Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

A combined repeated dose toxicity study with reproduction/developmental toxicity screening test was performed according to OECD 422 and in accordance with GLP principles. Administration of Diazo PW 980 by once daily oral gavage was well tolerated in rats at levels of 1000 mg/kg bw/day without in-life observations of toxicity or histopathological findings in the F0 or F1 generation. There was no evidence of test item related changes to the mating and fertility, maternal behavior or pre-weaning viability. Mean litter weights and pre-weaning physical development were similar in all dose groups. Based on these results, the no-observed-effect level (NOEL) of parental, reproduction (up to and including implantation) and developmental (from implantation onwards) was considered to be 1000 mg/kg bw/day.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 July 2019 - 9 June 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Qualifier:
according to guideline
Guideline:
other: The United States Environmental Protection Agency (EPA) Health Effects Test Guidelines, OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test.
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient condition, protected from light
- Stability: The stability of the bulk test item was not determined during the course of this study. Stability analyses performed previously in conjunction with study 20190830 demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.
Species:
rat
Strain:
other: CRL:WI (Han)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River UK, Margate, Kent, UK
- Age at study initiation: 11 to 12 weeks
- Weight at study initiation: 249 - 392 g (males); 174 - 247 g (females)
- Housing: Animals were initially housed 2 or 3 per cage by sex (unless reduced by mortality) in appropriately sized suspended polycarbonate cages with stainless steel grid tops and solid bottoms. Bedding material was sterilised white wood shavings. A few days prior to mating, males were transferred to individual cages with solid bottoms. Mated females were transferred to individual solid bottomed cages. White paper tissue was supplied as nesting material from Gestation Day (GD) 20. Females with litters
were retained in this type of cage until termination. After mating, the males were re-housed with their original cages mates.
- Diet: SDS VRF-1 breeder diet, ad libitum (except during designated procedures)
- Water: tap water, ad libitum
- Acclimation period: 5 weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 18 to 26°C (actual range)
- Humidity (%): 28 to 89% (actual range)
- Air changes: Ten or greater air changes per hour
- Photoperiod: 12-hour light/12-hour dark cycle

IN-LIFE DATES: From: 22 July 2019 To: 29 September 2019

Deviations: The protocol stated that the target environmental conditions would be 19 to 23°C for temperature and 40 to 70% for humidity. The actual ranges were 18 to 26°C and 28 to 90% respectively. Animal welfare checks (morning and afternoon) were performed on all the study animals during these dates and all animals appeared unaffected from the transient change in environmental conditions. Therefore, this deviation had no impact on the outcome of the study.
Route of administration:
oral: gavage
Vehicle:
other: 0.25% Methocel KM4 premium in water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations were prepared based on a method established at the Test Facility at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared at least weekly, stored in a refrigerator set to maintain 4˚C, protected from light, or transferred to animal unit immediately and dispensed daily.

VEHICLE
- Concentration in vehicle: 10.7 mg/L; 32.1 mg/mL; 107 mg/mL
- Amount of vehicle: 10 mL/kg
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: maximum of 14 nights
- Proof of pregnancy: vaginal plug and/or 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:
Analyses were performed by ultra-performance liquid chromatography-ultra violet (UPLC-UV) using a validated analytical procedure (20190830).
Duplicate top, middle and bottom (middle only for control) samples for each sampling time point were sent to the analytical laboratory. Triplicate top, middle and bottom (middle only for control) samples were collected for each sampling occasion as backup. Week 2 and 5 backup samples were shipped to the analytical laboratory for analysis and Week 1 back up samples were retained at the Test Facility. Sample volumes were 1 mL for Day 1 and Week 5 collections and 0.5 mL for Week 2 collections. Concentration results were considered acceptable if sample concentration results were within or equal to ±10% of theoretical concentration. Each individual sample concentration result was considered acceptable if it was within or equal to ± 15%.
Homogeneity results were considered acceptable if the relative standard deviation (RSD) of the mean value at each sampling location was ≤ 5%.
Duration of treatment / exposure:
The males were dosed for at least 4 weeks overall, starting from 2 weeks prior to mating. The females were dosed from 2 weeks prior to mating, then continuing until the day prior to termination.
Frequency of treatment:
Once daily
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Group 2
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
Group 3
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
Group 4
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on information provided from the previous dose range finding study (490089) where the administration of the substance by once daily for 14 days was well tolerated in Han Wistar rats at dose levels up to 1000 mg/kg bw/day with no effects on clinical observations, body weights, body weight gains or food consumption.
- Fasting period before blood sampling for clinical biochemistry: no
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least weekly beginning Week -1

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed weekly from Week -1 and mated females were weighed on Gestation Day (GD) 0, 4, 7, 11, 14, 17 and 20 and on Lactation Day (LD) 0 (when possible for dosing purposes), 1, 4, 7, 10 and 13

FOOD CONSUMPTION:
- Time schedule: Food consumption was quantitively measured weekly for both sexes from Week -1 until pairing for mating, and on GD 0 to 4, 4 to 7, 7 to 11, 11 to 14, 14 to 17, and 17 to 20 and LD 1 to 4, 4 to 7, 7 to 10, and 10 to 13 for the mated females, where possible. Weekly food consumption resumed for the males following mating.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Water consumption was monitored on a regular basis throughout the study by visual inspection of the water bottles.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week 4 (males); Lactation day 11/12 (females)
- How many animals: 5 selected animals/sex/group
- Anaesthetic used for blood collection: Not specified
- Parameters checked: Red blood cell count, Haemoglobin, Haematocrit, Mean cell volume, Mean cell haemoglobin concentration, Mean cell haemoglobin Reticulocytes, Reticulocyte count (absolute), Red blood cell distribution width, Platelets, White blood cell count, Neutrophils count (absolute), Lymphocytes count (absolute), Monocytes count (absolute), Eosinophils count (absolute), Basophils count (absolute), Large unstained cells (absolute)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 4 (males); Lactation day 11/12 (females)
- Animals fasted: Not specified
- How many animals: Five males and 5 females per group
- Parameters checked: Urea, Glucose, Aspartate aminotransferase, Alanine aminotransferase, Alkaline phosphatase, Creatine phosphokinase, Lactate dehydrogenase, Sodium, Potassium, Chloride, Total protein, Albumin, Globulin, Albumin/globulin ratio, Cholesterol, Creatinine, Total bilirubin, Calcium Inorganic phosphate, Triglycerides, Total bile acids

TSH, T4 and T3: Yes
- Time schedule for collection of blood: Prior to necropsy
- Animals fasted: No
- How many animals: all males and females
- Parameters checked: TSH, T4 and T3

FUNCTIONAL OBSERVATIONS: Yes
- Time schedule for examinations: for 5 selected males per group (prior to blood sampling) once during Week 4 and for the first 5 females per group which reared their litter to at least LD 10 to 12 (prior to blood sampling) during lactation
- Battery of functions tested: functional observation battery included homecage observations (posture, temperature), handling observations, air righting, extensor thrust, observations in a standardized arena (2 minute observation), functional test including: reaction to sudden sound, reaction to touch, grip strength, pain perception, landing foot splay, motor activity.
Oestrous cyclicity (parental animals):
Over a 14 day period prior to test item administration, then continuing through pre-mating test item administration and mating period, vaginal lavages were taken early each morning and the stages of oestrous observed was recorded. Vaginal smears were examined on the morning of necropsy to determine the stage of oestrus cycle to allow correlation with histopathology of ovaries.
Sperm parameters (parental animals):
Testis weight, epididymis weight, staging of spermatogenesis
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- Maximum of 8 pups/litter (4/sex/litter as nearly as possible) was selected from each litter; excess pups were 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. Particular attention was paid to the external reproductive genitals which were examined for signs of altered development; gross evaluation of external genitalia.

BLOOD SAMPLES (or total Thyroxine (Total T4) determination)
- from 1 pup/sex/litter on PND 4 and 13

GROSS EXAMINATION OF DEAD PUPS:
- yes, for external abnormalities; any externally abnormal decedent pup was preserved; externally normal pups were discarded.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals (week 6)
- Maternal animals: All surviving animals (LD 13)

GROSS NECROPSY
- Gross necropsy consisted of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues.
The reproductive tracts of all females were examined for signs of implantation, the number of any implantation sites being recorded. The total number of corpora lutea graviditatis was recorded for each female.

HISTOPATHOLOGY
- Organs were collected, processed and evaluated in accordance with the applicable guideline.
- Full tissues of unscheduled deaths from all groups.
- Full tissues of the 5 male and 5 female Group 1 and Group 4 animals used for laboratory investigations.
- The reproductive organs (cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles, testes, uterus, and vagina) of all Group 1 and Group 4 animals
- Testes of all Group 1 and Group 4 males, examined for staging of spermatogenesis (qualitative evaluation).
- Gross lesions of all animals (all groups).

ORGAN WEIGHTS:
- Brain, Epididymis, Adrenal gland, Parathyroid gland, Pituitary gland, Prostate gland, Seminal vesicle gland, Coagulation gland, Thyroid gland, Heart, Kidney, Liver, Ovary, Spleen, Testis, Thymus, Uterus/Cervix
Postmortem examinations (offspring):
TERMINATION;
- Samples of any abnormal tissues were taken and fixed in neutral buffered 10% formalin. All pups were examined by open dissection for internal sex confirmation. Thyroid with parathyroid were collected from one pup of each sex per litter. The remaining carcasses were discarded.

ORGAN WEIGHTS:
- Thyroid gland (after fixation).

OTHER:
- The thyroid gland was examined microscopically from 1 male and 1 female F1 generation pup per litter from Groups 1 and 4 only.
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and are reported at the 1% and 5% levels, unless otherwise noted. The pairwise comparisons of interest are listed below:
- Group 2 vs. Group 1
- Group 3 vs. Group 1
- Group 4 vs. Group 1

Levene’s test was used to assess the homogeneity of group variances. The groups were compared using an overall one-way ANOVA F-test if Levene’s test was not significant or the Kruskal-Wallis test if it was significant. If the overall F-test or Kruskal-Wallis test was found to be significant, then pairwise comparisons were conducted using Dunnett’s or Dunn’s test, respectively.
Reproductive indices:
Fertility Index (male) = Number siring a litter/Number paired
Fertility Index (female) = Number pregnant/Number paired
Gestation Index = Number bearing live pups/Number pregnant
Offspring viability indices:
Birth Index = Total number of pups born (live and dead)/Number of implantation scars
Live Birth Index = Number of pups live on Day 0 of lactation/Total number born (live and dead)
Viability Index = Number of pups live on Day 4 of lactation/Number live on Day 0
Clinical signs:
no effects observed
Description (incidence and severity):
At dose levels up to and including 1000 mg/kg bw/day, there were no adverse clinical observations considered to be related to treatment. Abnormal coloured faeces (yellow) were observed in the males and females of the high dose group. However, this finding was considered not to be adverse. All other clinical observations were considered to be normal background findings typical of rats of this strain and were observed across all the dose groups, including controls. Therefore, considered unrelated to treatment.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Two control females were euthanised unscheduled on Gestation Day 11 (Day 27 of the study) due to clinical signs of decreased muscle tone in both forelimbs and swollen abdomen resulting in an abnormal gait. One animal was also noted to have increased vocalisation. There appeared to be no effects on body weights or food consumption. One animal dark red discoloration of the thymus was observed during gross necropsy examination. This finding correlated microscopically with agonal haemorrhage. Moderate axonal degeneration was also noted microscopically in the lumbar spinal nerves attached to the lumbar spinal cord segment and this was associated with mild, regionally extensive myofiber atrophy and replacement by adipose tissue in skeletal muscle from the hindlimb. The second control animal was euthanised there were no gross necropsy findings, however, mild axonal degeneration was also noted microscopically in the lumbar spinal nerves accompanied by mild, regionally extensive hindlimb skeletal muscle myofiber atrophy and replacement by adipose tissue, similar to that seen in the first control animal that was euthanised. Although lumbar spinal nerve degeneration and secondary skeletal muscle myofiber atrophy would be expected to be associated with clinical signs in the hindlimbs (e.g. abnormal gait and decreased muscle tone), given the nature of these microscopic changes it is considered likely that this may be indicative of a more generalised/multifocal condition which could account for the gait/forelimb changes described clinically in these animals. Irrespective, as both animals were in Group 1 (Control) and had not received any test item, these findings cannot be associated with the treatment.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There were minor changes that occasionally achieved statistical significance such as Day 8 to 15, Group 4 males at 1000 mg/kg bw/day body weight gain was noted to be 48% lower in comparison to the Control male group with an overall 18% lower body weight gain from Day 1 to 42. In addition, on Day 15 to 22 Group 2 males at 100 mg/kg bw/day body weight gain was 62% lower in comparison to the Control male group. However, due to the low incidence and lack of a dose related pattern, these findings were considered sporadic and unrelated to treatment.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were minor changes that achieved statistical significance however, noted changes were considered to be within normal biological variation and/or due to a small magnitude of difference. They were therefore considered incidental and unrelated to treatment.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were minor changes that were considered to be within normal biological variation and/or due to a small magnitude of difference. They were therefore considered incidental and unrelated to treatment.
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
At dose levels up to 1000 mg/kg bw/day, there were no test item-related microscopic findings in F0 generation adults.
Other effects:
no effects observed
Description (incidence and severity):
At dose levels up to and including 1000 mg/kg bw/day, there were no treatment related changes in the thyroid hormone parameters noted in the F0 animals.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
At dose levels up to and including 1000 mg/kg bw/day, there were no treatment related changes in the duration or pattern of the oestrus cycle.
Reproductive performance:
no effects observed
Description (incidence and severity):
At dose levels up to and including 1000 mg/kg bw/day, there were no treatment related changes in the mating performance.
At dose levels up to and including 1000 mg/kg bw/day, there were no treatment related effects on mating performance. There were no effects on gestation length, group means ranged from 21.9 to 22.3 days (typical for this strain of rat). All pregnant females produced a live litter, giving a gestation index of 100%. The mean number of live pups per litter was also similar over all groups. A higher total number of pups born and higher male: female pup ratio was observed in all treated groups when compared with the controls. This was a result of two control animals being euthanised early.
Key result
Dose descriptor:
NOAEL
Remarks:
Parental
Effect level:
1 000 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: based on the absence of adverse effects up to and including the highest dose levels tested.
Key result
Dose descriptor:
NOAEL
Remarks:
Fertility
Effect level:
1 000 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: based on the absence of adverse effects up to and including the highest dose level tested.
Key result
Critical effects observed:
no
Mortality / viability:
no mortality observed
Description (incidence and severity):
Survival rates for the pups were typical of this strain of rat and there was no test item effect on the viability index of the pups over Lactation Day 0 to 4 or, following litter standardisation, between Lactation Days 4 to 13.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The mean litter weight and the mean litter-mean pup weight of males and females was similar over all groups.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were no treatment related changes in the thyroid hormone parameters in PND 4 pooled samples or the PND 13 individual samples. However, majority of thyroid stimulation hormone (TSH), including in the control group were found to be
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
At dose levels up to 1000 mg/kg bw/day, there were no test item differences in the anogenital distance of male or female pups on PND 1.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
At dose levels up to 1000 mg/kg bw/day, there were no test item differences in nipple retention present for males on PND 13.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related thyroid gland weight differences in F1 generation pups. There were individual thyroid gland weight values that were different from their respective controls. There were, however, no patterns or correlating data to suggest these values were related to treatment.
Gross pathological findings:
no effects observed
Description (incidence and severity):
At dose levels up to and including 1000 mg/kg bw/day, there were no treatment related gross findings in F1 generation pups.
Histopathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related microscopic findings in the thyroid gland of F1 pups. Minimal focal interstitial neutrophilic infiltration was present in the thyroid gland of a single animal (group 4) and was considered to be an incidental finding and not related to treatment.
Key result
Dose descriptor:
NOAEL
Remarks:
Developmental
Effect level:
1 000 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: based on the absence of adverse effects up to and including the highets dose level tested.
Key result
Reproductive effects observed:
no
Conclusions:
A combined repeated dose toxicity study with reproduction/developmental toxicity screening test was performed according to OECD 422 and in accordance with GLP principles. Administration of Diazo PW 980 by once daily oral gavage was well tolerated in rats at levels of 1000 mg/kg bw/day without in-life observations of toxicity or histopathological findings in the F0 or F1 generation. There was no evidence of test item related changes to the mating and fertility, maternal behavior or pre-weaning viability. Mean litter weights and pre-weaning physical development were similar in all dose groups. Based on these results, the no-observed-effect level (NOEL) of parental, reproduction (up to and including implantation) and developmental (from implantation onwards) was considered to be 1000 mg/kg bw/day.
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Experimental exposure time per week (hours/week):
56
Species:
rat
Quality of whole database:
The information for this endpoint study record was obtained from an experimental study. The OECD GLP criteria were met and the methods applied are fully compliant with OECD TG 422.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Effects on developmental toxicity

Description of key information

A combined repeated dose toxicity study with reproduction/developmental toxicity screening test was performed according to OECD 422 and in accordance with GLP principles. Administration of Diazo PW 980 by once daily oral gavage was well tolerated in rats at levels of 1000 mg/kg bw/day without in-life observations of toxicity or histopathological findings in the F0 or F1 generation. There was no evidence of test item related changes to the mating and fertility, maternal behavior or pre-weaning viability. Mean litter weights and pre-weaning physical development were similar in all dose groups. Based on these results, the no-observed-effect level (NOEL) of parental, reproduction (up to and including implantation) and developmental (from implantation onwards) was considered to be 1000 mg/kg bw/day.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 July 2019 - 9 June 2020
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:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Qualifier:
according to guideline
Guideline:
other: The United States Environmental Protection Agency (EPA) Health Effects Test Guidelines, OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test.
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient condition, protected from light
- Stability: The stability of the bulk test item was not determined during the course of this study. Stability analyses performed previously in conjunction with study 20190830 demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.
Species:
rat
Strain:
other: CRL:WI (Han)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River UK, Margate, Kent, UK
- Age at study initiation: 11 to 12 weeks
- Weight at study initiation: 249 - 392 g (males); 174 - 247 g (females)
- Housing: Animals were initially housed 2 or 3 per cage by sex (unless reduced by mortality) in appropriately sized suspended polycarbonate cages with stainless steel grid tops and solid bottoms. Bedding material was sterilised white wood shavings. A few days prior to mating, males were transferred to individual cages with solid bottoms. Mated females were transferred to individual solid bottomed cages. White paper tissue was supplied as nesting material from Gestation Day (GD) 20. Females with litters
were retained in this type of cage until termination. After mating, the males were re-housed with their original cages mates.
- Diet: SDS VRF-1 breeder diet, ad libitum (except during designated procedures)
- Water: tap water, ad libitum
- Acclimation period: 5 weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 18 to 26°C (actual range)
- Humidity (%): 28 to 89% (actual range)
- Air changes: Ten or greater air changes per hour
- Photoperiod: 12-hour light/12-hour dark cycle

IN-LIFE DATES: From: 22 July 2019 To: 29 September 2019

Deviations: The protocol stated that the target environmental conditions would be 19 to 23°C for temperature and 40 to 70% for humidity. The actual ranges were 18 to 26°C and 28 to 90% respectively. Animal welfare checks (morning and afternoon) were performed on all the study animals during these dates and all animals appeared unaffected from the transient change in environmental conditions. Therefore, this deviation had no impact on the outcome of the study.
Route of administration:
oral: gavage
Vehicle:
other: 0.25% Methocel KM4 premium in water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations were prepared based on a method established at the Test Facility at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared at least weekly, stored in a refrigerator set to maintain 4˚C, protected from light, or transferred to animal unit immediately and dispensed daily.

VEHICLE
- Concentration in vehicle: 10.7 mg/L; 32.1 mg/mL; 107 mg/mL
- Amount of vehicle: 10 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were performed by ultra-performance liquid chromatography-ultra violet (UPLC-UV) using a validated analytical procedure (20190830).
Duplicate top, middle and bottom (middle only for control) samples for each sampling time point were sent to the analytical laboratory. Triplicate top, middle and bottom (middle only for control) samples were collected for each sampling occasion as backup. Week 2 and 5 backup samples were shipped to the analytical laboratory for analysis and Week 1 back up samples were retained at the Test Facility. Sample volumes were 1 mL for Day 1 and Week 5 collections and 0.5 mL for Week 2 collections. Concentration results were considered acceptable if sample concentration results were within or equal to ±10% of theoretical concentration. Each individual sample concentration result was considered acceptable if it was within or equal to ± 15%.
Homogeneity results were considered acceptable if the relative standard deviation (RSD) of the mean value at each sampling location was ≤ 5%.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: maximum of 14 nights
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
The males were dosed for at least 4 weeks overall, starting from 2 weeks prior to mating. The females were dosed from 2 weeks prior to mating, then continuing until the day prior to termination.
Frequency of treatment:
Once daily
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Group 2
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
Group 3
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
Group 4
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on information provided from the previous dose range finding study (490089) where the administration of the substance by once daily for 14 days was well tolerated in Han Wistar rats at dose levels up to 1000 mg/kg bw/day with no effects on clinical observations, body weights, body weight gains or food consumption.
- Fasting period before blood sampling for clinical biochemistry: no
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least weekly beginning Week -1

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed weekly from Week -1 and mated females were weighed on Gestation Day (GD) 0, 4, 7, 11, 14, 17 and 20 and on Lactation Day (LD) 0 (when possible for dosing purposes), 1, 4, 7, 10 and 13

FOOD CONSUMPTION:
- Time schedule: Food consumption was quantitively measured weekly for both sexes from Week -1 until pairing for mating, and on GD 0 to 4, 4 to 7, 7 to 11, 11 to 14, 14 to 17, and 17 to 20 and LD 1 to 4, 4 to 7, 7 to 10, and 10 to 13 for the mated females, where possible.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Water consumption was monitored on a regular basis throughout the study by visual inspection of the water bottles.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Lactation day 11/12
- How many animals: 5 selected animals/sex/group
- Anaesthetic used for blood collection: Not specified
- Parameters checked: Red blood cell count, Haemoglobin, Haematocrit, Mean cell volume, Mean cell haemoglobin concentration, Mean cell haemoglobin Reticulocytes, Reticulocyte count (absolute), Red blood cell distribution width, Platelets, White blood cell count, Neutrophils count (absolute), Lymphocytes count (absolute), Monocytes count (absolute), Eosinophils count (absolute), Basophils count (absolute), Large unstained cells (absolute)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Lactation day 11/12
- Animals fasted: Not specified
- How many animals: 5 females per group
- Parameters checked: Urea, Glucose, Aspartate aminotransferase, Alanine aminotransferase, Alkaline phosphatase, Creatine phosphokinase, Lactate dehydrogenase, Sodium, Potassium, Chloride, Total protein, Albumin, Globulin, Albumin/globulin ratio, Cholesterol, Creatinine, Total bilirubin, Calcium Inorganic phosphate, Triglycerides, Total bile acids

TSH, T4 and T3: Yes
- Time schedule for collection of blood: Prior to necropsy
- Animals fasted: No
- How many animals: all animals
- Parameters checked: TSH, T4 and T3

FUNCTIONAL OBSERVATIONS: Yes
- Time schedule for examinations: for the first 5 females per group which reared their litter to at least LD 10 to 12 (priorto blood sampling) during lactation
- Battery of functions tested: functional observation battery included homecage observations (posture, temperature), handling observations, air righting, extensor thrust, observations in a standardized arena (2 minute observation), functional test including: reaction to sudden sound, reaction to touch, grip strength, pain perception, landing foot splay, motor activity.

OESTROUS CYCLE:
Over a 14 day period prior to test item administration, then continuing through pre-mating test item administration and mating period, vaginal lavages were taken early each morning and the stages of oestrous observed was recorded. Vaginal smears were examined on the morning of necropsy to determine the stage of oestrus cycle to allow correlation with histopathology of ovaries.

SACRIFICE
- Maternal animals: All surviving animals (LD 13)

GROSS NECROPSY
- Gross necropsy consisted of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues. The reproductive tracts of all females were examined for signs of implantation, the number of any implantation sites being recorded. The total number of corpora lutea graviditatis was recorded for each female.

HISTOPATHOLOGY
- Organs were collected, processed and evaluated in accordance with the applicable guideline.
- Full tissues of unscheduled deaths from all groups.
- Full tissues of the 5 female Group 1 and Group 4 animals used for laboratory investigations.
- The reproductive organs (cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles, testes, uterus, and vagina) of all Group 1 and Group 4 animals
- Gross lesions of all animals (all groups).

ORGAN WEIGHTS:
- Brain, Epididymis, Adrenal gland, Parathyroid gland, Pituitary gland, Seminal vesicle gland, Coagulation gland, Thyroid gland, Heart, Kidney, Liver, Ovary, Spleen, Thymus, Uterus/Cervix
Fetal examinations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- Maximum of 8 pups/litter (4/sex/litter as nearly as possible) was selected from each litter; excess pups were killed and discarded.

PARAMETERS EXAMINED
Birth Index = Total number of pups born (live and dead)/Number of implantation scars
Live Birth Index = Number of pups live on Day 0 of lactation/Total number born (live and dead)
Viability Index = Number of pups live on Day 4 of lactation/Number live on Day 0

BLOOD SAMPLES (or total Thyroxine (Total T4) determination)
- from 1 pup/sex/litter on PND 4 and 13

GROSS EXAMINATION OF DEAD PUPS:
- yes, for external abnormalities; any externally abnormal decedent pup was preserved; externally normal pups were discarded.

TERMINATION;
- Samples of any abnormal tissues were taken and fixed in neutral buffered 10% formalin. All pups were examined by open dissection for internal sex confirmation. Thyroid with parathyroid were collected from one pup of each sex per litter. The remaining carcasses were discarded.

ORGAN WEIGHTS:
- Thyroid gland (after fixation)
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and are reported at the 1% and 5% levels, unless otherwise noted. The pairwise comparisons of interest are listed below:
- Group 2 vs. Group 1
- Group 3 vs. Group 1
- Group 4 vs. Group 1

Levene’s test was used to assess the homogeneity of group variances. The groups were compared using an overall one-way ANOVA F-test if Levene’s test was not significant or the Kruskal-Wallis test if it was significant. If the overall F-test or Kruskal-Wallis test was found to be significant, then pairwise comparisons were conducted using Dunnett’s or Dunn’s test, respectively.
Indices:
Fertility Index (female) = Number pregnant/Number paired
Gestation Index = Number bearing live pups/Number pregnant
Birth Index = Total number of pups born (live and dead)/Number of implantation scars
Live Birth Index = Number of pups live on Day 0 of lactation/Total number born (live and dead)
Viability Index = Number of pups live on Day 4 of lactation/Number live on Day 0
Clinical signs:
no effects observed
Description (incidence and severity):
At dose levels up to and including 1000 mg/kg bw/day, there were no adverse clinical observations considered to be related to treatment. Abnormal coloured faeces (yellow) were observed in the animals of the high dose group. However, this finding was considered not to be adverse. All other clinical observations were considered to be normal background findings typical of rats of this strain and were observed across all the dose groups, including controls. Therefore, considered unrelated to the treatment.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Two control females were euthanised unscheduled on Gestation Day 11 (Day 27 of the study) due to clinical signs of decreased muscle tone in both forelimbs and swollen abdomen resulting in an abnormal gait. One animal was also noted to have increased vocalisation. There appeared to be no effects on body weights or food consumption. In one animal dark red discoloration of the thymus was observed during gross necropsy examination. This finding correlated microscopically with agonal haemorrhage. Moderate axonal degeneration was also noted microscopically in the lumbar spinal nerves attached to the lumbar spinal cord segment and this was associated with mild, regionally extensive myofiber atrophy and replacement by adipose tissue in skeletal muscle from the hindlimb. In the second animal that was euthanised there were no gross necropsy findings, however, mild axonal degeneration was also noted microscopically in the lumbar spinal nerves accompanied by mild, regionally extensive hindlimb skeletal muscle myofiber atrophy and replacement by adipose tissue, similar to that seen in the first control animal that was euthanised. Although lumbar spinal nerve degeneration and secondary skeletal muscle myofiber atrophy would be expected to be associated with clinical signs in the hindlimbs (e.g. abnormal gait and decreased muscle tone), given the nature of these microscopic changes it is considered likely that this may be indicative of a more generalised/multifocal condition which could account for the gait/forelimb changes described clinically in these animals. Irrespective, as both animals were in Group 1 (Control) and had not received any test item, these findings cannot be associated with treatment.
Body weight and weight changes:
no effects observed
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were minor changes that achieved statistical significance however, noted changes were considered to be within normal biological variation and/or due to a small magnitude of difference. They were therefore considered incidental and unrelated to treatment.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were minor changes that were considered to be within normal biological variation and/or due to a small magnitude of difference. They were therefore considered incidental and unrelated to treatment.
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Compared to controls, absolute and relative (to both body weight and brain weight) thymus weights were slightly lower in Group 2, 3 and 4 females (although these differences were not statistically significant). In two group 4 animals this correlated microscopically with minimal decreased cellularity in the cortex. However, this microscopic finding was also present in one control animal, with an individual absolute thymus weight lower than that of one group 4 animal.As these differences were small, not statistically significant and lacked any compatible gross finding (e.g. small thymus) and given that a correlating microscopic finding occurred in only one sex (females) and was also present in a control animal, they wereconsidered not to be related to treatment and were considered most likely to be a spontaneous occurrence or related to pregnancy/lactation.
Gross pathological findings:
no effects observed
Description (incidence and severity):
All gross findings observed were considered incidental, of the nature commonly observed in this strain and age of rat or occurred at a similar incidence in control and treated animals, and therefore, were considered not to be related to treatment.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
At dose levels up to 1000 mg/kg bw/day, there were no test item-related microscopic findings in F0 generation adults.
Other effects:
no effects observed
Description (incidence and severity):
At dose levels up to and including 1000 mg/kg/day, there were no treatment related changes in the thyroid hormone parameters noted in the F0 animals.
Dead fetuses:
no effects observed
Description (incidence and severity):
All pregnant females produced a live litter, giving a gestation index of 100%. The mean number of live pups per litter was also similar over all groups. Survival rates for the pups were also typical of this strain of rat and there was no test item effect on the viability index of the pups over Lactation Day 0 to 4 or, following litter standardisation, between Lactation Days 4 to 13.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
At dose levels up to and including 1000 mg/kg bw/day, there were no treatment related effects on gestation length. Group means ranged from 21.9 to 22.3 days (typical for this strain of rat).
Key result
Dose descriptor:
NOAEL
Remarks:
maternal
Effect level:
1 000 mg/kg bw/day (actual dose received)
Basis for effect level:
other: based on the absence of effects up to and including the highest dose level tested.
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
effects observed, non-treatment-related
Description (incidence and severity):
A higher total number of males born: female pup ratio was observed in all treated groups when compared with the controls. This was a result of two control animals being euthanised early.
Changes in litter size and weights:
effects observed, non-treatment-related
Description (incidence and severity):
The mean litter weight and the mean litter-mean pup weight of males and females was similar over all groups. A higher total number of pups born. This was a result of two control animals being euthanised early.
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
Survival rates for the pups were also typical of this strain of rat and there was no test item effect on the viability index of the pups over Lactation Day 0 to 4 or, following litter standardisation, between Lactation Days 4 to 13.
Other effects:
no effects observed
Description (incidence and severity):
There were no treatmen related changes in the thyroid hormone parameters in PND 4 pooled samples or the PND 13 individual samples. However, majority of thyroid stimulation hormone (TSH), including in the control group were found to be The thyroid gland was examined microscopically from 1 male and 1 female F1 generation pup per litter from Groups 1 and 4 only. There were no test item-related microscopic findings in the thyroid gland of F1 pups.
Key result
Dose descriptor:
NOAEL
Remarks:
developmental
Effect level:
1 000 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: based on the absence of effects up to and including the highest dose level tested.
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

Dose Formulation Analyses

No test item was detected in the Group 1 control samples.

Formulation analysis demonstrated that Week 1 samples did not meet the specified acceptance criteria. Out of specification investigation concluded that samples were out of specification due to being unprotected from light. As a result, an additional sampling occasion was performed (Week 2) on samples protected from light. Formulation analysis demonstrated that the Week 2 and 5 prepared formulation samples were accurate and homogenous. Initial analysis of Group 2 and Group 3 samples failed to meet the criterion, therefore back up samples were analysed and were within the specified acceptance criteria:

- Mean sample concentration results within or equal to ± 10% of theoretical concentration (week 2: 91-98%; week 5: 88-95%)

- Each individual sample concentration result within or equal to ± 15% (week 2: 88-106%; week 5: 85-96%)

- A relative standard deviation (RSD) of concentrations of ≤ 5% for each group (week 2: 1.0-4.2; week 5: 1.5-2.1).

Conclusions:
A combined repeated dose toxicity study with reproduction/developtmental toxicity screening test was performed according to OECD 422 and in accordance with GLP principles. Administration of Diazo PW 980 by once daily oral gavage was well tolerated in rats at levels of 1000 mg/kg bw/day without inlife observations of toxicity or histopathological findings in the F0 or F1 generation.There was no evidence of test item related changes to the mating and fertility, maternal behaviour or pre-weaning viability. Mean litter weights and pre-weaning physical development were similar in all dose groups. Based on these results, the no-observed-effect level (NOEL) of parental, reproduction (up to and including implantation) and developmental (from implantation onwards) was considered to be 1000 mg/kg bw/day.
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
Experimental exposure time per week (hours/week):
56
Species:
rat
Quality of whole database:
The information for this endpoint study record was obtained from an experimental study. The OECD GLP criteria were met and the methods applied are fully compliant with OECD TG 422.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the provided information there is no need for classification according to the EU Regulation (EC) No 1272/2008 or updates on Classification,Labelling and Packaging of Substances and Mixtures. 

Additional information

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