Bis[O,O-bis(2-ethylhexyl) dithiophosphorato-S,S']dioxodi-μ-thioxodimolybdenum

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2016

Deviations:

yes

Remarks:

, all were minor deviations not having an effect on the study outcome

GLP compliance:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 10-11 weeks (males), 13-14 weeks (females)
- Weight at study initiation: 253-307 g (males), 206-253 g (females)
- Fasting period before study: no
- Housing: group housing up to 5 animals of the same sex and dosing group ; mated females were housed individually
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 6-7 days prior to start of dosing

DETAILS OF FOOD AND WATER QUALITY: pelleted rodent diet (SM R/M-Z from SNIFF Spezialdiäten GmbH, Germany), drinking water

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-22
- Humidity (%): 44-67
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To:

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:

VEHICLE
- Justification for use and choice of vehicle (if other than water): suitable vehicle
- Concentration in vehicle: 5, 20, 80 mg/ml
- Amount of vehicle (if gavage): 5 ml/kg body weight
- Lot/batch no. (if required):
- Purity:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

the concentrations of the test substance analysed in the formulations by HPLC/MS were in agreement with target concentrations and were homogeneous.

Duration of treatment / exposure:

Males were dosed for 29 days. Females that delivered offspring were dosed for 51-62 days. Additional animals in the recovery groups (high dose) were treated similarly (males for 29 days, females for 55 days) and were terminated after a treatment-free period of 29 days.

Frequency of treatment:

once daily

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on results of a dose range finding study
- Rationale for animal assignment (if not random): random
- Fasting period before blood sampling for clinical biochemistry: yes (overnight)
- Rationale for selecting satellite groups: based on the results of the dose range finding study
- Post-exposure recovery period in satellite groups: 29 days
- Section schedule rationale (if not random):
- Other:

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:twice daily
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually on the first day of treatment (prior to dosing), and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / Not specified

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / Not specified
- Time schedule for examinations:

OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations:
- Dose groups that were examined:

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood of Main F0-animals was collected at the end of the treatment period on the day of scheduled necropsy. Blood of Recovery animals was collected at the end of the treatment period and on the day of scheduled necropsy at the end of the recovery period.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: 5/sex/dose
- Parameters checked in table [No.?] were examined. see table on haematology attached

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: as for haematology
- Animals fasted: Yes
- How many animals: 5/sex/dose
- Parameters checked in table [No.?] were examined. see table on blood chemistry attached

URINALYSIS: No
- Time schedule for collection of urine:
- Metabolism cages used for collection of urine: Yes / No / Not specified
- Animals fasted: Yes / No / Not specified
- Parameters checked in table [No.?] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: males: during week 4, females: during the last week of lactation
- Dose groups that were examined: all dose groups
- Battery of functions tested: sensory activity / grip strength / motor activity / other:

IMMUNOLOGY: No
- Time schedule for examinations:
- How many animals:
- Dose groups that were examined:
- Parameters checked in table [No.?] were examined.

OTHER:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes

Statistics:

All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible.
Parametric: Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
Non-Parametric: Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test). The motor activity data set was compared using an overall Kruskal-Wallis.
Incidence: An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

All males and females at 400 mg/kg showed pale and soft faeces starting at Treatment Day 4. In males, the faeces appeared normal from Day 7 of the recovery period onwards. In females, soft faeces persisted until about one week before the end of the treatment period, while faeces remained pale until the end of treatment. Faeces of females looked normal throughout the recovery period.
Hunched posture occurred in all 400 mg/kg males and females from Treatment Day 10. In males, the clinical sign persisted until Day 3 of the recovery period. In females, hunched posture persisted until about one week before the end of the treatment period in most animals. (4/9 lactating females showed hunched posture until the end of treatment).
Piloerection was noted in all 400 mg/kg males and females between Treatment Days 10-16 and in a single female (no. 90) at 400 mg/kg on Treatment Day 17.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

one animal of the mid-dose group died after 4 days due to a gavage dosing accidient

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

see Tables on body weight attached as background material

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

see Tables on food consumption attached as background material

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

see Tables on haematology summaries attached as background material

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The following statistically significant changes, mostly at 400 mg/kg, distinguished treated animals from control animals. Differences (fold-change) from the control group means are indicated between parentheses.
• Higher alanine aminotransferase (ALAT) in males (1.40x of control) and Recovery females (1.48x of control) at 400 mg/kg at the end of the treatment period. ALAT was no longer increased at the end of the recovery period.
• Lower alkaline phosphatase (ALP) in 400 mg/kg Recovery females (0.60x of control) at the end of the treatment period. Lower ALP values were also noted in males (0.72x of control) and Main females (0.56x of control) at 400 mg/kg at the end of treatment, but statistical significance was not achieved. ALP was no longer decreased at the end of the recovery period.
• Higher bile acids in males (1.68x of control) and Main females (2.32x of control) at 400 mg/kg at the end of the treatment period. Mean bile acids was also higher in 400 mg/kg Recovery females at the end of the treatment period (1.74x of control) but not statistically significantly. Bile acids were still increased (1.51x of control) in Recovery females at the end of the recovery period, although not statistically significant.
• Lower total bilirubin in males at 100 and 400 mg/kg (0.28 and 0.39x of control, respectively) and in 400 mg/kg Recovery females (0.11x of control) at the end of the treatment period. Total bilirubin was no longer reduced at the end of the recovery period.
• Lower cholesterol in 400 mg/kg Recovery females (0.69x of control) at the end of the treatment period. Cholesterol in these females was still lower (0.77x of control) at the end of the recovery period, but no longer statistically significantly.
• Higher urea in 400 mg/kg Recovery females (1.43x of control) at the end of the treatment period. Except in female no. 94, urea values had returned to normal at the end of the recovery period.
• Lower potassium in Main females at 400 mg/kg (0.86x of control). Potassium in 400 mg/kg Recovery females was similar to that in Recovery controls.
Mean ALAT and urea in 400 mg/kg animals slightly exceeded the P95-limit of the historical control ranges. Bilirubin values in all males at 100 and 400 mg/kg and all Recovery 400 mg/kg females were far below the historical ranges (below the detection limit in several animals). Mean values for ALP, bile acids, cholesterol and potassium remained within the historical control ranges.
Serum levels of T4 in F0 males were considered not to be affected by treatment. Although not reaching statistical significance serum levels of TSH in F0 males were increased at 400 mg/kg, (2.41x of control). In the absence of a clear dose-response relationship and as values remained within the historical control range, this finding was considered not toxicologically relevant.
Serum levels of T4 in F0 females were considered not to be affected by treatment. Similar to what was observed in F0 males, serum levels of TSH in F0 females were increased at 100 and 400 mg/kg, (1.94x and 1.74x of control, respectively). As all values remained well within the historical control range, this finding was considered not toxicologically relevant

see Tables on clinical biochemistry attached as background material

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

The variation in motor activity did not indicate a relation with treatment.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

At the end of the treatment period, test item-related higher weights of the liver and kidneys were noted in both sexes. Liver weights (absolute and relative to body weight) were increased in 400 mg/kg Main males and females. Kidney weights were increased in Main males at 100 and 400 mg/kg (absolute and relative to body weight) and in Main females at 400 mg/kg (absolute).
At the end of the recovery period, organ weights were back within normal limits. Organ weights of treated Recovery females showed no remarkable differences from those of recovery controls.

see Tables on organ weights attached as background material

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Description (incidence and severity):

Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals.
Grip strength values of treated animals (fore and hind limbs) at 400 mg/kg/d were 13-20% lower than controls but remained within the historical control ranges.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Hepatocellular hypertrophy, mainly centrilobular, was present at 400 mg/kg in Main males at minimal degree and in Main females at minimal to slight degree. This correlated with the increased liver weight. After the 28-day treatment-free recovery period, there were no test item-related liver findings.
Follicular cell hypertrophy of the thyroid gland was present at increased incidence and severity in Main females at 400 mg/kg up to slight degree. After the 28-day treatment-free recovery period, this finding was within normal background values.
There were no other test item-related histologic changes.

Histopathological findings: neoplastic:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

other: discomfort/general toxicity

Organ:

other: discomfort, general toxicity

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Conclusions:

Clinical signs of discomfort/toxicity (hunched posture, piloerection) were observed in both sexes at the highest dose level of 400 mg/kg body weight/day. In addition, several effects were observed at study termination (28 days for males and 51-62 days for females having offspring) that all were reversible after a 28 day recovery period without dosing.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2016

Deviations:

yes

Remarks:

all were minor deviations not having an effect on study outcome

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Bis[O,O-bis(2-ethylhexyl) dithiophosphorato-S,S']dioxodi-μ-thioxodimolybdenum

EC Number:

273-381-6

EC Name:

Bis[O,O-bis(2-ethylhexyl) dithiophosphorato-S,S']dioxodi-μ-thioxodimolybdenum

Cas Number:

68958-92-9

Molecular formula:

C32H68Mo2O6P2S6

IUPAC Name:

Bis[O,O-bis(2-ethylhexyl) dithiophosphorato-S,S']dioxodi-μ-thioxodimolybdenum

Test material form:

liquid

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 10-11 weeks (males), 13-14 weeks (females)
- Weight at study initiation: 253-307 g (males), 206-253 g (females)
- Fasting period before study: no
- Housing: group housing up to 5 animals of the same sex and dosing group
; mated females were housed individually
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 6-7 days prior to start of dosing

DETAILS OF FOOD AND WATER QUALITY: pelleted rodent diet (SM R/M-Z from SNIFF Spezialdiäten GmbH, Germany), drinking water

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-22
- Humidity (%): 44-67
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light):
12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:

VEHICLE
- Justification for use and choice of vehicle (if other than water): suitable vehicle
- Concentration in vehicle: 5, 20, 80 mg/ml
- Amount of vehicle (if gavage): 5 mg/kg body weight
- Lot/batch no. (if required):
- Purity:

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: max. 14 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility. not applicable
- Further matings after two unsuccessful attempts: [no / yes (explain)]
- After successful mating each pregnant female was caged (how): indivually
- Any other deviations from standard protocol:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

the concentrations of the test substance analysed in the formulations by HPLC/MS were in agreement with target concentrations and were homogeneous.

Duration of treatment / exposure:

Males were dosed for 29 days. Females that delivered offspring were dosed for 51-62 days.

Frequency of treatment:

once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on results of a dose range finding study
- Rationale for animal assignment (if not random): random
- Fasting period before blood sampling for clinical biochemistry: yes (overnight)
- Other: animals of satelite groups for a post-exposure recovery period were not mated

Positive control:

no

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:twice daily
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily

BODY WEIGHT: Yes
- Time schedule for examinations:
Animals were weighed individually on the first day of treatment (prior to dosing), and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / Not specified

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / Not specified
- Time schedule for examinations:

Oestrous cyclicity (parental animals):

Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage.
Daily vaginal lavage was performed for all females (Main and Recovery) during 14 days prior to treatment (pretest period) and the first 14 days of treatment. For Main females, daily vaginal lavage was continued during mating until evidence of copulation was observed.

Sperm parameters (parental animals):

For the testes of all selected Main males of Groups 1 and 4 and all Main males that failed to sire, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were used for blood sampling, killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups,

GROSS EXAMINATION OF DEAD PUPS: no

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals after 28 days of dosing

- Maternal animals: All surviving animals on PND 14-16

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring was sacrificed on PND 14-16
- These animals were subjected to postmortem examinations (macroscopic examination) as follows:
On PND 4, the surplus pups (> 8 pups per litter) were euthanized by decapitation. From two surplus pups per litter, blood was collected, if possible. All remaining pups were euthanized on PND 14-16. Sex was determined both externally and internally. Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development.
In addition, blood was collected from two pups per litter, and the thyroid from two pups per litter (one male and one female pup) was preserved in 10% buffered formalin. The pups selected for blood sampling were the same pups as selected for thyroid preservation.

GROSS NECROPSY
- Gross necropsy consisted of external examinations

HISTOPATHOLOGY / ORGAN WEIGTHS: not performed

Statistics:

All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible.
Parametric: Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
Non-Parametric: Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test). The motor activity data set was compared using an overall Kruskal-Wallis.
Incidence: An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant

Reproductive indices:

see "reproduction and viability indices" attached as background material

Offspring viability indices:

see "reproduction and viability indices" attached as background material

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

All males and females at 400 mg/kg showed pale and soft faeces starting at Treatment Day 4. In males, the faeces appeared normal from Day 7 of the recovery period onwards. In females, soft faeces persisted until about one week before the end of the treatment period, while faeces remained pale until the end of treatment. Faeces of females looked normal throughout the recovery period.
Hunched posture occurred in all 400 mg/kg males and females from Treatment Day 10. In males, the clinical sign persisted until Day 3 of the recovery period. In females, hunched posture persisted until about one week before the end of the treatment period in most animals. (4/9 lactating females showed hunched posture until the end of treatment).
Piloerection was noted in all 400 mg/kg males and females between Treatment Days 10-16 and in a single female (no. 90) at 400 mg/kg on Treatment Day 17.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

one animal of the mid-dose group after 4 days due to a gavage dosing accident

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

see respective Table in endpoint study record of repeated dose toxicity study attached as background material

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

see respective Table in endpoint study record of repeated dose toxicity study attached as background material

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

see respective Table in endpoint study record of repeated dose toxicity study attached as background material

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

see respective Table in endpoint study record of repeated dose toxicity study attached as background material

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

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Hepatocellular hypertrophy, mainly centrilobular, was present at 400 mg/kg in Main males at minimal degree and in Main females at minimal to slight degree. This correlated with the increased liver weight. After the 28-day treatment-free recovery period, there were no test item-related liver findings.
Follicular cell hypertrophy of the thyroid gland was present at increased incidence and severity in Main females at 400 mg/kg up to slight degree. After the 28-day treatment-free recovery period, this finding was within normal background values.
There were no other test item-related histologic changes.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

see Table on estrous cycles attached as background material

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Description (incidence and severity):

see Tables on reproduction data attached as background material

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

Remarks on result:

other: discomfort/toxicity (hunched posture, piloerection)

2

Key result

Dose descriptor:

NOAEL

Remarks:

for reproductive function

Effect level:

> 400 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: no effects at highest dose tested

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

System:

other: only general, clinical toxicity was observed, no specific organ toxicity

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was considered not to be affected by treatment. Viability indices were 99, 100, 98 and 97% for the control, 25, 100 and 400 mg/kg groups, respectively. One pup of the control group, two pups at 100 mg/kg (from two litters) and four pups at 400 mg/kg (from four litters) went missing (presumed cannibalized) on PND 2, 3 or 4. This post-natal loss was regarded as unrelated to treatment due to the incidental occurrence (within the range considered normal for pups of this age).

Body weight and weight changes:

no effects observed

Description (incidence and severity):

see Table on body weights of pups attached as background material

Food consumption and compound intake (if feeding study):

not examined

Description (incidence and severity):

pups were killed on PND 14-16

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):

In female PND 14-16 pups there was a trend towards an increase in total T4 with increasing dose levels. The relative differences from controls were 4, 10 and 18% at 25, 100 and 400 mg/kg, respectively (statistical significance was not achieved). Mean T4 values at 100 and 400 mg/kg approached the P95-limit of the historical control range (whereas the concurrent control mean was close to the historical mean) and about half of the individual values at 100 and 400 mg/kg exceeded the historical range. TSH levels in female PND 14-16 pups were decreased at 400 mg/kg (0.85x of control), although not reaching statistical significance. As all values remained within the range of the historical control data, this finding was considered not toxicologically relevant.
Serum T4 and TSH levels in male PND 14-16 pups were considered not to be affected by
treatment.
Serum levels of T4 in PND 4 pups were considered not to be affected by treatment.

see Table on T4 and TSH levels of PND 14-16 pups attached as background material

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

see Table on anogenital distance and nipple retention of pups attached as background material

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

see Table on anogenital distance and nipple retention of pups attached as background material

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Other effects:

no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

> 400 mg/kg bw/day (actual dose received)

Based on:

test mat.

Remarks:

dams were dosed

Sex:

male/female

Remarks on result:

other: no effects at highest dose tested

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

Clinical signs of discomfort/toxicity (hunched posture, piloerection) were observed in parental animals of both sexes at the highest dose level of 400 mg/kg body weight/day. In addition, several effects were observed at study termination (28 days for males and 51-62 days for females having offspring) that all were reversible after a 28 day recovery period without dosing.
Despite parental toxicity observed at the highest dose level of 400 mg/kg/d no effects on reproductive function and on development of offspring until PND 14-16 was observed at the highest dose level.