Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

OECD 422 (oral, rat): NOAEL = 350 mg/kg bw/day for fertility

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 Nov 2016 - 21 Jul 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted Jul 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
The Department of Health of the Government of the United Kingdom
Limit test:
no
Species:
rat
Strain:
Wistar
Details on species / strain selection:
The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The RccHan™;WIST (Han Wistar) strain was used because of the historical control data available at this laboratory.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo (RMS) UK Limited
- Age at study initiation: Females: 98 - 104 days; Males: 84 - 90 days
- Weight at study initiation: Females: 192 - 232 g; Males: 323 - 362 g
- Housing: Up to 5 animals/sex were housed during pre-pairing, up to 5 males after mating. During gestation and lactation, one female (+ litter) was housed per cage. Solid (polycarbonate) bottom cages were used during the acclimatization, pre-pairing, gestation, littering and lactation periods. Solid bottom cages contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week. Grid bottomed polypropylene cages were used during pairing, when one male and one female were co-housed. These were suspended above absorbent paper which was changed daily during pairing.
- Diet: SDS VRF1 Certified pelleted (GLP Certificated rat and mouse breeding diet), ad libitum
- Water: Potable water, ad libitum
- Acclimation period: Females: 20 days; Males: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 40 - 70
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 24 Nov 2016 To: 21 Jul 2017
Route of administration:
oral: gavage
Vehicle:
other: 1% methylcellulose in water
Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
The required amount of test material was ground in a mortar using a pestle to a fine powder and mixed with a small amount of the vehicle to form a paste. Any agglomerates were broken down. Further amounts of vehicle were gradually added and mixed to produce a smooth, pourable suspension. The suspension was transferred to a measuring cylinder which had been wetted with vehicle, the mortar was rinsed with vehicle and this was added to the measuring cylinder. Vehicle was added to achieve the final volume and the suspension was transferred to a beaker and mixed using a high shear homogenizer. The suspension was transferred to the final containers, via syringe whilst magnetically stirring. A series of formulations at the required concentrations were prepared by dilution of individual weighings of the test material.

- VEHICLE
- Amount of vehicle: 10 mL/kg bw
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: Up to two weeks
- Proof of pregnancy: Ejected copulation plugs in cage tray and sperm in the vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): During gestation and lactation, one female (+ litter) was housed per cage.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentrations of test material in test formulations were analysed gravimetrically for the study, confirming accurate formulation. Homogeneity was confirmed gravimetrically. The mean analysed concentration for the three samples (5, 13.5 and 35 mg/mL) remained within 10% of the mean value and the variation was less than 10%.
Duration of treatment / exposure:
Males: 5 weeks; Females: Two weeks before pairing, throughout pairing, gestation and until Day 13 of lactation
Frequency of treatment:
Once daily, 7 days/week
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
equivalent to: 45.2 mg/kg bw/day as active ingredient
Dose / conc.:
135 mg/kg bw/day (nominal)
Remarks:
equivalent to: 122.1 mg/kg bw/day as active ingredient
Dose / conc.:
350 mg/kg bw/day (nominal)
Remarks:
equivalent to: 316.6 mg/kg bw/day as active ingredient
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were chosen based on the results of a 2-week preliminary toxicity study in the Han Wistar rat conducted at the same testing facility. In that study, dose levels of 50, 125 and 300 mg/kg bw/day were well tolerated with no clinical signs or abnormal macroscopic findings. There was a marked initial reduction in male body weight gain at 300 mg/kg bw/day and an overall marked reduction of weight gain over Days 1-15 of study in females receiving 300 mg/kg bw/day, compared with animals receiving 50 mg/kg bw/day. As the LD50 has been reported as 500 mg/kg (tesed at the same testing facility), a high dose level of 350 mg/kg bw/day (as active ingredient) was selected for use on this study. The low dose remained at 50 mg/kg bw/day. The geometric mean was 132.3 mg/kg bw/day, so 135 mg/kg bw/day was selected as the mid dose level to allow for assessment of a dose response.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes, animals were inspected visually for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s).
- Time schedule: at least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes, detailed observations, detailed physical examination and arena observations were performed in parental animals.
- Time schedule: Males were inspected daily in Week 1 and thereafter once each week. Females were inspected daily in Week 1, once in Week 2, on Days 0, 7, 14 and 20 during gestation phase and on Days 1, 6 and 12 during lactation phase. Sensory reactivity and grip strength assessments were performed and motor activity were measured (all before dosing) on the five lowest numbered surviving males in each group during Week 5 of treatment and on the first five lactating females in each group at Day 7-9 of lactation.

BODY WEIGHT: Yes, the body weight of all animals was recorded
- Time schedule for examinations: For males, before dosing on the day of treatment and weekly thereafter, and on the day prior to necopsy and on the day of necropsy. For females, before dosing on the day that treatment commenced (Day 1) and weekly before pairing, on Day 0, 7, 14 and 20 after mating, on Day 1, 4, 7 and 13 of lactation and on the da prior to necropsy and on the day of necropsy.

FOOD CONSUMPTION: The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded.
- the mean weekly or daily consumption per animal (g/animal/week or g/animal/day) was calculated for each phase.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at termination
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, overnight
- How many animals: The five lowest numbered surviving males and the first five females with a surviving litter, in each dose group.
- Parameters checked: Haematocrit, haemoglobin concentration, erythrocyte count, absolute reticulocyte count, mean cell hemoglobin, mean cell haemoglobin concentration, mean cell volume, red cell distribution width, total leucocyte count, differential leucocyte count (neutrophils, lymphocytes, basophils, monocytes, large unstained cells), plateled count, prothrombin time, activated partial thromplastin time and thyroid hormone analysis

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at termination
- Animals fasted: Yes, overnight
- How many animals: The five lowest numbered surviving males and females per group.
- Parameters checked: alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, total bilirubin, bile acids, urea, creatinine, glucose, total cholesterol, tryglycerides, sodium, potassium, chloride, calcium, inorganic phosphorus, total protein, albumin
and albumin/globulin ratio
Oestrous cyclicity (parental animals):
Dry smears were taken for 15 days before pairing using cotton swabs.
Wet smears were taken using pipette lavage during the following phases:
- For 14 days before treatment (all females including spares); animals that failed to exhibit 4-5 day cycles were not allocated to study
- After pairing until mating
- For four days before scheduled termination
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, live births, weight gain, anogenital distance (AGD), presence of nipples/areolae in male pups

GROSS EXAMINATION OF DEAD PUPS:
Macroscopic examination of offspring that either died prematurely or at scheduled termination on Day 13 of age was performed.
Postmortem examinations (parental animals):
GROSS PATHOLOGY: All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative. Males were sacrificed after Week 5 investigations were completed. Females were sacrificed either on Day 25 after mating, if the females failed to produce a viable litter, or on or after the day the last offspring died, if the litter died before Day 13, or on Day 14 of lactation following terminal blood sampling.
- Organs checked: Adrenals, bone marrow smear, brain (including cerebrum, cerebellum and pons), cecum, colon, duodenum, epididymides, eyes, heart (including auricular and ventricular regions), ileum , jejunum, kidneys, liver (section from two lobes), lungs (section from two major lobes including bronchi), lymph nodes (left axillary and mesenteric) , ovaries, Peyer’s Patch, pituitary, prostate, sciatic nerve, seminal vesicles with coagulating glands, skeletal muscle, skin with mammary glands (inguinal area), spinal cord (transverse and longitudinal sections at the cervical level), spleen, sternum (with marrow), stomach, testes, thymus, thyroid, trachea, urinary bladder, uterus (including cervix and oviducts) and vagina

HISTOPATHOLOGY: Histopathological examination was performed for 5 surviving males and 5 lactating femals with surviving litter each of the control and of the highest dose group, respectively, and for all animals in case of abnormalities occurred. Dehydrated tissue samlples were embebbed in paraffin wax
- Organs checked: Adrenals, brain (including cerebrum, cerebellum and pons), cecum, colon, duodenum, epididymides, eyes, heart (including auricular and ventricular regions), ileum , jejunum, kidneys, liver (section from two lobes), lungs (section from two major lobes including bronchi), lymph nodes (left axillary and mesenteric) , ovaries, Peyer’s Patch, prostate, sciatic nerve, seminal vesicles with coagulating glands, skeletal muscle, skin with mammary glands (inguinal area), spinal cord (transverse and longitudinal sections at the cervical level), spleen, sternum (with marrow), stomach, testes, thymus, thyroid, trachea, urinary bladder, uterus (including cervix and oviducts) and vagina
Postmortem examinations (offspring):
SACRIFICE
- F1 offspring was sacrificed on Day 13 of age and selected offspring for thyroid hormone analysis was sacrificed on Day 4 of age.

GROSS NECROPSY
- F1 offspring on Day 4 of age: For blood sampling for thyroid hormone analysis and external examination. Externally abnormal offspring examined, and retained pending possible future examination.
- F1 offspring on Day 13 of age: For blood sampling for thyroid hormone analysis and all animals were subject to an external macroscopic examination; particular attention was paid to the external genitalia. Animals observed with external abnormalities were retained pending possible future examination. Thyroid glands were preserved from one male and one female in each litter.
Statistics:
For grip strength, motor activity, clinical pathology, litter size and survival indices, if 75% of the data (across all groups) were the same value, for example c, Fisher’s exact tests (Fisher 1973) were performed. Treatment groups were compared using pairwise comparisons of each dose group against the control both for i) values c, as applicable.
For gestation length an exact two-tailed Linear-by-linear test (Cytel 1995), with equally spaced scores, was applied to all groups. If the test was statistically significant (p<0.05), the highest dose group was excluded and the test re-applied. This ‘step-down’ process was repeated until the test was no longer statistically significant (p≥0.05). If the exact version of the Linear-by-linear test could not be calculated (due to the size of the table containing the
data), then the asymptotic version was used instead.
Sex ratio was analyzed by generalized mixed linear model with binomial errors, a logit link function and litter as a random effect (Lipsitz 1991). Each treated group was compared to control using a Wald chi-square test. For sex ratio, the numerator was Number of males, the denominator was Number of live fetuses.
For organ weight data, analysis of covariance was performed using terminal body weight as covariate (Angervall and Carlstrom, 1963), unless non-parametric methods were applied. The treatment comparisons were made on adjusted group means in order to allow for differences in body weight which might influence the organ weights. Similarly, for the litter average ano-genital distance, analysis of covariance was performed using the average pup body weight for each litter as the covariate.
Reproductive indices:
- Fertility Index [%] = Number of animals achieving pregnancy/Animals pairing x 100
- Gestation Index [%] = Number of litters born/Number of pregnant rats x 100
- Conception rate [%] = Number of animals achieving pregnancy/Animals mated x 100
- Mating index [%] = Number of animals mating/Animals paired x 100
Offspring viability indices:
- Post-implantation survival index [%] = Total number of offspring born/Total number of uterine implantation sites x 100
- Live Birth Index [%] = Number of live offspring on Day 1 after littering/Total number born (live + dead) x 100
- Viability Index [%] = Number of live offspring on Day 4 after littering/Number of live offspring on Day 1 (after blood sampling) x 100
- Lactation index [%] = Number of live offspring on Day 13 after littering/Number of live offspring on Day 4 (after blood sampling) x 100
- Percentage males = Number of males in litter/Total number of offspring in litter x 100
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Sporadical salivation and/or post dose salivation was observed in 6/10 males and in 6/10 females of the mid dose group, and in 8/10 males and all females of the highest dose group, respectively. In 1/10 high-dosed males, piloerection and underactivity were observed after the first dose administration, which disappeared thereafter.
At the routine weekly examination, vocalisation was observed in 3/10 females receiving 50 mg/kg bw/day, 2/10 males and 3/10 females receiving 135 mg/kg bw/day, and 5/10 males and 1/10 females receiving 350 mg/kg bw/day when compared with 1/10 control males and 3/10 control females.
The only other clinical sign observed was ventral body hair loss in one female receiving 135 mg/kg/day during lactation; this was not considered to be related to treatment with the test substance according to the study director.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant reduced bodyweight gain was observed during Days 1 - 8 (62% lower), 8 - 15 (37% lower), 15 - 22 (92% lower), 1 - 15 (54% lower) and 1 - 36 (43% lower) of treatment for males receiving 350 mg/kg bw/day, when compared with controls. Group mean bodyweight and bodyweight change for females receiving the test substance was similar to controls prior to pairing, however bodyweight change was statistically significantly lower during Day 0 - 7 (27% lower) and 7 - 14 (30% lower) of gestation for females receiving 350 mg/kg bw/day when compared with controls. Bodyweight gain was lower (75% lower) during Days 1 - 4 of lactation for females receiving 350 mg/kg bw/day. However, during Days 7 - 13 of lactation bodyweight gain was statistically significantly higher for females receiving 350 mg/kg bw/day when compared with controls.

Please refer to Table 1 and Table 2 under "any other information on results incl. tables".
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Group mean food consumption was low for males receiving 350 mg/kg bw/day during Week 1 of treatment (20% lower) and to a lesser extent in Week 2 (12% lower) when compared to controls.
At 350 mg/kg bw/day food intake of females was marginally low during Week 1 of treatment (15% lower) and marginally but statistically significantly low during gestation (11% lower during Days 7-12 and 10% lower during Days 13-19) and was 17% lower (but not statistically significant) during Days 1 to 3 of lactation. Food consumption was similar to controls for females receiving 50 or 135 mg/kg bw/day of the test substance prior to pairing, during gestation and lactation.

Please refer to Table 3 under "any other information on results incl. tables".
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Haematological examination at termination, following five weeks of treatment for males and seven weeks for females revealed statistically slightly lower male haematocrit levels and lower female prothrombin time for all treatment groups when compared with controls.
Haemoglobin levels were statistically lower and lymphocyte counts were statistically higher for males receiving 135 or 350 mg/kg bw/day when compared with controls. Mean cell haemoglobin, mean cell volume and activated partial thromboplastin time were all statistically reduced and white blood cell, neutrophil and eosinophil counts were statistically higher for males receiving 350 mg/kg bw/day when compared with controls.

Please refer to Table 4 and Table 5 under "any other information on results incl. tables".
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
When compared with the controls, glucose (79 and 78%, respectively) and creatinine (88 and 83%, respectively) levels were decreased for males receiving 135 or 350 mg/kg bw/day. Potassium (111 and 113%, respectively) and phosphorus (118 and 131%, respectively) levels were statistically significantly higher for males receiving 135 mg/kg bw/day or 350 mg/kg bw/day; phosphorus (126%) levels were also statistically significantly higher for females receiving 350 mg/kg bw/day.
Chloride (98 and 94% for males and females, respectively) levels were statistically significantly lower for animals receiving 350 mg/kg bw/day and total protein (90%) and albumin (92%) levels were statistically significantly lower for males receiving 350 mg/kg bw/day.
Plasma levels of bile acids for males and females treated the test substance were highly variable, and statistical significance was not attained, these findings were considered due to individual variation.
All other inter-group differences from controls were minor, were confined to one sex or lacked a dose-relationship; these values were considered fortuitous and were therefore considered of no toxicological importance.

Please refer to Table 4 and Table 5 under "any other information on results incl. tables".
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Group mean hindlimb grip strength values for females receiving 350 mg/kg bw/day were statistically significantly low when compared with controls.
The majority of group mean activity scores for both high and low beams for all groups of treated males and females were low compared with controls, and statistical significance was achieved for males receiving 135 mg/kg bw/day at the 42-minute interval and the total score and for males receiving 350 mg/kg bw/day at the 6, 24 and 42-minute interval and the total score (all high beam). The low beam total score for females receiving 350 mg/kg bw/day has attained statistical significance as has the 18 and 42-minute interval and total score for females receiving 350 mg/kg bw/day (also low beam). There was a clear dose-related reduction at both high and low beam activity as seen by the total scores for both males and females.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Organ weights for males killed after 5 weeks of treatment revealed significantly lower terminal bodyweight and the group mean unadjusted (78%) and adjusted (70%) thymus weights were lower for males that received 350 mg/kg bw/day, when compared with controls although the difference was not statistically significant. Adjusted kidney weights (112%) were marginally but statistically higher in high-dosed males than in controls.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Dark areas were seen in the glandular mucosa of the stomach of animals treated at all dose levels and in controls:
Control: 0/10 males and 2/10 females
50 mg/kg bw/day: 1/10 males and 1/10 females
135 mg/kg bw/day: 0/10 males and 3/10 females
350 mg/kg bw/day: 5/10 males and 1/10 females
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Stomach
Inflammatory cells, predominantly neutrophils, were scattered throughout the glandular mucosa in both sexes, all dose levels and one control female, mostly at minimal severity. In most males and three females given 350 mg/kg bw/day, this was accompanied by minimal to moderate eosinophilic globules in mucous neck cells. This same change was seen at minimal severity in four males given 135 mg/kg bw/day and one male given 50 mg/kg bw/day. These two changes were occasionally associated with a minimal degree of apoptosis of the mucous neck cells. A submucosal inflammatory cell infiltrate, mostly lymphocytic, was present in five males and one female given 350 mg/kg bw/day, and in one male given 50 mg/kg bw/day. Mucosal congestion was seen in four males given 350 mg/kg bw/day, in two females given 135 mg/kg bw/day and in two control females, and correlated in most cases with dark areas seen at necropsy. Minimal to slight mucosal erosion was seen at low incidence in males given 350 mg/kg bw/day and females at all dose levels. Overall, the changes in the stomach were seen at a higher incidence and severity in males.

Thymus
Involution/atrophy, which can be background change, was seen at minimal severity but higher incidence in males given 350 mg/kg bw/day and correlated with decreased absolute and adjusted thymus weight.

Prostate
Mixed inflammatory cell infiltrate was seen in the dorsolateral lobe in most males given 350 mg/kg bw/day at minimal to moderate severity and at minimal to slight severity in six males given 135 mg/kg bw/day.

Epididymides
Increase in luminal cell debris was seen at minimal to slight severity in eight males given 350 mg/kg bw/day and at minimal severity in three males given 135 mg/kg bw/day, one male given 50 mg/kg bw/day and one control.

Please refer to Table 6 under "any other information on results incl. tables".
Histopathological findings: neoplastic:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
One Control female and one female receiving 350 mg/kg bw/day were not pregnant, and one female receiving 350 mg/kg bw/day had total litter loss on Day 6 of gestation.
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks:
equivalent to 45.2 mg/kg bw/day as active ingredient
Sex:
male/female
Basis for effect level:
other: No adverse and treatment-related effects were observed at 50 mg/kg bw/day
Key result
Dose descriptor:
NOAEL
Remarks:
reproduction
Effect level:
135 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: marked reduction in mean offspring body weights on Day 1 of age at 350 mg/kg bw/day
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There was a marked reduction in offspring bodyweights on Day 1 of age with parents dosed at 350 mg/kg bw/day. Subsequently, bodyweight and bodyweight gain of F1 Offspring males and females at this level were slightly low when compared to concurrent controls.
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:
no effects observed
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
effects observed, treatment-related
Description (incidence and severity):
The mean adjusted ano-genital distance in male offspring in the 350 mg/kg bw/day was marginally but statistically significantly higher than in the controls; this direction of difference is considered to be of no toxicological significance.
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Other effects:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Remarks:
developmental
Generation:
F1
Effect level:
135 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: marked reduction in mean offspring body weights on Day 1 of age at 350 mg/kg bw/day
Key result
Reproductive effects observed:
no

Table 1: Body weight and body weight change - group mean values (g) for offspring (F1)

Dose level (mg/kg bw/day)   Day of age (before blood sampling) Day of age (after blood sampling) Change
  1 4 4 7 13 1-4 4-7 7-13 1-13
MALES
Control Mean 6.6 9.7 9.7 14.6 25.7 3.1 4.9 11.1 19.2
SD 0.5 0.7 0.7 1.2 3.1 0.3 0.6 2.1 2.8
N 9 9 9 9 9 9 9 9 9
50 Mean 6.5 9.3 9.3 13.9 25.1 2.9 4.6 11.1 18.6
SD 0.3 0.9 0.9 1.3 2.5 0.6 0.6 1.5 2.3
N 10 10 10 10 10 10 10 10 10
135 Mean 6.5 10.0 10.0 14.9 26.7 3.6 4.9 11.7 20.2
SD 0.8 1.5 1.5 2.0 2.7 0.8 0.6 1.0 2.1
N 10 10 10 10 10 10 10 10 10
350 Mean 5.3** 7.3** 7.3** 11.2** 21.3** 2.0** 3.7** 10.2 16.0*
SD 0.4 1.0 1.0 1.6 2.7 0.7 0.8 1.3 2.4
N 9 9 9 8 8 9 8 8 8
FEMALES
Control Mean 6.2 9.3 9.4 14.1 25.1 3.2 4.7 11.1 19.0
SD 0.5 0.7 0.7 1.1 3.1 0.3 0.6 2.2 2.9
N 9 9 9 9 9 9 9 9 9
50 Mean 6.2 9.1 9.1 13.6 24.4 2.9 4.5 10.8 18.2
SD 0.4 0.9 1.0 1.6 2.8 0.7 0.8 1.5 2.5
N 10 10 10 10 10 10 10 10 10
135 Mean 6.2 9.8 9.8 14.7 26.5 3.5 4.9 11.7 20.2
SD 0.8 1.6 1.5 2.0 2.8 0.8 0.6 1.1 2.2
N 10 10 10 10 10 10 10 10 10
350 Mean 5.1** 7.1** 7.0** 10.7** 20.7** 1.9** 3.5** 10.0 15.7*
SD 0.5 1.0 1.0 1.6 2.7 0.7 0.7 1.2 2.3
N 9 9 9 8 8 9 8 8 8
Conclusions:
The NOAEL for reproductive and developmental toxicity is 135 mg/kg bw/day (122.1 mg/kg bw/day as active ingredient) due to the marked reduction in mean offspring body weights on Day 1 of age at 350 mg/kg bw/day. However, body weights of pups of dams administered the test substance at 350 mg/kg bw/day were significantly lower than controls at birth through to Day 13 of age and may have resulted from reduced food consumption in these dams during gestation and early lactation.
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
135 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The available information comprises an adequate and reliable study, and is thus sufficient to fulfill the standard information requirements set out in Annex VIII-IX, 8.6, of Regulation (EC) No 1907/2006.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

In a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test according to OECD 422 and under GLP conditions, 10 male and 10 female Wistar rats received Zinc, bis[O,O-bis(1-methylethyl) phosphorodithioato-.kappa.S]bis(cyclohexanamine)-, (T-4)- (CAS 52585-16-7) by gavage at dose levels of 50, 135 and 350 mg/kg bw/day (equivalent to 45.2, 122.1 and 316.6 mg/kg bw/day active ingredient); dose selection was based on the results of a preliminary range-finding study at the same testing facility (Envigo CSR, 2017). Males were exposed for 5 weeks, and females were exposed for two weeks before pairing, throughout pairing, gestation and until Day 13 of lactation. Parental animals were observed at least twice daily for mortality and clinical signs. Body weights and food consumption were recorded weekly. Wet and dry smears were taken to investigate estrous cyclicity in females. All adult animals were subject to a detailed necropsy. At necropsy, haematological, clinical biochemistry parameters and organ weights were determined. Histopathological examination was performed for 5 surviving males and 5 lactating females with surviving litter each of the control and of the highest dose group, respectively, and for all animals in case of abnormalities occurred.

Based on the results of this study the NOAEL for reproductive toxicity is 135 mg/kg bw/day due to the marked reduction in mean offspring body weights on Day 1 at the highest tested dose level, no adverse effects regarding toxicity to reproduction were noted. Regarding systemic toxicity, the NOAEL is set at 50 mg/kg bw/day (45.2 mg/kg bw/day as active ingredient) (please refer to 7.5 for more details).

Effects on developmental toxicity

Description of key information

OECD 422 (oral, rat): NOAEL = 135 mg/kg bw/day for developmental toxicity

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
135 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The available information comprises an adequate and reliable study, and is thus sufficient to fulfill the standard information requirements set out in Annex VIII-IX, 8.6, of Regulation (EC) No 1907/2006.
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 Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test according to OECD 422 and under GLP conditions, 10 male and 10 female Wistar rats received Zinc, bis[O,O-bis(1-methylethyl) phosphorodithioato-.kappa.S]bis(cyclohexanamine)-, (T-4)- (CAS 52585-16-7) by gavage at dose levels of 50, 135 and 350 mg/kg bw/day (equivalent to 45.2, 122.1 and 316.6 mg/kg bw/day active ingredient); dose selection was based on the results of a preliminary range-finding study at the same testing facility (Envigo CSR, 2017). Males were exposed for 5 weeks, and females were exposed for two weeks before pairing, throughout pairing, gestation and until Day 13 of lactation. The number and sex of pups, live births, weight gain, anogenital distance (AGD), presence of nipples/areolae in male pups were examined for the F1 generation. Macroscopic examination of offspring that either died prematurely or at scheduled termination on Day 13 of age was performed. The mean adjusted ano-genital distance in male offspring in the 350 mg/kg bw/day was marginally but statistically significantly higher than in the controls; this direction of difference is considered to be of no toxicological significance. There was a marked reduction in offspring bodyweights on Day 1 of age with parents dosed at 350 mg/kg bw/day. Subsequently, bodyweight and bodyweight gain of F1 Offspring males and females at this level were slightly low when compared to concurrent controls. Based on these results, the NOAEL was set at 135 mg/kg bw/day for the offspring. However, since 50 mg/kg bw/day (45.2 mg/kg bw/day as active ingredient) can be considered as NOAEL for systemic toxicity and for local effects in parental animals (please refer to 7.5 for more details), the effects on body weight in the offspring can be regarded as secondary effect.

Justification for classification or non-classification

The available data on toxicity to reproduction with the registered substance do not meet the criteria for classification according to Regulation (EC) No 1272/2008, however since no pre-natal developmental toxicity study and/or no extended one-generation toxicity study is available, the conclusion is data lacking.