal concentrations. The stability of the test substance in 0.5% CMC suspension in drinking water over a period of 7 days at room temperature was demonstrated. The homogeneous distribution of the test substance in the vehicle (0.5% CMC suspension in drinking water) was demonstrated.

Details on mating procedure:

The animals were paired by the breeder (“time-mated”); the day of evidence of mating (= detection of vaginal plug/sperm) was referred to as GD 0.

Duration of treatment / exposure:

From GD 6 to GD 19

Frequency of treatment:

once daily

Duration of test:

From implantation to one day prior to expected day of parturition

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:

25

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: At the request of the sponsor
- Rationale for animal assignment (if not random): random (manually)
- Route selection rationale: The oral route was selected since this has proven to be suitable for the detection of a toxicological hazard.

Maternal examinations:

OBSERVATIONS OF THE DAMS: Yes
- Time schedule: Twice daily
- Checked for mortality

CLINICAL SYMPTOMS: Yes
- Time schedule: once daily
- Prameters checked: signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on GD 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20.

FOOD CONSUMPTION: Yes
- Time schedule: The consumption of food was 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.

WATER CONSUMPTION: No:

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: reproductive organs (uterus), liver, thyroid glands
- Histopathology: Yes, thyroid glands

ORGAN WEIGHTS
- The following weights were determined in all dams sacrificed on schedule: liver, thyroid glands

HEMATOLOGY: Yes
- Time schedule for collection of blood: On GD 20
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Not specified
- How many animals: All females
- Parameters checked: 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 (RETA)

CLINICAL PTATHOLOGY: Yes
- Time schedule for collection of blood: On GD 20
- Animals fasted: Not specified
- How many animals: all females
- Parameters checked: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), γ-Glutamyltransferase (GGT), Inorganic phosphate, Calcium, Urea, Creatinine, Glucose, Total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol
- In addition: Thyroid hormones were checked (T3, T4, and TSH)

Ovaries and uterine content:

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

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half litter

At necropsy each fetus was weighed, sexed, and external tissues and all orifices were examined macroscopically. The sex was determined by observing the distance between the anus and the base of the genitalia. Furthermore, the viability of the fetuses and the condition of placentas, umbilical cords, fetal membranes, and fluids were examined. The placentas were weighed and their individual weights were recorded. Thereafter, the fetuses were sacrificed by a subcutaneous injection of pentobarbital. The anogenital distance (defined as the distance from the center of the anal opening to the base of the genital tubercle) measurements were conducted, using a measuring ocular, on all liveborn fetuses. After these examinations, approximately one half of the fetuses per dam were eviscerated, skinned and fixed in ethanol; the other half was placed in Harrison’s fluid for fixation.

Statistics:

- Dunnett's test, Fisher's exact test, Wilcoxon test, Kruskal-Wallis test

Indices:

Conception rate, preimplantation loss, postimplantation loss, anogenital index

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any female of the test groups 0-3 (0, 100, 300 or 1000 mg/kg bw/d).

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were no test substance-related or spontaneous mortalities in any females of all test groups.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The mean body weights and the average body weight gain of the low-, mid- and high-dose dams (100, 300 and 1000 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

Description (incidence and severity):

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

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes among hematological parameters were observed. At gestation day 21 in dams of test groups 1, 2 and 3 (100, 300 and 1000 mg/kg bw/d) absolute eosinophil counts were significantly decreased. Additionally, in dams of test groups 2 and 3, relative eosinophil counts were significantly decreased. However, the values were within historical control ranges (dams, absolute eosinophils 0.05-0.09 Giga/L; relative eosinophils 0.8-1.7 %). In dams of test group 1 (100 mg/kg bw/d) absolute monocyte counts were significantly decreased, but the alteration was not dose-dependent. Therefore, the mentioned changes were regarded as incidental and not treatment-related.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

In dams of test group 3 (1000 mg/kg bw/d), significantly decreased T3 values were measured. However, the values were within the historical control range (dams, T3: 0.56-1.06 nmol/L), whereas the study control T3 values were at the upper border of this range. Therefore, this change was regarded as incidental and not treatment related.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Uterus: The mean gravid uterus weights of the animals of test groups 1-3 (100, 300 and 1000 mg/kg bw/d) were not influenced by the test substance. The differences between these groups and the control group revealed no dose-dependency and were assessed to be without biological relevance.
Thyroid: When compared to control group 0 (=100%), the mean absolute weight of the thyroids was significantly decreased in females treated with 300 mg/kg. Since this change had no dose-dependent relationship, it was regarded as not treatment-related.
Liver: When compared to control group 0 (=100%), the mean relative weight of the liver was significantly increased in females treated with 300 (103%) and 1000 mg/kg (108%). This increase is regarded as treatment-related but not adverse as no clinical chemical alterations of the hepatic parameters were noted.

Gross pathological findings:

no effects observed

Description (incidence and severity):

One individual finding occurred and was considered incidental or spontaneous in origin and without any relation to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

A minimal follicular hypertrophy/hyperplasia occurred in all test groups with a minimal dose-dependent increased incidence in test groups 2 and 3 (300 and 1000 mg/kg bw/day). These changes were of low magnitude and were not consistent with hormonal changes. Therefore, histopathological findings in dams of test groups 2 (300 mg/kg bw/d) and 3 (1000 mg/kg bw/d) were assumed to be possibly treatment-related but not adverse.

Histopathological findings: neoplastic:

no effects observed

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

The conception rate reached 96% in the test groups 0 and 1000 mg/kg bw/d and 100 % in the test groups 100 and 300 mg/kg bw/d.

Other effects:

no effects observed

Description (incidence and severity):

There were no test substance related and/or biologically relevant differences between the test groups 0-3 in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and post-implantation losses, the number of resorptions and viable fetuses. All observed differences are considered to reflect the normal range of fluctuations for animals of this strain and age.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: no adverse effects observed

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The mean fetal weights of test groups 1, 2 and 3 were not influenced by the test substance and did not show any biologically relevant differences in comparison to the control group.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The sex distribution of the fetuses in test groups 1-3 (100, 300 and 1000 mg/kg bw/d) was comparable to the control fetuses.

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

External malformations were detected in 3 fetuses of three different litters exclusively in test group 2 (300 mg/kg bw/d), all were associated with skeletal malformations. These events added up to a statistically significantly increased total incidence of external malformations in test group 2. In absence of a dose-relationship, none of these malformations were assessed as treatment-related. One external variation, i.e. limb hyperextension, was recorded in two control fetuses. There were no external variations in any of the treated groups.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Skeletal malformations were detected in five fetuses of 300 mg/kg bw/d and in one single fetus of 1000 mg/kg bw/d. However, findings can be found in historical control data at comparable incidences, they showed no dose-response relationship and thus, were assessed as a spontaneous, isolated event.
Observed skeletal variations were related to several parts of fetal skeletons and appeared without a relation to dose. The overall incidences of skeletal variations were comparable to the historical control data.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

- 1000 mg/kg bw/day: Only one fetus showed multiple visceral malformations. However, these findings were isolated events and were well within the range of historical control data, hence, were assessed as not treatment-related. Three soft tissue variations were detected, however, findings were within historical control data and were not considered to be treatment-related.

Other effects:

no effects observed

Description (incidence and severity):

The mean placental weights of test groups 1-3 were comparable to the concurrent control group. Neither on anogenital distance nor on anogenital index test substance-related effects were noted in all fetuses of test groups 1-3.

Details on embryotoxic / teratogenic effects:

Overall, there was no evidence for toxicologically relevant adverse effects of the test substance on fetal morphology at any dose. Under the conditions of this study the test item is not teratogenic.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Abnormalities:

no effects observed

Developmental effects observed:

no

Organ weights

Absolute weights	Females
Test group	1	2	3
(mg/kg bw/d)	(100)	(300)	(1000)
Thyroid glands	95%	88%**	97%

* : p <= 0.05, **: p <= 0.01

Relative weights	Females
Test group	1	2	3
(mg/kg bw/d)	(100)	(300)	(1000)
Liver	100%	103%*	108%**

* : p <= 0.05, **: p <= 0.01

All other mean relative weight parameters did not show significant differences when compared to the control group 0. The significantly decreased absolute weight of the thyroid glands in females of test group 2 had no dose-dependent relationship and was therefore considered not treatment-related. The significantly increased relative weight of the liver in females of test group 3 (4.845%) was marginally above the historical control range (4.343 – 4.801%) and was regarded as treatment-related, whereas the relative weight increase in test group 2 (4.621%) was within the control range and was considered not treatment-related.

Summary of histopathology

	Female animals
Test group (mg/kg bw/d)	0 (0)	1 (100)	2 (300)	3 (1000)
No. of dams	24	25	25	24
Hypertrophy/hyperplasia, follicular	4	2	6	9
·       Grade1	4	2	6	9

A minimal follicular hypertrophy/hyperplasia occurred in all test groups with a minimal dose-dependent increased incidencein test groups 2 and 3. These changes were of low magnitude and were not consistent with hormonal changes. Therefore, histopathological findings in dams of test groups 2 (300 mg/kg bw/d) and 3 (1000 mg/kg bw/d) were assumed to be possibly treatment-related but not adverse. All other 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.

External Examnation of fetuses

Individual fetal external malformations:

Test group	Dam No.-Fetus No., Sex	Finding
0 (0 mg/kg bw/d)	none
1 (100 mg/kg bw/d)	none
2 (300 mg/kg bw/d)	54-08 F^a)	exencephaly, microphthalmia
	63-13 M^a)	multiple external malformations
	70-06 F^a)	exencephaly, macroglossia, microphthalmia
3 (1000 mg/kg bw/d)	none

No. = number; M = male; F = female

^a) fetus with additional skeletal malformation (see below)

Total external malformations:

		Test group 0 0 mg/kg	Test group 1 100 mg/kg	Test group 2 300 mg/kg	Test group 3 1000 mg/kg
Litter	N	24	25	25	24
Fetuses	N	268	287	266	263
Fetal incidence	N (%)	0	0	3 (1.1)	0
Litter incidence	N (%)	0	0	3 (12)	0
Affected fetuses/litter	Mean%	0	0	1.0*	0

N = number; % = per cent, * = p <= 0.05 (Wilcoxon-test [one-sided])

External malformations were detected in 3 fetuses of three different litters exclusively in test group 2 (300 mg/kg bw/d), all were associated with skeletal malformations. These events added up to a statistically significantly increased total incidence of external malformations in test group 2. In absence of a dose-relationship, none of these malformations were assessed as treatment-related. One external variation, i.e. limb hyperextension, was recorded in two control fetuses. There were no external variations in any of the treated groups. No external unclassified observations were recorded.

Soft Tissue Examination

Individual fetal soft tissue malformations:

Test group	Dam No.-Fetus No., Sex	Finding
0 (0 mg/kg bw/d)	none
1 (100 mg/kg bw/d)	none
2 (300 mg/kg bw/d)	none
3 (1000 mg/kg bw/d)	99-12 M	multiple visceral malformations

No. = number; M = male; F = female^a)

Total soft tissue malformations:

		Test group 0 0 mg/kg	Test group 1 100 mg/kg	Test group 2 300 mg/kg	Test group 3 1000 mg/k
Litter	N	24	25	25	24
Fetuses	N	130	136	122	126
Fetal incidence	N (%)	0	0	0	1 (0.8)
Litter incidence	N (%)	0	0	0	1 (4.3)
Affected fetuses/litter	Mean%	0	0	0	0.6

N = number; % = per cent

One fetus of test group 3 (1000 mg/kg bw/d) had multiple visceral malformations, such as torsion of stomach, aortic arch atresia and abnormal lung lobation. However, these findings were isolated events in one single fetus and no cluster of any of these individual malformations were seen in the other offspring of this or the other treated groups. Furthermore, the value of affected fetuses per litter of this finding in test group 3 (0.6 %) was well within the range of the historical control data (affected fetuses per litter: 0.0 – 0.7 %). Thus, it was not assessed as treatment-related. The total incidence of soft tissue malformations in treated animals did not differ significantly from the concurrent control group and was covered by the historical control data.

Total soft tissue variations:

		Test group 0 0 mg/kg	Test group 1 100 mg/kg	Test group 2 300 mg/kg	Test group 3 1000 mg/kg
Litter	N	24	25	25	24
Fetuses	N	130	136	122	126
Fetal incidence	N (%)	3 (2.3)	3 (2.2)	2 (1.6)	10 (7.9)
Litter incidence	N (%)	2 (8.3)	3 (12)	2 (8.3)	8 (35)^Fi*
Affected fetuses/litter	Mean%	2.9	2.3	1.5	7.9^Wi*

N = number; % = per cent

^Fi* = p <= 0.05 (Fisher’s exact test [one-sided])

^Wi* = p <= 0.05 (Wilcoxon-test [one-sided])

Three soft tissue variations were detected, i.e. short innominate in test group 3, dilated renal pelvis and dilated ureter in all test groups. The incidences of ‘dilated renal pelvis’ (2.9/1.6/1.5/7.2* mean% affected fetuses/litter) and ‘dilated ureter’ (2.1/1.5/1.5/6.5* mean% affected fetuses/litter) were statistically significantly higher in test group 3 (1000 mg/kg bw/d). For both findings, the mean values of affected fetuses per litter were well within the range of the historical control data (affected fetuses per litter, dilated renal pelvis: range: 0.7 - 11.5 %; dilated ureter: 0.0 - 6.9 %). Consequently, the findings summed up to a statistically significantly higher total incidence of fetal soft tissue variations in the high-dose group. However, the mean value was well within the range of the historical control data (total soft tissue variations: mean: 0.7 - 12.6 %). Overall, based on the discussion above, these findings in test group 3 were not assessed as treatment-related. No soft tissue unclassified observations were recorded.

Skeletal examination of the fetuses

Individual fetal skeletal malformations:

Test group	Dam No.-Fetus No., Sex	Finding
0 (0 mg/kg bw/d)	none
1 (100 mg/kg bw/d)	none
2 (300 mg/kg bw/d)	51-05 M	cleft sternum
	54-08 F^a)	severely malformed skull bones, severely malformed vertebral column
	63-13 M^a)	severely malformed skull bones, severely malformed vertebral column
	70-06 F^a)	severely malformed skull bones
	75-09 M	shortened humerus
3 (1000 mg/kg bw/d)	77-07 M	shortened scapula, shortened humerus

No. = number; M = male; F = female

^a)fetus with additional external malformation (see above)

Total skeletal malformations:

		Test group 0 0 mg/kg	Test group 1 100 mg/kg	Test group 2 300 mg/kg	Test group 3 1000 mg/kg
Litter	N	24	25	25	24
Fetuses	N	138	151	144	137
Fetal incidence	N (%)	0	0	5 (3.5)	1 (0.7)
Litter incidence	N (%)	0	0	5 (20)^Fi*	1 (4.2)
Affected fetuses/litter	Mean%	0	0	3.0^Wi*	1

N = number; % = per cent

^Fi* = p <=0.05 (Fisher’s exact test [one-sided])

^Wi* = p <=0.05 (Wilcoxon-test [one-sided])

Skeletal malformations were detected in five fetuses of test group 2 (300 mg/kg bw/d) and in one single fetus of test group 3 (1000 mg/kg bw/d). In test group 2, different findings without relation to dose were observed: three fetuses (No. 54-08, No. 63-13, No. 70-06) of test group 2 were multiple malformed and had additional external malformations. The finding ‘severely malformed skull bones’ which occurred only in these three multiple malformed fetuses, was statistically significantly increased in test group 2. However, the finding can be found in the historical control data at comparable incidences and it occurred without any relation to dose. Therefore, it was not assessed as treatment-related. The same was true for the finding ‘severely malformed vertebral column’ showing no dose-response-relationship.

The finding cleft sternum in one single fetus of the mid-dose group was assessed as incidental, isolated event since it occurred without relation to dose. The malformations in the remaining fetuses (‘shortened humerus’ and ‘shortened scapula’) of mid- and high-dose are not assessed as treatment-related since they were isolated events in single fetuses without any relation to dose. All of them were well within the range of the historical control data (e.g. shortened humerus: affected fetuses per litter: range of 0.0 – 1.3%). The different findings in the mid-dose summed up to a statistically significant increase of total incidence of skeletal malformations in test group 2. However, consequently to the above- mentioned discussion of the individual findings, this increase occurred without a relation to dose and was well within the historical control range (total skeletal malformations: 0.0 - 3.1%). Therefore, this was not assessed as treatment-related.

Total fetal skeletal variations:

		Test group 0 0 mg/kg	Test group 1 100 mg/kg	Test group 2 300 mg/kg	Test group 3 1000 mg/kg
Litter	N	24	25	25	24
Fetuses	N	138	151	144	137
Fetal incidence	N (%)	133 (96)	147 (97)	136 (94)	135 (99)
Litter incidence	N (%)	24 (100)	25 (100)	25 (100)	24 (100)
Affected fetuses/litter	Mean%	96.6	97.3	94.9	98.6

N = number; % = per cent

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 without a relation to dose. 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 are compiled in the table below. All incidences were expressed on a fetus per litter basis.

Occurrence of statistically significantly increased fetal skeletal variations:

Finding	Test group 0 0 mg/kg	Test group 1 100 mg/kg	Test group 2 300 mg/kg	Test group 3 1000 mg/kg	HCD Mean % (range)
Incomplete ossification of parietal; unchanged cartilage	2.2	6.2	4.9	7.2*	8.1 (0.7 - 20.0)
Dumbbell ossification of thoracic centrum; dumbbell-shaped cartilage of centrum	0	0	3.2*	0.7	2.7 (0.0 - 10.1)

HCD = Historical control data; % = per cent

* = p <= 0.05 (Wilcoxon-test [one-sided])

Concerning the statistically significant findings, an association to the test substance and a toxicological relevance is not assumed. As described in the table above, the mean value of the finding in the high-dose group was well within the range of the historical control data. The finding in the mid-dose group was without relation to dose and well within the historical control range.

Additionally, some isolated cartilage findings without impact on the respective bony structures, which were designated as unclassified cartilage observations,occurred in all test groups. The observed unclassified cartilage findings were related to the skull, the vertebral column, the ribs and the sternum and did not show any relation to dosing.

Assessment of all fetal external, soft tissue and skeletal observations

There were noted external, soft tissue and skeletal malformations in five fetuses of test group 2 and two fetuses of test group 3. Three fetuses of test group 2 and two fetuses of test group 3 had more than one malformation or were multiple-malformed across the different examination areas. Other malformations, such as cleft sternum and shortened humerus were scattered observations in individual fetuses of test group 2. They were not dose-related and all of them can be found in the historical control data at comparable or higher frequency. In test group 3, the observed malformations were either single isolated events without any hints of a cluster seen in the other offspring (e.g. ‘multiple visceral malformations’) and/or without relation to dose (e.g. ‘shortened humerus’). All of them were well within the historical control data. Therefore, they are not assessed as treatment-related, adverse events. In test group 2, three fetuses were multiple malformed. The findings showed no relation to dose and were not assessed as treatment-related. The different findings in test group 2 summed up to statistically significant incidence of total malformations showing no relation to dose. The mean value of affected fetuses per litter was well within the historical control range (HCD total malformations: 0.00 - 2.03 %). Therefore, it is not assessed as treatment-related.

Total malformations:

		Test group 0 0 mg/kg	Test group 1 100 mg/kg	Test group 2 300 mg/kg	Test group 3 1000 mg/kg
Litter	N	24	25	25	24
Fetuses	N	268	287	266	263
Fetal incidence	N (%)	0	0	5 (1.9)	2 (0.8)
Litter incidence	N (%)	0	0	5 (20)^Fi*	2 (8.3)
Affected fetuses/litter	Mean%	0	0	1.7^Wi*	0.8

N = number; % = per cent

^Fi* = p <= 0.05 (Fisher’s exact test [one-sided])

^Wi* = p <= 0.05 (Wilcoxon-test [one-sided])

One external variation, three soft tissue variations and a range of skeletal variations were noted in all test groups including the controls. The majority of individual variations are equally distributed across the different test groups, if normal biological variation is taken into account, and can be found in the historical control data at a comparable frequency. In test group 3, the incidences of two soft tissue variations, ‘dilated renal pelvis’ and ‘dilated ureter’ were statistically significantly higher in test group 3 (1000 mg/kg bw/d). For both findings, the mean values of affected fetuses per litter were well within the range of the historical control data (see above). Consequently, the findings summed up to a statistically significantly higher incidence in total fetal soft tissue variations in the high-dose group. However, this mean value was also well within the range of the historical control data. Overall, as discussed above, these findings in test group 3 were not assessed as treatment-related.

Total fetal variations:

		Test group 0 0 mg/kg	Test group 1 100 mg/kg	Test group 2 300 mg/kg	Test group 3 1000 mg/kg
Litter	N	24	25	25	24
Fetuses	N	268	287	266	263
Fetal incidence	N (%)	136 (51)	150 (52)	138 (52)	145 (55)
Litter incidence	N (%)	24 (100)	25 (100)	25 (100)	24 (100)
Affected fetuses/litter	Mean%	51.1	52.3	53.6	56.7**

N= number; % = per cent

** = p <= 0.01 (Wilcoxon-test [one-sided])

Unclassified external and unclassified soft tissue observations did not occur in any of the fetuses in this study. A spontaneous origin is assumed for the unclassified skeletal cartilage observations which were observed in several fetuses of all test groups (0, 100, 300 and 1000 mg/kg bw/d). The distribution and type of these findings do not suggest any relation to treatment. Finally, fetal examinations revealed that there is no effect of the compound on the respective morphological structures up to 1000 mg/kg bw/d.

Conclusions:

Under the conditions of this prenatal developmental toxicity study, the oral administration of the test item to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) caused no treatment-related, adverse findings up to the highest dose level of 1000 mg/kg bw/d. In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is the highest dose level of 1000 mg/kg bw/d. There was no evidence for toxicologically relevant adverse effects of the test substance on fetal morphology at any dose. Under the conditions of this study the test item is not teratogenic. In conclusion, the no observed adverse effect level (NOAEL) for prenatal developmental toxicity is the highest 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 doses 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 drinking 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, 24-25 females per group had implantation sites. Food consumption and body weights of the animals were recorded regularly throughout the study period. The state of health of the animals was checked each day. On GD 20, blood samples were obtained from all females by retrobulbar venous puncture following isoflurane anesthesia. After blood sampling all females were sacrificed by decapitation (under isoflurane anesthesia) and assessed by gross pathology (including sampling of thyroid glands (with parathyroid glands) and weight determinations of the liver, thyroid glands (with parathyroid glands), unopened uterus and placentas). For each dam, corpora lutea were counted and number and distribution of implantation sites (differentiated between resorptions, live and dead fetuses) were determined. The fetuses were removed from the uterus, sexed, weighed and further investigated for external findings. Anogenital distance measurements were conducted on all liveborn fetuses. Thereafter, one half of the fetuses of each litter were examined for soft tissue findings and the remaining fetuses for skeletal (inclusive cartilage) findings. Under the conditions of this prenatal developmental toxicity study, the oral administration of the test item to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) caused no treatment-related, adverse findings up to the highest dose level of 1000 mg/kg bw/d. In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is the highest dose level of 1000 mg/kg bw/d. There was no evidence for toxicologically relevant adverse effects of the test substance on fetal morphology at any dose. Under the conditions of this study the test item is not teratogenic. In conclusion, the no observed adverse effect level (NOAEL) for prenatal developmental toxicity is the highest dose of 1000 mg/kg bw/d.

Effect on developmental toxicity: 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 developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

In a prenatal developmental toxicity study, the test substance was administered to pregnant Wistar rats daily by gavage from implantation to one day prior to the expected day of parturition (GD 6-19) to evaluate its potential maternal and prenatal developmental toxicity. In general, analyses confirmed the correctness of the prepared concentrations, their homogeneous distribution and the stability of the test substance in the vehicle. Concerning clinical examination, no findings in clinical observation, food consumption and body weight (change) were observed up to the highest tested dose level of 1000 mg/kg bw/d. Concerning clinical pathology, no treatment-related and adverse effect was observed up to the highest dose level of 1000 mg/kg bw/d. Regarding pathology, the relative weight of the liver was significantly but minimally in-creased (+8%) in dams of test group 3 (1000 mg/kg bw/d), which was regarded treatment-related but not adverse as no clinical chemical alterations of the hepatic parameters were noted. Histopathology of the thyroid glands revealed in all test groups a minimal follicular hyper-trophy/hyperplasia with a minimal dose-dependently increased incidence in test groups 2 (6 out of 25 dams) and 3 (9 out of 24 dams, compared to 4 out of 23 in control dams). These changes were of low magnitude and were not consistent with hormonal changes. Therefore, histopathological findings in dams of test groups 2 (300 mg/kg bw/d) and 3 (1000 mg/kg bw/d) were assumed to be possibly treatment-related but not adverse. All other histopathological findings in the thyroid glands 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. No differences of toxicological relevance between the control and the treated groups (100, 300 or 1000 mg/kg bw/d) were determined for any reproductive parameters, such as conception rate, mean number of corpora lutea, mean number of implantations, as well as pre- and post-implantation loss. Similarly, no toxicologically relevant influence of the test substance on sex distribution and anogenital distance/index of the fetuses was noted at any dose. Overall, there was no evidence for toxicologically relevant adverse effects of the test substance on fetal morphology at any dose.

Under the conditions of this prenatal developmental toxicity study, the oral administration of O,O,O-Triphenyl phosphorothioate to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) caused no treatment-related, adverse findings up to the highest dose level of 1000 mg/kg bw/d. In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is the highest dose level of 1000 mg/kg bw/d. There was no evidence for toxicologically relevant adverse effects of the test substance on fetal morphology at any dose. Under the conditions of this study the test item is not teratogenic. In conclusion, the no observed adverse effect level (NOAEL) for prenatal developmental toxicity is the highest dose of 1000 mg/kg bw/d.

Justification for classification or non-classification

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

The available screening study is reliable. It is considered suitable for classification purposes under Regulation 1272/2008 and as a result, the substance is not considered to be classified for reproductive toxicity under Regulation (EC) No. 1272/2008.