Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

- In a screening reproductive toxicity study (according to OECD guideline 421; Calvert Laboratories Inc, 2011; key; Klimisch 1), rats were exposed to 3, 10, 30 mg/kg (nominal in water) via topical application. No systemic, reprotoxic or developmental effects were observed: a NOAEL of 30 mg/kg bw/day was derived.

- In a key, K1 extended one-generation reproductive toxicity study (performed according to OECD guideline 443 and compliant to GLP requirements), rats were exposed to 50, 150 and 450 mg/kg bw/day via oral gavage (Barnett, 2020). Based on draft results, the NOAEL for general, systemic toxicity for the P and F1 generation is considered to be 150 mg/kg bw/day. The reproductive and developmental NOAEL was set at 450 mg/kg bw/day.

Link to relevant study records

Referenceopen allclose all

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-10-11 to 2010-12-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
- Name of test material (as cited in study report): Poly[oxy(methyl-1,2-ethanediyl)], alpha-(2-aminomethylethyl)-omega-(2-aminomethylethoxy)-
- Physical state: clear colorless liquid
- Lot/batch No.: OF502
- Storage condition of test material: at room temp (10-24.6°C)
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan
- Age at study initiation: (P) x a minimum of 13 weeks at initiation of cohabitation
- Weight at study initiation: (P) Males: >/=300 g; Females: >/=200 g at initiation of cohabitation
- Fasting period before study:
- Housing: Upon arrival and until randomization, males and females were group-housed, sexes separate. Following randomization and until cohabitation, males and females were individually housed. During cohabitation, one female was placed with a male breeder from the same group. Following cohabitation, males and females were housed individually.
- Use of restrainers for preventing ingestion (if dermal): yes/no
- Diet (e.g. ad libitum): All animals had access to Harlan Teklad Rodent Diet (certified) or equivalent ad libitum.
- Water (e.g. ad libitum): Water was available ad libitum via an automatic watering device.
- Acclimation period: Study animals were acclimated to their housing for a minimum of 7 days prior to the first day of dosing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26
- Humidity (%): 30-70
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark, except when room lights were turned on during the dark cycle to accommodate blood sampling or other study procedures.

IN-LIFE DATES: From: To:
Route of administration:
dermal
Vehicle:
other: deionized water
Details on exposure:
TEST SITE
- Area of exposure: 4cm x 4cm
- % coverage:
- Type of wrap if used:
- Time intervals for shavings or clipplings: The pelage covering the interscapular and dorsal thoracic regions of the body of the study animals were clipped at least 48 hours prior to the first topical dose and additionally as needed.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): In case of excessive test substance buildup the application site was cleansed after consultation with the sponsor.

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 5 ml/kg (concentrations of 20, 50 and 80 mg/ml) of the test substance was distributed with a syringe as evenly as possible over the exposure area of the intact skin of each animal.
- Constant volume or concentration used: yes

VEHICLE
- Amount(s) applied (volume or weight with unit): 5 ml/kg

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes; During the dosing period the rats were fitted with BiteNot collars 8 hours a day to prevent the animal from licking the test substance.
Details on mating procedure:
- M/F ratio per cage: 1 male to 1 female from the same group
- Length of cohabitation: Males and females remained together until evidence of copulation was noted or for a maximum of three weeks.
- Proof of pregnancy: Day 0 of gestation was determined by evidence of copulation which was determined by the examination of vaginal smears made daily to determine if sperm were present in a smear of vaginal contents or by the presence of a copulatory plug in situ. Examinations of vaginal smears were performed at approximately the same time each day (early morning) through the cohabitation period.
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility: After three weeks of cohabitation, the Study director was able to elect to move certain females with other males from the same dose group, in an attempt to expedite mating.
- Further matings after two unsuccessful attempts: [no / yes (explain)]
- After successful mating each pregnant female was caged (how): Following cohabitation, the females were housed individually.
- Any other deviations from standard protocol:
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
On the first day of dosing, at the beginning of cohabitation and at the last day of dosing, duplicate 1-ml samples were obtained from top, middle and bottom of each formulation, including the vehicle control, to determine the concentration and homogeneity of the test substance in vehicle. In addition, samples were tested for stability. These samples were stored at room temperature, approximately 10 to 30 degrees C.
Duration of treatment / exposure:
The males were treated once daily for a minimum of four weeks (starting two weeks prior to cohabitation). Treatment continued during the same-group cohabitation period and until the day before sacrifice (Day 28/euthanasia Day 29). Females were treated once daily for a minimum of 15 days prior to cohabitation, during cohabitation and from presumed Gestation Days 0 through Day 19 of gestation.
Frequency of treatment:
once daily
Details on study schedule:
- F1 parental animals not mated until [...] weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were [...] days of age.
- Age at mating of the mated animals in the study: [...] weeks
Dose / conc.:
3 mg/kg bw/day (nominal)
Dose / conc.:
10 mg/kg bw/day (nominal)
Dose / conc.:
30 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10/sex/group (80 animals)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected by the Sponsor in consultation with the Study Director based upon previously conducted toxicity studies. A dermal dose range finding study was performed by Calvert labs (report 0325RH11.001, dated March 21, 2011). Test article was dosed by topical application to 80 SD rats (40 males, 40 females) approximately 11 weeks old at initiation. Four dose levels used: control, 100 mg/kg, 250 mg/kg, 400 mg/kg. Due to necrosis and thickening of the skin at the test article application site, dosing for group 3 and 4 was discontinued on day 2 and animals were euthanised. On day 4, similar effects were observed in the dosing group 2. Due to these severe skin effects observed in the 100-400 mg/kg/d dose groups, 24 additional animals were added to the study in order to evaluate dermal effects at lower dose levels: 8 animals (4 male/4 female) were divided over 3 dose groups: 10 mg/kg, 25 mg/kg and 75 mg/kg. The dose was applied once daily for 7 days. Dose administration was discontinued for the 75 mg/kg dose group on day 3 (necrosis). Surviving animals (dose group 10 and 25 mg/kg) were sacrificed on day 7.
Gross necropsy was performed (examination of the external surface of the body, all orifices, and the cranial, thoracic and abdominal cavities and their contents; no tissues were retained).
Clinical signs in animals dosed at 25 mg/kg were limited to very slight to well defined erythema and fissuring at the dosing site of some male and female animals. The NOAEL was considered to be 10 mg/kg.

- Rationale for animal assignment: Using a random number generator, study animals were assigned a random number, sorted based upon the random number assignments, and then assigned to groups. Using SYSTAT version 9.0.1, an Analysis of Variance followed by Dunnett's test was performed to confirm that there were no significant differences in mean body weights between the study groups. If the group assignments showed statistical significane, animals were allowed to be reassigned within the order to improve the similarity of group mean body weights.

Positive control:
N/A
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Mortality was observed twice daily (a.m. and p.m.), once prior to scheduled sacrifice. Clinical observations were made throughout the treatment phase, a minimum of twice daily, prior to dose administration and a minimum of once following dosing. On non-dosing days, observations were made a minimum of once daily.

BODY WEIGHT: Yes
- Time schedule for examinations: Males were weighed once weekly prior to initiation of cohabitation, during cohabitation and at terminal sacrifice. A final body weight was also obtained for all males sacrificed moribund. Females were weighed once weekly prior to initiation of cohabitation, on Gestation Days 0, 4, 7, 14 and 20, and on Day 0 and 4 of lactation. A final body weight was also obtained for all females showing signs of premature delivery or sacrificed moribund.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Full feeder weights and/or feeder weight backs were recorded once weekly, except during mating.

Oestrous cyclicity (parental animals):
Estrous cycle evaluation was performed daily for 2 weeks prior to treatment initiation and daily during the treatment and cohabitation periods.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals were sacrificed by CO2 asphyxiation 2 weeks post-mating.
- Maternal animals: All surviving animals were sacrificed by CO2 asphyxiation on Day 4 of lactation.

GROSS NECROPSY
For all males and females sacrificed moribund or found dead prior to scheduled sacrifice, a complete gross necropsy was performed. The necropsy included the examination of the external surface of the body, all orifices, and the cranial, thoracic and abdominal cavities and their contents. The ear tag, all gross lesions, ovaries, uterus, cervix, vagina and prostate were retained in 10% neutral buffered formalin for possible histopathological evaluation. Additionally, for male rats, the testes and epididymides were retained in modified Davidson's fixative for possible histopathological evaluation. Where applicable, the total number of implantation sites and the total number of corpora lutea for each ovary were recorded. Where applicable, the number of viable and non-viable fetuses were also recorded. All fetuses were discarded.
For the terminally sacrificed males (2 weeks post-mating), necropsy included the examination of the external body surface, all orifices and the cranial, thoracic and abdominal cavities and their contents.
For the terminally sacrificed females (Day 4 of lactation), necropsy included the examination of the external body surface, all orifices and the cranial, thoracic and abdominal cavities and their contents. The total number of corpora lutea were determined for each ovary, and the total number of implantation sites, viable and non-viable fetuses, and early or late resorptions were also determined. For presumed non-gravid females, the uterus was stained with 10% ammonium sulfide to assess the presence of implantation sites.

HISTOPATHOLOGY / ORGAN WEIGHTS
The epididymides and testes were weighed before fixation, after dissection of excess fat and other excess tissues, for all males surviving until the scheduled sacrificed day. Organ to body weight ratios were calculated (using the final body weight obtained prior to necropsy), as well as organ to brain weight ratios.
All tissues for all animals were examined. For all animals necropsied, the following tissues were preserved in 10% neutral buffered formalin (except for the epididymides and testes that were retained in modified Davidson's fixative for optimum fixation): ovaries, uterus, cervix, vagina, testes, epididymides, prostate, seminal vesicles, and gross findings. Special emphasis on stages of spermatogenesis and histopathology of the interstitial testicular structure were give.
Tissues for evaluation were processed to paraffin blocks and prepared to slides. Slides were stained with hematoylin and eosin. Occasionally, other stains may have been required to aid in the diagnosis of lesions; these were documented when used.
Slides were prepared for all tissues for all rats in the vehicle and high dose groups and for all animals that died early. If test substance related lesions were noted, additional slides were prepared on those tissues from the low and mid dose groups.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were euthanized by an intraperitoneal injection of a barbiturate overdose on day 4 of lactation.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

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

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively.
Statistics:
Statistical analysis was performed on In-Life, Clinical Pathology, and Necropsy data when 3 or more animals were present in 2 or more dose groups. Statistical analyses were not performed if N<3 animals per group. For In-Life and Clinical Pathology parameters, the homogeneity of the data was determines by Bartlett¿s Test. If the data was homogeneous, a one-way analysis of variance was performed to assess statistical significance. If statistically significant differences between the means were found, Dunnett¿s test was used to determine the degree of significance from the control means (p<0.05 and p<0.01). If the data was non-homogeneous, the Kruskal-Wallis non-parametric analysis was performed to assess statistical significance. If statistically significant differences between the means were found (p<0.05, p<0.01), the Mann-Whitney U-Test was used to determine the degree of significance from the control means (p<0.05 and p<0.01). If only 2 dose groups were present for evaluation, the Mann-Whitney U-test was used to assess statistical significance between the 2 groups. For necropsy organ weight data, the evaluation of the equality of means were made by a one-way analysis of variance using the F distribution to assess statistical significance. If statistically significant differences between the means were found, Dunnett¿s test was used to determine the degree of significance from the control means (p<0.05 and p<0.01).
Reproductive indices:
1. Pre-coital interval (in days): sum of days until successful copulation/# of presumed pregnant animals
2. Copulation index (%): # of presumed pregnant animals/# of paired animals x 100
3. Fertility index (%): # of pregnant animals/# of presumed pregnant animals x 100
4. Preimplantation loss (%): # of corpora lutea-# of implantations/# of corpora lutea x 100
No effects were observed, except some local irritating effects at the site of administration at 30 mg/kg bw.
Key result
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: male and female reproductive performance
Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: parental clinical signs
Critical effects observed:
no
No effects were observed.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
30 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: There were no treatment-related effects on pregnancy status; gestation, viability and related parameters; fetal weights and fetal morphological observations. There were no test article related changes in fetal sex ratios.
Critical effects observed:
no
Key result
Reproductive effects observed:
no
Conclusions:
No systemic effects were observed at the highest dose administrated. Therefore a NOAEL of 30 mg/kg bw/d is derived for male and female reproductive performance. The NOAEL for parental clinical signs was considered to be 10 mg/kg bw/d. There were no treatment-related effects on pregnancy status; gestation, viability and related parameters; fetal weights and fetal morphological observations.
Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2019-12-03 to final report date (for more explanation see attachment to section "attached full study report").
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Version / remarks:
2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Justification for study design:
SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS:

- Premating exposure duration for parental (P0) animals: 10 weeks (70 days), to cover the full spermatogenesis and folliculogenesis before mating (assessment of effects on fertility). There is no other substance specific information supporting shorter premating exposure time.

- Basis for dose level selection: Dose level selections were made based on the results of the OECD 421 study (Charles River Study No. 20209537). Mortality, adverse clinical signs, reduced body weight gain and food consumption were observed in the female rats at 600 mg/kg/day. Therefore, the dose of 450 mg/kg/day was selected to show some signs of toxicity. The highest dose level aims to induce systemic toxicity, but not death or severe suffering of the animals, to allow comparison of reproductive and systemic toxicity. The intermediate and low dose were selected in anticipation of showing dose response relationships.

- Inclusion/exclusion of extension of Cohort 1B: extension of cohort 1B is excluded as this is the standard information requirement laid down in column 1 of 8.7.3, Annex X of the REACH regulation. Criteria to extend the cohort 1B are not met.

- Route of administration and dose levels: The oral (gavage) route was selected for use because this is the intended route of human exposure

- Test system and number of animals: The test system was selected because: 1) it is a standard species accepted for use in fertility and early embryonic development studies; 2) this species and strain has been demonstrated to be sensitive to reproductive toxicants; and 3) historical data and experience exist at the Testing Facility.
The number of animals chosen for this study is the smallest number considered necessary to provide the minimum number of pregnancies recommended by the applicable guidelines.
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Chemical name: Reaction products of di-, tri- and tetra-propoxylated propane-1,2-diol with ammonia
- Source and lot/batch number of test material: 9D410
- Expiration date of the lot/batch: 2021-07-31
- Appearance: clear, colorless liquid
- Purity: 99.6%
- Correction factor: 1

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: kept at controlled room temperature, blanketed under nitrogen
- Stability under test and storage conditions: not specified
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: test substance is stable in R.O. deionized water when prepared and stored under the same conditions at concentrations bracketing those used in the present study.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: not specified

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The test system was selected because it is a standard species accepted for use in fertility and early embryonic development studies. This species and strain has been demonstrated to be sensitive to reproductive toxicants. Historical data and experience exist at the Testing Facility.
The number of animals chosen for this study is the smallest numbered considered necessary to provide the minimum number of pregnancies recommended by the applicable guidelines.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Crl:CD(SD) Sprague-Dawley rats from Charles River Laboratories, Inc
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at animal arrival: approx 52 days
- Weight at study initiation: (P) Males: 179 - 245 g; Females: 139 - 191 g
- Housing:
- P generation: Animals were socially housed (where possible) in solid-bottomed cages by dose group (no more than 3 per cage/sex) until cohabitation, unless deemed inappropriate by the Study Director and/or Clinical Veterinarian. During the cohabitation period, one male rat and one female rat were pair housed in the male rat’s solid-bottomed cage. After cohabitation, female rats were individually housed until delivery or scheduled euthanasia. Male rats were returned to the same sex pair, where possible, that was established prior to the mating period. Each dam and delivered litter were housed in a common nesting box. Control group animals were housed on a separate rack from the test substance-treated animals.
- F1 Generation: After weaning, the F1 generation rats were socially housed in solid-bottomed cages by dose group throughout the study period (no more than 3 per cage per sex), unless deemed inappropriate by the Study Director and/or Clinical Veterinarian. Control group animals where housed on a separate rack from the test substance-treated animals.
- Nesting material was provided. Nesting material was changed as often as necessary to keep the aimals dry and clean. Analyses for possible contamination are conducted on each lot of nesting material. There were no known contaminants in the nesting material considered to interfere with the objectives of the study.
- For psychological enrichment, animals were provided with items and/or a chewing object. Analysis for possible contamination are conducted on each lot of enrichment devices. There were no known contaminants in the enrichment devices considered to interfere with the objectives of the study.
- Diet (e.g. ad libitum): ad libitum, pelleted Certified Rodent Diet, available from individual feeders. The food is analyzed for environmental contaminants. Rats selected for clinical pathology blood collection were fasted on the day before scheduled euthanasia (no longer than 24 hours; water was available ad libitum). There were no known contaminants in the food considered to interfere with the objectives of the study.
- Water (e.g. ad libitum): ad libitum, from an automatic watering access system and/or individual bottles attached to the nesting boxes. All water was from a local source and passed through a reverse osmosis membrane before use. Chlorine was added to the processed water as a bacteriostat; processed water was expected to contain no more than 1.2 ppm chlorine at the time of analysis. There were no known contaminants in the water considered to interfere with the objectives of the study.
- Acclimation period: at least 5 days prior to initiation of dose administration (male rats) or estrous cycle evaluation (female rats).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19°C to 25°C (actual range 18°C -25°C)
- Humidity (%): 30% to 70% (actual range as low as 20%)
- Air changes (per hr): at least 10 changes per hour
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark

IN-LIFE DATES: From: 2019-12-03 To: 2020-08-30
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
R.O. deionized water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- Preparation of control substance: The control substance was dispensed daily for administration to Groups 1 and 5 animals. An adequate amount of the control substance was prepared at least once weekly and dispensed into daily aliquots, which were stored in a refrigerator set to maintain 4 °C until use. The aliquots were removed from the refrigerator and allowed to warm to room temperature and stirred for at least 30 minutes before dosing. The dosing formulation was also stirred continuously during dosing.
- Preparation of test substance: The dosing formulations were prepared at least once weekly, blanketed under nitrogen, stored in a refrigerator set to maintain 4°C, and dispensed daily. The dosing formulations were removed from the refrigerator and stirred for at least 30 minutes before dosing. The dosing formulation was also stirred continuously during dosing.

VEHICLE
- Justification for use and choice of vehicle (if other than water): R.O. deionized water
- Storage conditions: Ambient
- Concentration in vehicle: 0,10, 30, 90 mg/ml for control, G1, G2, G3, G4 groups respectively.
- Amount of vehicle (if gavage): 5 mL/kg
Details on mating procedure:

- M/F ratio per cage: one male per one female. Within each dose group, consecutive order was used to assign rats to cohabitation (i.e., pairing)
- Length of cohabitation: maximum of 14 days
- Proof of pregnancy: Females with spermatozoa observed in a smear of the vaginal contents and/or a copulatory plug observed in situ were considered to be at DG 0 and assigned to individual housing.
- After 7 days of unsuccessful pairing replacement of first male by another male with proven fertility: Females that did not mate with a male within the first 7 days of cohabitation were assigned an alternate male that did mate with a female (same dose group) and remained in cohabitation for a maximum of 7 additional days.
- Further matings after two unsuccessful attempts: Females not mated after completion of the 14-day cohabitation period were considered to be at DG 0 on the last day of cohabitation and assigned to individual housing.
- After successful mating each pregnant female was caged (how): individual
- Any other deviations from standard protocol: not specified
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The following dose formulation samples were collected for analysis:
- P generation First preparation: concentration (all groups), homogeneity (groups 2 and 4)
- P generation Last preparation: concentration (all groups)
- F1 generation First preparation: concentration (all groups), homogeneity (groups 6 and 8)
- F1 generation Last preparation: concentration (all groups)
The homogeneity results obtained from the top, middle and bottom for the groups 2 and 4 or groups 6 and 8 preparations were averaged and utilized as the concentration results.
Analyses were performed by GC using a validated analytical procedure.
Duplicate (1 mL each) top, middle, and bottom sets of samples (duplicate middle only for Groups 1, 3, 5 and 7) for each sampling time point were sent to the analytical laboratory. On days when only concentration analysis was required, the formulations were only sampled from the middle. Triplicate top, middle, and bottom sets of samples were also collected in the same manner and were retained and stored in a refrigerator set to maintain 4°C, blanketed under nitrogen at the Testing Facility as backup samples. Concentration results were considered acceptable if mean 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 at a relative standard deviation (RSD) of concentrations of < = 5% for each group. After acceptance of the analytical results, remaining backup samples will be discarded.
Duration of treatment / exposure:
Males: 113 -116 days
Females: 104 -108 days
Frequency of treatment:
daily
Details on study schedule:
Age at mating of the mated animals in the study: approx 17 weeks
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
P generation - G1 (Control vehicle)
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
P generation - G2 (low dose)
Dose / conc.:
150 mg/kg bw/day (nominal)
Remarks:
P generation - G3 (mid dose)
Dose / conc.:
450 mg/kg bw/day (nominal)
Remarks:
P generation - G4 (high dose)
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
F1 generation - G5 (Control vehicle)
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
F1 generation - G6 (low dose)
Dose / conc.:
150 mg/kg bw/day (nominal)
Remarks:
F1 generation - G7 (mid dose)
Dose / conc.:
450 mg/kg bw/day (nominal)
Remarks:
F1 generation - G8 (high dose)
No. of animals per sex per dose:
25 males and 25 females per dose group
Control animals:
yes, concurrent vehicle
Details on study design:
- Justification of route: The oral (gavage) route was selected for use because this is the intended route of human exposure
- Justification of dose levels: Dose level selections were made based on the results of the OECD 421 study (Charles River Study No. 20209537). Mortality, adverse clinical signs, reduced body weight gain and food consumption were observed in the female rats at 600 mg/kg/day. Therefore, the dose of 450 mg/kg/day was selected to show some signs of toxicity. The intermediate and low dose were selected in anticipation of showing dose response relationships.
Positive control:
not applicable
Parental animals: Observations and examinations:
VIABILITY CHECKS: Yes
- Time schedule: At least twice daily during the study

CAGE SIDE AND DETAILED OBSERVATIONS: Yes
- General Appearance: At least weekly during the acclimation period and daily during the dose and postdose periods.
- Postdose Observations: between 1 to 2 hours after dose administration.
- Maternal Observations: Daily during the postpartum period.

BODY WEIGHT: Yes
Frequency: At least weekly during acclimation and daily during the dose and postdose periods.

FOOD CONSUMPTION: Yes
Frequency
- Males: food consumption values were recorded at least twice weekly during the dose period up until the initiation of cohabitation (food left value).
- Females: At least twice weekly during the dose period up until the initiation of cohabitation, on DGs 0, 4, 7, 11, 14, 17, 20 and 25 (as applicable) and on DL 0, 4, 7, 10 and 13.

CLINICAL PATHOLOGY
- Sample collection: On the day of scheduled euthanasia, blood samples were collected from 10 rat/sex/dose group Blood samples were collected from the inferior vena cava while under isoflurane/oxygen anesthesia. Rats were euthanized via exsanguination, following blood sample collection.
- timing of sample collection: DS 114-115 for males groups 1-4, DL22 for females groups 1-4
- Animals fasted: Animals were fasted on the day before scheduled euthanasia (no longer than 24 hours; water was available ad libitum).

- Hematology: Blood samples (0.5 mL each) were analyzed for the following parameters: Red blood cell count, Hemoglobin concentration, Hematocrit, Mean corpuscular volume, Red Blood Cell Distribution Width, Mean corpuscular hemoglobin concentration, Mean corpuscular hemoglobin, Reticulocyte count (absolute), Platelet count, White blood cell count, Neutrophil count (absolute), Lymphocyte count (absolute), Monocyte count (absolute), Eosinophil count (absolute), Basophil count (absolute), Large unstained cells, Other cells (as appropriate).
Blood smears were prepared from each hematology sample. Two slides per animal were prepared at the Testing Facility and stained by CR-ASH. Slide review was only performed on samples that met flagging criteria to confirm accurate hematology data.
- Coagulation: Blood samples (0.9 mL each) were processed for plasma, and plasma was analyzed for the following parameters: Activated partial thromboplastin time, Fibrinogen, Prothrombin time.
- Clinical Chemistry: Blood samples (0.5 mL each) were processed for serum, and the serum was analyzed for the following parameters: Alanine aminotransferase, Aspartate aminotransferase
Alkaline phosphatase, Gamma-glutamyltransferase, Creatine kinase, Total bilirubin (When total bilirubin was >0.5 mg/dL, direct bilirubin was also measured and indirect bilirubin was calculated), Urea nitrogen, Creatinine, Calcium, Phosphorus, Total protein, Albumin, Globulin (calculated), Albumin/globulin ratio, Glucose, Cholesterol, Triglycerides, Sodium, Potassium, Chloride, Sample Quality (appearance includes a degree of hemolysis, lipemia, and icterus.

URINALYSIS:
- Sample collection: Urine samples were collected overnight for evaluation on the night prior to scheduled euthanasia.
- Samples were maintained at ambient conditions until analysis at the Testing Facility for the following parameters: Color, Appearance/Clarity, Specific gravity, Total Volume, pH, Protein, Glucose, Bilirubin, Ketones, Blood.

Thyroid Analyses:
- Blood samples (1.5 mL each) were collected from the same male and female rats selected for clinical pathology samples. Blood samples were collected from the inferior vena cava while under isoflurane/oxygen anesthesia for analysis of thyroxine (T4), and TSH. Rats were euthanized via exsanguination, following blood sample collection.
- Blood samples were placed into serum separator tubes, allowed to clot for at least 20 minutes (not exceeding 1 hour) and centrifuged at room temperature at 3500 rpm for 15 minutes. The resulting serum was separated and divided into two aliquots with 250µL of serum for TSH analysis and the remaining for T4 analysis, and frozen immediately on dry ice. Samples designated for TSH analysis were stored in a freezer set to maintain -20ºC. Samples designated for T4 analysis were stored in a freezer set to maintain -80°C until shipment for analysis.
- Analyses: Total serum thyroxine (T4) was measured in rat srrum using a validated analytical method. The thyroid stimulating hormone (TSH) was measured in rat serum using a validated analytical method.

OTHER:
- Natural delivery observations: Beginning on DG 17, the dam will be observed at least twice daily.

Oestrous cyclicity (parental animals):
- Frequency: Samples were collected for 14 consecutive days before initiation of the cohabitation period and then until spermatozoa was observed in a smear of the vaginal contents and/or a copulatory plug is observed in situ during the cohabitation period.
Samples were collected prior to scheduled euthanasia on DG 25, DL 21 or DL 22.
- Procedure: Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage.
Sperm parameters (parental animals):
To assess the potential toxicity of the test substance on male reproductive system, the endpoints listed were evaluated for each male that survived to scheduled euthanasia
- Sperm motility: evaluated using computer-assisted sperm analysis (CASA). Motility was evaluated following dispersion, into an appropriate medium, of sperm from the left vas deferens.
- Sperm concentration: A homogenate was prepared from the left cauda epididymis for evaluation to determine sperm concentration (sperm per gram of tissue weight). Sperm concentration was evaluated using computer-assisted sperm analysis (CASA).
Litter observations:
VIABILITY CHECKS
- Preweaning: litters were observed for dead pups at least twice daily and the pups in each litter were counted once daily during the preweaning period starting on Day 0 postpartum
- Postweaning: at least twice daily during the study

CLINICAL OBSERVATIONS
- Preweaning: At least once daily starting on Day 0 postpartum
- Postweaning: general appearance once daily, postdose observations between 1 to 2 hours after dose administration

BODY WEIGHTS
- Preweaning: Days 0 (birth) 4, 7, 10, 13, 17 and 21 postpartum
- Postweaning: Daily

ANOGENITAL DISTANCE
- Preweaning: on day 0 postpartum (birth)
Procedure: using a calibrated stereomicroscope, micrometer and ruler. The anogenital distance was measured from the cranial edge of the anus, which comes to a point, to the base of the genital tubercle.

NIPPLE PRESENCE - ALL MALE ANIMALS
- Preweaning: On Day 12 postpartum, nipple presence and number were evaluated and recorded for all F1 generation male pups.

FOOD CONSUMPTION
- Frequency: At least once weekly.
- Procedure: Food consumption will be quantitatively measured per cage, where appropriate

SEXUAL MATURATION
- Once daily beginning on Day 27 postpartum (females) or once daily beginning on Day 38 postpartum (males) until the criterion was achieved.
- A body weight was recorded on the day that sexual maturation was achieved

ESTROUS CYCLE EVALUATIONS in F1 generation
Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage. Samples were collected on the day after the vagina is observed to be patent continuing until 1 estrous stage has been identified. Samples were also be collected for at least 14 consecutive days beginning on Day 74 postpartum through the day of scheduled necropsy.

CLINICAL PATHOLOGY
- Sample collection: On PNDs 88 to 91, blood samples were collected from 10 rats/sex/dose group. Blood samples were collected from the inferior vena cava while under isoflurane/oxygen anesthesia. Rats were euthanized via exsanguination, following blood sample collection.
- Rats were fasted on the day before scheduled euthanasia (no longer than 24 hours; water was available ad libitum).
- Hematology: Blood samples were analyzed for the following parameters: Red blood cell count, Hemoglobin concentration, Hematocrit, Mean corpuscular volume, Red Blood Cell Distribution Width, Mean corpuscular hemoglobin concentration, Mean corpuscular hemoglobin, Reticulocyte count (absolute), Platelet count, White blood cell count, Neutrophil count (absolute), Lymphocyte count (absolute), Monocyte count (absolute), Eosinophil count (absolute), Basophil count (absolute), Large unstained cells, Other cells (as appropriate). Blood smears were prepared from each hematology sample. Two slides per animal were prepared at the Testing Facility and stained by CR-ASH. Slide review was only performed on samples that met flagging criteria to confirm accurate hematology data.
- Coagulation: Blood samples (1.8 mL each) were processed for plasma, and plasma was analyzed for the following parameters: Activated partial thromboplastin time, Fibrinogen, Prothrombin time.
- Clinical Chemistry: Blood samples (0.5 mL each) were processed for serum, and the serum was analyzed for the following parameters: Alanine aminotransferase, Aspartate aminotransferase
Alkaline phosphatase, Gamma-glutamyltransferase, Creatine kinase, Total bilirubin (When total bilirubin was >0.5 mg/dL, direct bilirubin was also measured and indirect bilirubin was calculated), Urea nitrogen, Creatinine, Calcium, Phosphorus, Total protein, Albumin, Globulin (calculated), Albumin/globulin ratio, Glucose, Cholesterol, Triglycerides, Sodium, Potassium, Chloride, Sample Quality (appearance includes a degree of hemolysis, lipemia, and icterus.

URINALYSIS
Urine samples were collected overnight for evaluation from rats on the night prior to scheduled euthanasia. Samples were maintained at ambient conditions until analysis at the Testing Facility for the following parameters: Color, Appearance/Clarity, Specific gravity, Total Volume, pH, Protein, Glucose, Bilirubin, Ketones, Blood.

Thyroid Analyses:
- On PND 4 postpartum, blood samples were collected via cardiac puncture following euthanasia from all of the culled pups and pooled by litter.
- On PND 21, blood samples were collected via vena cava following euthanasia from 9 to 11 pups/sex/group not selected for continued observation and dosing.
- On PND 88-91 day, blood samples were collected from the same male and female rats selected for clinical pathology samples. Blood samples were collected from the inferior vena cava while under isoflurane/oxygen anesthesia. Rats were euthanized via exsanguination, following blood sample collection.
- Target Volume: As much as possible, when possible
- Anticoagulant: None (Serum separator tubes)
- Blood was collected and immediately placed into serum separator tubes, allowed to clot at room temperature for at least 10 minutes and then centrifuged for approximately 10 minutes in a refrigerated centrifuge set to maintain 4°C at 3000 g within 30 minutes of collection. Sera samples were split into two approximately equal aliquots. Samples designated for TSH analysis were stored in a freezer set to maintain -20ºC. Samples designated for T4 analysis were stored in a freezer set to maintain -80°C until shipment for analysis.
- T4 and TSH were measured in rat serum using a validated analytical method
Postmortem examinations (parental animals):
SACRIFICE:
- Sacrifice / method of euthanasia: P generation rats selected for clinical pathology evaluation and/or hormone analysis were euthanized via exsanguination following blood collection under isoflurane/oxygen anesthesia. All other P generation rats were euthanized by carbon dioxide asphyxiation.
- Unscheduled death: Rats that died or were euthanized before scheduled termination were examined for the cause of death or condition as soon as possible after the observation was made. The rats were examined for gross lesions.
- Scheduled euthanasia:
Female rats
- Females that did not deliver a litter were euthanized on DG 25 and were examined in Necropsy, Ovarian and Uterine Examinations. Organs were collected, weighted and preserved as described below.
- Dams with no surviving pups were euthanized after the last pup was found dead, missing (presumed cannibalized), or euthanized. The rats were examined in Necropsy, Ovarian and Uterine Examinations. Organs were collected, weighted and preserved as described below.
- On Day 21 postpartum, P generation female rats not selected for blood sample collection were euthanized and examined in Necropsy and Ovarian, Uterine Examinations. Organs were collected, weighted and preserved as described below.
- On Day 22 postpartum, P generation female rats selected for blood sample collection had blood samples collected as described above, and were euthanized. The rats were examined in Necropsy and Ovarian, Uterine Examinations. Organs were collected, weighted and preserved as described below.
Male rats:
- On DS 114 to 117, P generation male rats had blood samples collected as described above and/or were euthanized. The rats were examined in Necropsy, organ weights, and histology. Organs were collected, weighted and preserved as described below.

OVARIAN AND UTERINE EXAMINATIONS
The reproductive tract was dissected from the abdominal cavity. The number and distribution of implantation sites and corpora lutea was recorded. Uteri of apparently nonpregnant animals were examined while being pressed between glass plates to confirm the absence of implantation sites. Uteri and ovaries from all female rats were retained in 10% neutral buffered formalin.

NECROPSY
The rats were examined for gross lesions and were subjected to a gross (or complete necropsy for some male and female rats) necropsy of the thoracic, abdominal, and pelvic viscera. Images were generated for illustration of for consultation on gross observations.

ORGAN WEIGHTS
The organs listed were weighed at necropsy for all scheduled euthanasia rats. Organ weights were not recorded for rats found dead or euthanized in poor condition or in extremis. Paired organs were weighed together, unless otherwise indicated. Organ to body weight ratio (using the terminal body weight) and organ to brain weight ratios were calculated.
The following organs were weighed: Brain, Cervix, Epididymis, Epididymis - Cauda Left, Gland adrenals, Gland coagulating, Gland parathyroid, Gland pituitary, Gland prostate, Gland seminal vesicles, Gland thyroid, Heart, Kidneys, Liver, Ovaries, Oviducts, Pancreas, Spleen, Testes, Thymus, Uterus

TISSUE COLLECTION AND PRESERVATION
Representative samples of the following tissues were collected from all rats and preserved in 10% neutral buffered formalin: Bone marrow, Brain, Cervix, Epididymis, Epididymis - Cauda Left, Esophagus, Eye, Gland adrenals, Gland coagulating, Gland mammary, Gland parathyroid, Gland pituitary, Gland prostate, Gland seminal vesicles, Gland thyroid, Gross lesions/masses, Heart, Kidneys, Large intestine cecum, Large intestine colon, Large intestine, rectum, Liver, Lungs, Muscle skeletal, Nerve peripheral, Nerve optic, Ovaries, Oviducts, Pancreas, Small intestine, duodenum, Small intestine, ileum, Small intestine, jejunum, Stomach, Spleen, Testes, Thymus, Trachea, Urinary bladder, Uterus, Vagina, Vas deferens.

HISTOLOGY
Tissues listed above were collected from rats that died or were euthanized before scheduled termination, all P generation rats in Groups 1 and 4, and target tissues for all rats, and rats with gross lesions were trimmed, embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin.

HISTOPATHOLOGY
Histopathological evaluation was performed by a board-certified veterinary pathologist.
Histological examination was performed on any rats that died or were euthanized before scheduled termination, all P generation rats in Groups 1 and 4, and all gross lesions from all rats. If lesions attributed to the test substance as determined by the Study Director and/or Veterinary Pathologist were observed in the rats exposed to the high test substance concentration, the same organs were examined histologically in all the rats exposed to the lower test substance concentrations until a no-effect level was established.
Postmortem examinations (offspring):
SACRIFICE:
- Method of Euthanasia:
- F1 generation rats/pups > PND 15 were euthanized by carbon dioxide asphyxiation.
- F1 generation pups ≤ PND 15 were euthanized by an intraperitoneal injection of sodium pentobarbital (390 mg/mL).
- F1 generation rats selected for clinical pathology evaluation were euthanized via exsanguination, following blood sample collection.
- Unscheduled death:
- Days 0 to 21 Postpartum: Pups that were found dead during delivery or at the completion of littering (Day 0 postpartum) were evaluated for vital status at birth. The lungs were removed and immersed in water. Pups with lungs that sink were identified as stillborn; pups with lungs that float were identified as liveborn and to have died shortly after birth. Pups with gross lesions were preserved in Bouin's solution for possible future evaluation. Pups that die (Days 1 to 21 postpartum) or were euthanized (Days 0 to 21 postpartum) before scheduled termination were examined for gross lesions and the cause of death or condition as soon as possible after the observation is made. Pups with gross lesions euthanized or found dead on Days 0 to 3 postpartum were preserved in Bouin's solution for possible future evaluation. Gross lesions from pups found on Days 4 to 21 postpartum were preserved in 10% neutral buffered formalin.
- Postweaning (> Day 21 Postpartum): Rats that died or were euthanized before scheduled termination were examined for the cause of death or condition as soon as possible after the observation is made. The rats were examined for gross lesions.
- Scheduled Euthanasia:
- Pups not selected for continued observation were euthanised on Day 21 postpartum. 9 to 11 pups/sex/group had blood samples collected. A gross necropsy of the thoracic, abdominal and pelvic viscera was performed and carcasses were retained in 10% neutral buffered formalin for 10 pups/sex/group (one pup per litter, when possible) and any pups with gross lesions; all other tissues/carcasses will be discarded.
- Female rats: On Day 88-91 postpartum, F1 generation female rats had blood samples collected and/or were euthanized. The rats were examined as described in the sections Necropsy and Organ Weights. Tissues were collected, weighed, retained and/or evaluated; all other tissues were discarded.
- Male rats: On Day 90 postpartum ± 1 day, F1 generation male rats had blood samples collected and/or were euthanized. The rats were examined as described in the sections Necropsy, and Organ Weights. Tissues were collected, weighed, retained and/or evaluated; all other tissues were discarded.
Assessment of the male reproductive system was performed. A similar proportion of males from each group, as appropriate, were euthanized on any one day.

MALE REPRODUCTIVE ASSESSMENT
To assess the potential toxicity of the test substance on the male reproductive system, the endpoints listed below were evaluated for each male that survived to scheduled euthanasia.
- Sperm motility: Sperm motility was evaluated using computer-assisted sperm analysis (CASA). Motility was evaluated following dispersion, into an appropriate medium, of sperm from the left vas deferens.
- Sperm concentration: A homogenate was prepared from the left cauda epididymis for evaluation to determine sperm concentration (sperm per gram of tissue weight). Sperm concentration was evaluated using computer-assisted sperm analysis (CASA).

NECROPSY
The rats were examined for gross lesions, and were subjected to a gross necropsy (or complete necropsy for some male and female rats) of the thoracic, abdominal, and pelvic viscera.
Images were generated for illustration of or consultation on gross observations.

ORGAN WEIGHTS
- The following organs were weighed at necropsy for all scheduled euthanasia rats: Brain, Cervix, Epididymis, Epididymis - Cauda Left, Gland - adrenals, Gland - coagulating, Gland - parathyroid, Gland - pituitary, Gland - prostate, Gland - seminal vesicles, Gland - thyroid, Heart, Kidneys, Liver, Ovaries, Oviducts, Spleen, Testes, Thymus, Uterus
- Organ weights were not recorded for rats found dead or euthanized in poor condition or in extremis. Paired organs were weighed together, unless otherwise indicated. Organ to body weight ratio (using the terminal body weight) and organ to brain weight ratios were calculated.

TISSUE COLLECTION AND PRESERVATION
- Representative samples of the tissues listed were collected from all rats and preserved in 10% neutral buffered formalin, unless otherwise indicated.
- in addition, F1 male rat 4079 (group 8, unscheduled euthanasia) head was retained when not required and F1 male rat 4004 (group 5, PND89) subcutis was retained in 10% NBF and examined when not required.
- list of tissues: Bone marrow, Brain, Cervix, Epididymis, Epididymis - Cauda Left, Esophagus, Eye, Gland - adrenals, Gland - coagulating, Gland - mammary, Gland - parathyroid, Gland - pituitary, Gland - prostate, Gland - seminal vesicles, Gland - thyroid, Gross lesions, Heart, Kidneys, Large intestine cecum, Large intestine - colon, Large intestine - rectum, Liver, Lungs, Muscle - skeletal, Nerve - peripheral, Nerve - optic, Ovaries, Oviducts, Pancreas, Small intestine - duodenum, Small intestine - ileum, Small intestine - jejunum, Stomach, Spleen, Testes, Thymus, Trachea, Urinary bladder, Uterus, Vagina, Vas deferens.

HISTOLOGY 
Tissues listed above were collected from rats that died or were euthanized before scheduled termination, all F1 generation rats in Groups 5 and 8, target tissues for all rats, and rats with gross lesions were trimmed, embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin.
Entire left and right ovaries for the F1 generation rats assigned to Groups 5, 6, 7 and 8 were embedded in separate paraffin blocks. Consistency in orientation of each ovary was imperative, such that each ovary was placed into its own designated block. Each ovary was sectioned beginning at 200 microns within the ovary. The first section was collected and designated as section 1 for follicle counts. After advancing the microtome 100 microns, the next section was collected as section 2. This process continued until a total of five sections from each ovary were collected for follicle counts, plus one section collected (between follicle count sections 3 and 4) on a separate slide for routine histopathological evaluation by the study pathologist. The five sections were placed on one slide for each ovary. Section 1 was placed closest to the slide label, continuing in a linear pattern, until five sections were on the same slide (section 5 being most distal to the slide label).
Each section was quantitatively evaluated for primordial follicles to include small growing follicles. Presence or absence of corpora lutea was recorded for each rat.  Group means and standard deviations were calculated for each rat (left, right, and both ovaries combined). Where the sample size was appropriate (N> 2), group mean values were compared in order to detect any significant differences in the number of primordial follicles in left ovaries, right ovaries, and both ovaries combined. The rats assigned to Group 8 counts were ± 15% (number of follicles and/or presence of corpora lutea) of the control animals and were not statistically significant, therefore, no further evaluation was considered necessary.

HISTOPATHOLOGY
Histopathological evaluation was performed by a board-certified veterinary pathologist.
Histological examination was performed on any rats that died or were euthanized before scheduled termination, all F1 generation rats in Groups 5 and 8, and all gross lesions from all rats. If lesions attributed to the test substance as determined by the Study Director and/or Veterinary Pathologist were observed in the rats exposed to the high test substance concentration, the same organs were examined histologically in all the rats exposed to the lower test substance concentrations until a no-effect level was established.
Statistics:
Any data collected during the predose period was not tabulated, summarized or statistically analyzed. All statistical analyses were performed within the respective study phase, unless otherwise noted.
Clinical and necropsy observations data were summarized but no inferential statistical analysis was performed.
Numerical data collected on scheduled occasions were summarized and statistically analyzed as indicated below according to sex and occasion or by litter.
- descriptive statistical analyses: means, standard deviations, percentages, numbers and/or incidences were reported as appropriate by dataset
- interferential statistical methods: 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% and 5% levels, unless otherwise noted.
The pairwise comparisons of interest were: group 2 vs 1, group 3 vs 1, group 4 vs 1, group 6 vs 5, group 7 vs 5, group 8 vs 5
More details are described in the field 'Any other information on materials and methods'
Reproductive indices:
Gestation Length: The gestation length is calculated from DG 0 to the day the first pup is observed.
Female Pregnancy Index: Number of Pregnant Females / Number of Females Paired
Gestation Index: Percentage of pregnancies that result in birth of live litters
(Number of Animals with Live Offspring/Number of Animals Pregnant )x 100
Offspring viability indices:
Number of offspring per litter: Live and dead pups
Number of implantation sites.
General condition of dam and litter during the postpartum period.
Live Birth Index: Percentage of pups born alive.
(Number of Live Newborn Pups/ Number of Newborn Pups ) x 100
Viability Index: Percentage of pups born that survive 21 days postpartum
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
- Male: An increased incidence of excess salivation and abnormal breathing sounds were observed at 450 mg/kg/day in comparison with the control group values.
All other clinical signs that were observed were considered unrelated to the test substance because: 1) the observations were not dose-dependent; and/or 2) the observations occurred in only one or two rats in a dose group and the clinical signs did not persist. These clinical signs included suspected dehydration, hunched posture, fur loss, fur staining, thin fur cover, scab on the skin, splayed limbs, thin body, hypersensitive, hypersensitive, abnormal consistency of the feces, red discharge, broken teeth. decreased activity and lack of or impaired righting reflex.
- Female: During the precohabitation, gestation and/or lactation periods, there was an increased incidence of suspected dehydration, labored breathing, abnormal breathing sounds, shallow breathing, hunched posture, thin fur cover erect fur, hyperreactivity and decreased activity at 450 mg/kg/day in comparison with the control group values.
All other clinical signs that were observed were considered unrelated to the the test substance because: 1) the observations were not dose-dependent; and/or 2) the observations occurred in only one or two rats in a dose group and the clinical signs did not persist. These clinical signs included salivation, bent tail, fur loss, fur staining, scab on the skin, thin body condition, low carriage, ungroomed fur, swollen muzzle and abnormal consistency of the feces.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
- Male: There were no test subtance-related mortalities observed in the P generation male rats. One male in the control group was euthanized due to adverse clinical signs on DS 82, a male at 150 mg/kg/day was found dead on DS 3 and a male at 450 mg/kg/day was euthanized due to adverse clinical signs on DS 36. Based on there being single occurrences in each dose group and there being no correlation in the death or moribundity, these deaths were not considered to be test substance related.
- Female: The only deaths that were considered to be test substance related occurred in the 450 mg/kg/day dose group. One female at 50 mg/kg/day and another at 150 mg/kg/day were euthanized due to no surviving pups. There were no test substance related clinical signs or effects on body weights observed in these females. At 450 mg/kg/day, two females were found dead and three females were euthanized due to adverse clinical signs. Common clinical signs in these females included hunched posture, decreased activity and respiratory issues (labored breathing and abnormal breathing signs). The females that were euthanized due to adverse clinical signs also experienced body weight loss prior to euthanasia. All other P generation female rats survived until scheduled euthanasia.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
- Male: Body weights and body weight gains were unaffected by the test substanceduring the dose period by doses up to and including 150 mg/kg/day. At 50 mg/kg/day, there was a statistically significant decrease (p≤0.01) in body weight gain observed on DSs 91 to 92 in comparison with the control group value. This difference was not considered to be test substance related because it was not dose dependent. There were statistically significant increases (p≤0.01) in body weight gain on DSs 94 to 95 and statistically significant (p≤0.05 or p≤0.01) body weight losses on DSs 95 to 96 observed in the 50, 150 and 450 mg/kg/day dose groups, respectively. These differences were not considered to be test substance related because they were single occurrences that did not persist. At 150 mg/kg/day, there was a statistically significant increase (p≤0.01) in body weight gain observed on DSs 30 to 31, a statistically significant (p≤0.01) body weight loss on DSs 43 to 44 and a statistically significant increase (p≤0.01) in body weight gain observed on DSs 44 to 45 in comparison with the control group values. These differences were not considered to be test substance related because they were not dose dependent.
At 450 mg/kg/day, there were reductions or statistically significant reductions (p≤0.05) in mean body weights observed beginning on DS 106 and continuing for the remainder of study in comparison with the control group values. At 450 mg/kg/day, there was a statistically significant reduction (p≤0.05) in body weight gain on DSs 7 to 8 and a statistically significant (p≤0.05) body weight loss on DSs 108 to 109 in comparison with the control group values.

- Female: Maternal body weights and body weight gains were unaffected by the test substance during the pre-mating, gestation and lactation periods by doses up to and including 150 mg/kg/day. At 50 mg/kg/day, there was a statistically significant decrease (p≤0.05) in body weight gain observed on DSs 48 to 49 in comparison with the control group value. There was also a statistically significant increase (p≤0.05) in body weight gain observed on DSs 66 to 67 and a statistically significant body weight loss (p≤0.05) at 50 mg/kg/day on DSs 68 to 69 in comparison with the control group values. At 450 mg/kg/day, there were statistically significant increases (p≤0.05 or p≤0.01) in body weight gain observed on DSs 18 to 19 and DLs 15 to 16 in comparison with the control group values. These differences were not considered to be test substance related because they were single occurrences that did not persist.
At 450 mg/kg/day, there were statistically significant reductions (p≤0.05) in mean body weights observed beginning on DS 20 and continuing for the remainder of the pre-mating period in comparison with the control group values. There were reductions or statistically significant reductions (p≤0.05) in mean body weights at 450 mg/kg/day on DGs 5, 19 and 21 in comparison with the control group values. There was a statistically significant (p≤0.01) body weight loss at 450 mg/kg/day on DSs 9 to 10 in comparison with the control group value.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
- Male: Food consumption was unaffected by doses of the test substance in the males at all intervals prior to cohabitation at doses up to and including 450 mg/kg/day. There were statistically significant increases (p≤0.05. or p≤0.01) in the mean food consumption values observed on DSs 22 to 25 at 50, 150 and 450 mg/kg/day in comparison with the control group value. There was a statistically significant decrease (p≤0.01) in the mean food consumption value on DSs 60 to 64 at 150 mg/kg/day in comparison with the control group value. There were statistically significant increases (p≤0.05 or p≤0.01) in the mean food consumption values on DSs 64 to 67 at 50 and 150 mg/kg/day and on DSs 67 to 71 at 150 mg/kg/day in comparison with the control group values.
- Female: Food consumption values were unaffected by the test substance during the precohabitation, gestation and lactation periods by doses up to and including 150 mg/kg/day. At 150 mg/kg/day, there was a statistically significant decrease (p≤0.05) in the mean food consumption value observed on DGs 17 to 20 in comparison with the control group value. This decrease was not considered to be test substance related because it was not dose dependent.
At 450 mg/kg/day, there were decreases or statistically significant decreases (p≤0.05 or p≤0.01) in the mean food consumption values observed on DSs 8 to 11, 11 to 15 and at all intervals between 18 to 22 and 46 to 50 in comparison with the control group values.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
Male and Female: No test substance-related differences in the hematology parameters occurred at any dose level. All differences were consistent with biological variation and were not considered to be test substance related.

Coagulation: No test substance-related differences in coagulation parameters occurred at any dose level in the F1 generation male or female rats. All differences in these parameters, regardless of statistical significance, were consistent with biological variation and were considered unrelated to the test substance
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Male and Female: No test item-related differences in the clinical chemistry occurred at any dose level. All differences were consistent with biological variation and were not considered to be test substance related.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
Male: No test substance-related changes in urinalysis values were observed in the P generation male rats. At 450 mg/kg/day, there was a statistically significant decrease (p≤0.01) in the pH of the urine in comparison with the control group value. This difference was not considered to be test substance related as it was consistent with biological variation for this parameter.

Female: No test substance-related changes in urinalysis values were observed in the P generation female rats at any dose. At 150 mg/kg/day, there was a statistically significant decrease (p≤0.05) in the volume of urine collected in comparison with the control group value. This difference was not considered to be test substance related because it was not dose dependent.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- Male: Inflammation was observed within the nasopharynx and multifocally within the nasal cavity of rats at 150 or 450 mg/kg/day. This inflammation was characterized by infiltrates of neutrophils and less frequently mononuclear cells within the epithelium and lamina propria, as well as within the lumen of the nasopharynx/nasal cavity. Segments of epithelium within areas of inflammation were occasionally necrotic/absent. The nasal respiratory epithelium was more frequently affected than the olfactory epithelium. In two male rats and three female rats at 450 mg/kg/day, segments of the nasopharyngeal epithelium were replaced by squamous metaplasia. Findings within the nasal cavity were considered test substance-related due to their presence in only treated animals and not controls. The described nasal cavity findings were considered to likely be secondary to irritation caused by nasal reflux of the administered test substance. Other microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rats, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to administration of the test substance.
- Female: Inflammation was seen within the nasopharynx and multifocally within the nasal cavity of rats at 150 or 450 mg/kg/day. This inflammation was characterized by infiltrates of neutrophils and less frequently mononuclear cells within the epithelium and lamina propria, as well as within the lumen of the nasopharynx/nasal cavity. Segments of epithelium within areas of inflammation were occasionally necrotic/absent. The nasal respiratory epithelium was more frequently affected than the olfactory epithelium. In three rats at 450 mg/kg/day, segments of the nasopharyngeal epithelium were replaced by squamous metaplasia. Findings within the nasal cavity were considered test substance-related due to their presence in only treated animals and not controls. The described nasal cavity findings were considered to likely be secondary to irritation caused by nasal reflux of the administered test substance. Other microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rats, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to administration of the test substance.
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Thyroid Hormone Evaluations:
- Male: There were no test substance-related differences observed in the T4 and TSH thyroid hormone levels in the adult males at doses up to and including 450 mg/kg/day. At 150 and 450 mg/kg/day, there were statistically significant increases (p≤0.01) in the T4 thyroid hormone levels in the adult males in comparison with the control group values. These differences were not considered to be test substance related as they were within Historical Control range for this parameter.
- Female: There were no test substance-related differences in T4 and TSH thyroid hormone levels in the LD 21 P generation females. There were also no test substance-related differences in T4 and TSH thyroid hormone levels in the PND 4 pooled samples and the PND 21 male and female pup samples. There were statistically significant reductions (p≤0.05) in mean TSH thyroid hormone levels at all dose levels for the male pups on PND 21 in comparison with the control group values. These differences were not considered to be test substance related because the differences were not dose dependent and they were within Historical Control range for this parameter.
Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test substance-related changes in the number of estrous stages per 14 day interval evaluated and the mean cycle lengths. At 150 mg/kg/day, there was a statistical significant increase (p≤0.05) in the mean cycle length (days) in comparison with the control group value. This increase was not considered to be test substance related because it was not dose dependent.
All additional fertility parameters were unaffected by doses of the test substance as high as 450 mg/kg/day.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
There were no statistically significant or biologically important differences in the mean percentage of motile sperm and the mean cauda epididymal sperm density parameters observed at any dose level.
Reproductive performance:
no effects observed
Description (incidence and severity):
- All male mating and fertility parameters were comparable among the dose groups and did not significantly differ from the control group values.
- Natural Delivery and Litter Observations: A total of 23/25 (92%), 23/25 (92%), 22/25 (88%) and 17/20 (85%) treated female rats were pregnant and delivered a liter in the 0 (Control), 50, 150 and 450 mg/kg/day dose groups, respectively.
Natural delivery and litter observations were not affected by doses of test substance as high as 450 mg/kg/day.
No parameters evaluated at natural delivery or during preweaning period was affected by the test substance at doses as high as 450 mg/kg/day. These parameters included duration of gestation, gestation index, females completing delivery, dams with liveborn pups, dams with no liveborn pups, dams with stillborn pups, number of stillborn pups per litter, number of stillborn pups, number of liveborn pups, live birth index (%), live male pups per litter (%), implantation sites and post-implantation loss per litter.
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Remarks:
reproduction
Effect level:
450 mg/kg bw/day (nominal)
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
not specified
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
From birth through weaning:
No clinical observations in the F1 generation pups were attributable to maternal doses of the test substance as high as 450 mg/kg/day.
All clinical signs that were observed were considered unrelated to the test substance because: 1) the observations were not dose dependent; and 2) the observations occurred in only one pup in a particular dose group. These clinical signs included thin appearance; pale skin; purple discoloration; a scab, lesion or laceration on the skin; swollen forelimb or hindlimb; ungroomed fur; thin fur cover; no milk band present; cold to touch; missing digit on the hindpaw; and mass present on the forelimb.

F1 generation:
There was an increased incidence in abnormal breathing sounds observed at 150 and 450 mg/kg/day in the F1 generation female rats and at 450 mg/kg/day in the F1 generation male rats in comparison with the control group values. There was also an increased incidence of suspected dehydration, excess salivation and labored breathing in the F1 generation male rats in comparison with the control group values.
All other clinical signs that were observed were considered unrelated to the test substance because: 1) the observations were not dose-dependent; and/or 2) the observations occurred in only one or two rats in a dose group and the clinical signs did not persist. These clinical signs included shallow breathing, hunched posture, thin fur, scab on the skin, abnormal fecal consistency, red or orange discharge, broken teeth and liquid discharge.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
The only deaths that occurred in the F1 generation rats occurred at 450 mg/kg/day. At 450 mg/kg/day one male and one female were found dead during the dose period and three males were euthanized due to adverse clinical signs. Common clinical signs in the rats included respiratory issues (labored breathing and abnormal breathing signs) and suspected dehydration. The three males that were euthanized due to adverse clinical signs also experienced body weight loss prior to euthanasia. Male 4094 was given a dosing holiday on PND 61 due to potential blockage observed during dosing. This male was euthanized on PND 62 due to adverse clinical signs and had necropsy observations indicative of an intubation error. All other F1 generation male and female rats survived until scheduled euthanasia.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
From birth through weaning:
No changes in body weights in the F1 generation pups were attributable to maternal doses of the test substance as high as 450 mg/kg/day. There was a statistically significant increase (p≤0.05) in mean male pup body weights on PND 10 in comparison with the control group value. This increase was not considered to be test substance related because it was not dose dependent.

F1 generation:
There were no test substance-related differences in body weight and body weight gain observed in the F1 generation male or female rats at doses up to and including 450 mg/kg/day.
There was a statistically significant reduction (p≤0.05) in body weight gain observed in the F1 generation male rats at 50 mg/kg/day on PNDs 80 to 81 in comparison with the control group value. In the F1 generation female rats at 50 mg/kg/day, there were statistically significant increases (p≤0.01) in body weight gain observed on PNDs 50 to 51, 70 to 71 and 74 to 75; statistically significant reductions (p≤0.05 or p≤0.01) in body weight gain observed on PNDs 60 to 61 and 71 to 72; and statistically significant (p≤0.01) body weight losses on PNDs 73 to 74 and 76 to 77 in comparison with the control group values. There were statistically significant reductions (p≤0.05 or p≤0.01) in body weight gains observed in the F1 generation male rats at 150 mg/kg/day on PNDs 51 to 52 and 67 to 68 in comparison with the control group values. In the F1 generation female rats at 150 mg/kg/day, there was a statistically significant increase (p≤0.05) in body weight gain observed on PNDs 37 to 38 and a statistically significant reduction (p≤0.01) in body weight gain observed on PNDs 67 to 68 in comparison with the control group values. These differences were not considered to be test substance-related because they were not dose dependent.
There was a statistically significant reduction (p≤0.05) in body weight gain observed in the F1 generation male rats at 450 mg/kg/day on PNDs 33 to 34 in comparison with the control group value. In the F1 generation male rats at 450 mg/kg/day, there was a statistically significant increase (p≤0.01) in body weight gain on PNDs 49 to 50 and a statistically significant reduction (p≤0.05) in body weight gain observed on PNDs 50 to 51 in comparison with the control group values. These differences were not considered to be test substance-related because they were single occurrences and did not persist.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no test substance-related differences in food consumption observed in the F1 generation male or female rats at doses up to and including 450 mg/kg/day.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
No test substance-related differences in the hematology parameters occurred at any dose level in the F1 generation male or female rats. All differences in these parameters, regardless of statistical significance, were consistent with biological variation and were considered unrelated to the test substance.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No test substance-related differences in the clinical chemistry parameters occurred at any dose level in the F1 generation male or female rats. All differences in these parameters, regardless of statistical significance, were consistent with biological variation and were considered unrelated to the test substance.
Urinalysis findings:
no effects observed
Description (incidence and severity):
There were no test substance-related differences in urinalysis observed in the F1 generation male or female rats at doses up to and including 450 mg/kg/day.
Sexual maturation:
no effects observed
Description (incidence and severity):
There were no test substance-related effects on male or female sexual maturation at doses as high as 450 mg/kg/day. The average day of preputial separation and vaginal patency on which these parameters were achieved did not differ significantly among the dose groups.

The day the first estrus occurred adter caginal patency, the number of estrous cycles per 14 day interval evaluated and the mean cycle lengths were all comparable among the groups.
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
The anogenital distances for the male and female pups were comparable across all dose groups on the day of delivery and there were no statistically significant differences.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
There were no male pups observed with the presence of nipples on study.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
No test substance-related organ weight changes were noted in the F1 generation male or female rats. There were isolated organ weight values that were statistically different from their respective controls. There were, however, no patterns, trends, or correlating data to suggest these values were toxicologically relevant. Thus, the organ weight differences observed were considered incidental and/or related to difference of sexual maturity and unrelated to administration of the test substance.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No test article-related gross findings were noted in the F1 generation male or female rats. The gross findings observed were considered incidental, of the nature commonly observed in this strain and age of rats, and/or were of similar incidence in control and treated animals and, therefore, were considered unrelated to administration of the test substance.
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
Inflammation was observed within the nasopharynx and multifocally within the nasal cavity of rats at 150 or 450 mg/kg/day. This inflammation was characterized by infiltrates of neutrophils and less frequently mononuclear cells within the epithelium and lamina propria, as well as within the lumen of the nasopharynx/nasal cavity. Segments of epithelium within areas of inflammation were occasionally necrotic/absent. The nasal respiratory epithelium was more frequently affected than the olfactory epithelium. Segments of the nasopharyngeal epithelium were replaced by squamous metaplasia in one female at 450 mg/kg/day. Findings within the nasal cavity were considered test substance related due to their presence in only treated animals and not controls. The described nasal cavity findings were considered to likely be secondary to irritation caused by nasal reflux of the administered test substance.
Other microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rats, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to administration of the test substance.
Other effects:
no effects observed
Description (incidence and severity):
Sperm evaluation: There were no statistically significant or biologically important differences in the mean percentage of motile sperm and the mean cauda epididymal sperm density parameters observed at any dose level in the F1 generation male rats.
Thyroid hormone levels: There were no test substance-related differences in T4 or TSH thyroid hormone levels in the F1 generation male or female rats at doses up to and including 450 mg/kg/day.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Dose descriptor:
NOAEL
Remarks:
development
Generation:
F1
Effect level:
450 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: viability and growth in the offspring
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Generation:
F1
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
histopathology: non-neoplastic
Critical effects observed:
not specified
Reproductive effects observed:
no

Dose formulation analyses

The dosing suspensions of the test substance prepared in R.O. deionized water were analyzed following the first and last preparations for the P generation and F1 generation and were found to be within the target concentration and homogeneous under the conditions of this study. 

The first preparation of the dosing suspensions of 10, 30 and 90mg/mL for the P generation were analyzed and found to be 112%, 101% and 95.6% of the target concentrations, respectively. The homogeneity values obtained for the first preparation were 1.3% and 1.6% RSD for the 10 and 90mg/mL formulations, respectively. Although the initial 10 mg/mL concentration was slightly above the protocol-specified ± 10% of the target concentration, this variation from the target concentration was slight and the 5 mg/mL dose preparation was homogeneous and considered to be acceptable for usage on study. The last preparation of the dosing suspensions of 10, 30 and 90mg/mL were analyzed and found to be 106%, 109% and 110% of the target concentrations, respectively. 

The first preparation of the dosing suspensions of 10, 30 and 90mg/mL for the F1 generation were analyzed and found to be 108%, 106% and 104% of the target concentrations, respectively. The homogeneity values obtained for the first preparation were 3.2% and 2.3% RSD for the 10 and 90mg/mL formulations, respectively. The last preparation of the dosing suspensions of 10, 30 and 90mg/mL were analyzed and found to be 110%, 104% and 106% of the target concentrations, respectively. 

Conclusions:
Based on the results of this study, the no-observed-adverse-effect-level (NOAEL) for general toxicity was considered 150 mg/kg/day for the P generation and F1 generation male and female rats. There was an increased incidence of microscopic findings in the nasal cavity of both the P generation and F1 generation rats at 150 or 450 mg/kg/day that included inflammation, neutrophilic exudates, and squamous metaplasia of the nasopharynx. Luminal hemorrhage was also observed in the nasal cavity of P generation rats at 450 mg/kg/day. There were abnormal breathing sounds and labored breathing observed in the P generation and F1 generation males and females that correlated with these microscopic findings. A link between the observed effects and the corrosive nature of the test substance cannot be excluded, so the NOAEL is established at 150 mg/kg bw/d.
Mortality was observed in the P generation females and F1 generation males and females. Reductions in body weight and body weight gain were observed in the P generation males and females at 450 mg/kg/day.
The reproductive NOAEL was 450 mg/kg/day as there were no test substance-related changes in estrous cycling in the female and mating and fertility in the males or females. The NOAEL for viability and growth in the offspring was also 450 mg/kg/day.
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
450 mg/kg bw/day
Study duration:
chronic
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
30 mg/kg bw/day
Study duration:
subacute
Species:
rat
Additional information

Screening study via topical application

In a screening reproductive toxicity study (according to OECD guideline 421), rats were exposed to 3, 10, 30 mg/kg (nominal in water) via topical application (Calvert Laboratories Inc, 2011; key study; Klimisch 1). No systemic effects were observed at the highest dose administered. Therefore, a NOAEL of 30 mg/kg bw/d is derived for male and female reproductive performance. The NOAEL for parental clinical signs was considered to be 10 mg/kg bw/d. There were no treatment-related effects on pregnancy status; gestation, viability and related parameters; fetal weights and fetal morphological observations.

Extended one-generation reproductive toxicity study

In a key, K1 extended one-generation reproductive toxicity study performed according to OECD guideline 443 and according to GLP requirements, the test substance was administered to rats at dose levels 50, 150, or 450 mg/kg bw/day via oral gavage (Barnett, 2020; draft report).

A total of 100 male and 100 female Crl:CD(SD) Sprague-Dawley rats (P generation) were randomly assigned to four dose groups of 25 rats per sex per group. Male rats were given the test substance and/or the control article (Reverse osmosis membrane-processed deionized water [R.O. deionized water]) formulations once daily via oral gavage beginning 70 days before cohabitation, during cohabitation and continuing through the day before euthanasia (a total of 113-116 consecutive doses). Female rats were given the test substance and/or the control substance formulations once daily oral gavage beginning 70 days before cohabitation, during cohabitation and continuing through Postpartum Day 20 (DL 20; rats that delivered a litter not selected for blood sample collection), DL 21 (rats that delivered a litter selected for blood sample collection), or DG 24 (rats that did not deliver a litter). Females not mated after completion of the 14-day cohabitation period were considered to be at DG 0 on the last day of cohabitation. The first day of dosing was designated as Day 1 of study (DS 1). The dose volume was 5 mL/kg based on the most recently recorded body weight, and doses were administered at approximately the same time each day. 

On the day of scheduled euthanasia (10 P generation rats/sex/dose group) or on Day 90 ± 1 postpartum (10 F1 generation rats/sex/dose group), blood samples were collected for clinical pathology and thyroid hormone evaluations.

The following parameters and end points were evaluated in this study for the P generation: viability, clinical signs, body weights, body weight changes, food consumption, mating and fertility, clinical pathology parameters (hematology, coagulation, clinical chemistry, and urinalysis), thyroid hormone analysis, gross necropsy findings, organ weights, and histopathologic examinations. Male rats were further evaluated for sperm motility, sperm concentration and reproductive organ weights, and female rats were further evaluated for estrous cycling and ovarian and uterine contents.  

The following parameters and end points were evaluated in this study for the F1 generation: viability, clinical signs, body weights, body weight changes, food consumption, anogenital distance, nipple presence (males), sexual maturation, clinical pathology parameters (hematology, coagulation, clinical chemistry, and urinalysis), thyroid hormone analysis, gross necropsy findings, organ weights, and histopathologic examinations. Male rats were further evaluated for sperm motility, sperm concentration and reproductive organ weights, and female rats were further evaluated for estrous cycling. 

P generation Male Rats

There were no test substance-related mortalities observed in the P generation male rats. One male in the control group was euthanized due to adverse clinical signs on DS 82, a male at 150 mg/kg/day was found dead on DS 3 and a male at 450 mg/kg/day was euthanized due to adverse clinical signs on DS 36. Based on there being single occurrences in each dose group and there being no correlation in the death or moribundity, these deaths were not considered to be test substance related.

An increased incidence of excess salivation and abnormal breathing sounds were observed in the P generation males at 450 mg/kg/day.

At 450 mg/kg/day, there were reductions or statistically significant reductions in mean body weights observed beginning on DS 106 and continuing for the remainder of study. At 450 mg/kg/day, there was a statistically significant reduction in body weight gain on DSs 7 to 8 and a statistically significant body weight loss on DSs 108 to 109. 

Food consumption was unaffected by doses of test substance in the males at all intervals prior to cohabitation at doses up to and including 450 mg/kg/day. 

All P generation male mating and fertility parameters were comparable among all dose levels. 

There were no test substance-related changes in the thyroid hormone levels observed in the P generation male rats. No hematologic, coagulation, clinical chemistry or urinalysis parameter was affected by doses of the test substance as high as 450 mg/kg/day. 

No effect on sperm (motility or count),gross necropsy and organ weights occurred in any dose group.

Inflammation was observed within the nasopharynx and multifocally within the nasal cavity of rats at 150 or 450 mg/kg/day. This inflammation was characterized by infiltrates of neutrophils and less frequently mononuclear cells within the epithelium and lamina propria, as well as within the lumen of the nasopharynx/nasal cavity. Segments of epithelium within areas of inflammation were occasionally necrotic/absent. The nasal respiratory epithelium was more frequently affected than the olfactory epithelium. In two rats at 450 mg/kg/day, segments of the nasopharyngeal epithelium were replaced by squamous metaplasia.  The described nasal cavity findings were considered to likely be secondary to irritation casued by nasal reflux of the gavaged test item.

P generation Female Rats

The only deaths that were considered to be test substance-related occurred in the 450 mg/kg/day dose group. One female at 50 mg/kg/day and another at 150 mg/kg/day were euthanized due to no surviving pups. There were no test substance related clinical signs or effects on body weights observed in these females. At 450 mg/kg/day, two females were found dead and three females were euthanized due to adverse clinical signs. Common clinical signs in these females included hunched posture, decreased activity and respiratory issues (labored breathing and abnormal breathing signs). The females that were euthanized due to adverse clinical signs also experienced body weight loss prior to euthanasia. 

During the precohabitation, gestation and/or lactation periods, there was an increased incidence of suspected dehydration, labored breathing, abnormal breathing sounds, shallow breathing, hunched posture, thin fur cover erect fur, hyperreactivity and decreased activity at 450 mg/kg/day.

At 450 mg/kg/day, there were statistically significant reductions in mean body weights observed beginning on DS 20 and continuing for the remainder of the pre-mating period. There were reductions or statistically significant reductions in mean body weights at 450 mg/kg/day on DGs 5, 19 and 21. There was a statistically significant body weight loss at 450 mg/kg/day on DSs 9 to 10.

At 450 mg/kg/day, there were decreases or statistically significant decreases in the mean food consumption values observed on DSs 8 to 11, 11 to 15 and at all intervals between 18 to 22 and 46 to 50. 

Estrous cycling, mating, fertility and natural delivery were not affected by doses of the test substance as high as 450 mg/kg/day.

Maternal doses as high as 450 mg/kg/day did not affect the anogenital distance as measured on the day of delivery. No male pup in any group had nipples observed on PND 11. There were no test substance-related adverse clinical signs or effects on pup weights during the pre-weaning period. 

There were no test substance-related changes in the thyroid hormone levels observed in the P generation female rats. No hematologic, coagulation, clinical chemistry or urinalysis parameter was affected by doses of the test substance as high as 450 mg/kg/day. 

No effect on gross necropsy and organ weights occurred in any dose group.

Inflammation was observed within the nasopharynx and multifocally within the nasal cavity of rats at 150 or 450 mg/kg/day. This inflammation was characterized by infiltrates of neutrophils and less frequently mononuclear cells within the epithelium and lamina propria, as well as within the lumen of the nasopharynx/nasal cavity. Segments of epithelium within areas of inflammation were occasionally necrotic/absent. The nasal respiratory epithelium was more frequently affected than the olfactory epithelium. In three females at 450 mg/kg/day, segments of the nasopharyngeal epithelium were replaced by squamous metaplasia.  The described nasal cavity findings were considered to likely be secondary to irritation caused by nasal reflux of the gavaged test item.

F1 Generation Male and Female Rats

The only deaths that occurred in the F1 generation rats occurred at 450 mg/kg/day. At 450 mg/kg/day, one male and one female were found dead during the dose period and three males were euthanized due to adverse clinical signs. Common clinical signs in the rats included respiratory issues (labored breathing and abnormal breathing signs) and suspected dehydration. The three males that were euthanized due to adverse clinical signs also experienced body weight loss prior to euthanasia. One male that was euthanized due to adverse clinical signs had necropsy observations that were indicative of an intubation error. 

There was an increased incidence in abnormal breathing sounds observed at 150 and 450 mg/kg/day in the F1 generation female rats and at 450 mg/kg/day in the F1 generation male rats. There was also an increased incidence of suspected dehydration, excess salivation and labored breathing in the F1 generation male rats. There were no test substance-related differences in body weights, body weight gains and food consumption observed in the F1 generation male or female rats at doses up to and including 450 mg/kg/day. There were no test substance-related effects on F1 generation male or female sexual maturation at doses as high as 450 mg/kg/day. There were no test substance-related effects on estrous cycling observed in the F1 generation female rats. There were no test substance-related effects on hematology, coagulation, clinical chemistry or urine analysis parameter at termination of the F1 generation male and female adult rats. Thyroid hormones were not affected by doses of the test substance as high as 450 mg/kg/day. 

No test substance-related gross findings or organ weight changes were observed in the F1 generation male and female rats. No effect on sperm (motility or count) occurred in the F1 generation males.

Inflammation was observed within the nasopharynx and multifocally within the nasal cavity of rats at 150 or 450 mg/kg/day. This inflammation was characterized by infiltrates of neutrophils and less frequently mononuclear cells within the epithelium and lamina propria, as well as within the lumen of the nasopharynx/nasal cavity. Segments of epithelium within areas of inflammation were occasionally necrotic/absent. The nasal respiratory epithelium was more frequently affected than the olfactory epithelium. Segments of the nasopharyngeal epithelium were replaced by squamous metaplasia in one F1 generation female at 450 mg/kg/day.  The described nasal cavity findings were considered to likely be secondary to irritation caused by nasal reflux of the gavaged test item.

Based on the results of this study, the no-observed-adverse-effect-level (NOAEL) for general toxicity was 150 mg/kg/day for the P generation and F1 generation male and female rats. Mortality was observed in the P generation females and F1 generation males and females. Reductions in body weight and body weight gain were observed in the P generation males and females at 450 mg/kg/day. There was an increased incidence of microscopic findings in the nasal cavity of both the P generation and F1 generation rats at 150 or 450 mg/kg/day that included inflammation, neutrophilic exudates, and squamous metaplasia of the nasopharynx. Luminal hemorrhage was also observed in the nasal cavity of P generation rats at 450 mg/kg/day. There were abnormal breathing sounds and labored breathing observed in the P generation and F1 generation males and females that correlated with these microscopic findings.  A link between the effects observed and the corrosive nature of the test substance cannot be excluded. Therefore, the NOAEL is considered to be 150 mg/kg bw/d.

The reproductive NOAEL was 450 mg/kg/day as there were no test substance-related changes in estrous cycling in the female and mating and fertility in the males or females. The NOAEL for viability and growth in the offspring was also 450 mg/kg/day.

Effects on developmental toxicity

Description of key information

The NOAEL for embryo-fetal survival, development and growth is set at 115 mg/kg/day when the test substance is administered during organogenesis in the rabbit. A key prenatal development study is performed according to OECD guideline 414 and according to GLP requirements (Renaut, 2016; Klimisch 1)

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-08-25 to 2016-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
Principles of method if other than guideline:
The study was also designed to meet the requirements of the following guideline: Japanese Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agricultural Chemicals, 12 Nohsan No. 8147, Agricultural Production Bureau, November 24, 2000
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
- Name of test material (as cited in study report): POPDA
- Substance type: yellow liquid
- Physical state: liquid
- Analytical purity: 100%
- Purity test date: no data
- Lot/batch No.: DR66700414
- Expiration date of the lot/batch: 2016-12-05
- Stability under test conditions: no data
- Storage condition of test material: at ambient temperature in the dark
- action of test substance: reaction products of propane-1,2-diol, propoxylated by amination of the terminal hydroxyl groups
- correction factor: none
- purity/weighing factor: none
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: 96 female (sexually mature, virgin) New Zealand White Rabbits, supplied by Charles River (France)
- Age at start of study (day 0 of gestation): 21-22 weeks
- Weight at day 0 (after mating): 3.17 - 4.62 kg
- Fasting period before study: no data
- Housing: One animal per cage (acclimatization and gestation) one stock male and one female per cage during mating; suspended cages fitted with perforated floor panels and mounted in batteries. Undertrays lined with absorbent paper were changed at least 3 times a week. The cages constituting each group were blocked by group and mounted in batteries.
- Diet (e.g. ad libitum): restricted (initially 150 g/animal/day during acclimatization up to one week prior to the onset of mating and 200 g/animal/day thereafter), Teklad diet. The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent. Should an individual show a significant non-treatment related reduced food consumption, moistened diet (50 g pelleted diet moistened with 50 mL of water) was offered, the consumption was recorded. In addition to this diet, a small supplement of autoclaved hay was given on a daily basis to promote gastric motility and a small amount of chopped fresh vegetables were given twice weekly.
- Water (e.g. ad libitum): ad libitum, potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals. Water bowls were also provided.
- Acclimation period: at least 19 days. Any individuals rejected during the acclimatization period were replaced with spare animals of suitable weight from the same batch.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16-20°C
- Humidity (%): 40-70%
- Air changes (per hr): filtered fresh air which was passed to atmosphere and not recirculated
- Photoperiod (hrs dark / hrs light): 10 hours dark/14 hours light, artificial lighting

IN-LIFE DATES: From: 2016-01-30 To: 2016-02-22 to 2016-02-26
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
purified
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- The required amount of test item for the highest concentration was mixed with sufficient vehicle to form a solution and mixed with a magnetic stirrer. The solution was made up to the required volume with vehicle and stirred again with a magnetic stirrer. The remaining groups were formulated by serial dilution with further quantities of vehicle.
- Individual dose volume was calculated from the most recently recorded scheduled body weight.
- Frequency of preparation: weekly
- A daily record of the usage of formulation was maintained based on weights. This balance was compared with the expected usage as a check of correct administration. No significant discrepancy was found.
- Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.


VEHICLE
- Concentration in vehicle: no data
- Amount of vehicle (if gavage): 5ml/kg body weight
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of each formulation prepared for administration in the first and last weeks of treatment were analyzed for achieved concentration of the test item.

The analytical method will involve the dilution of the test formulations with a suitable solvent followed by chromatographic assay.
The formulated samples will be analysed using a method transferred from the sponsor with respect to the determination of the specificity of analysis, limit of
detection, linearity of detector response, repeatability, method accuracy and precision.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused: no data
- M/F ratio per cage: 1:1, using identified stock New Zealand White bucks
- Length of cohabitation: no data
- Further matings after two unsuccessful attempts: no data
- Verification of same strain and source of both sexes: no data
- Proof of pregnancy: natural mating observed referred to as day 0 of pregnancy
- After mating each female was injected intravenously with 25 i.u. luteinising hormone.
- A colony of stud males was maintained specifically for the purpose of mating; these animals were not part of the study and were maintained as stock animals.
Duration of treatment / exposure:
days 6 to 28 after mating (22 days)
Frequency of treatment:
once daily, at approximately the same time each day
Duration of test:
30 days
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
15 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
115 mg/kg bw/day (nominal)
No. of animals per sex per dose:
22 females per dose
Control animals:
yes
Details on study design:
- Dose selection rationale: the dose levels were selected by the Sponsor based on a preliminary embryo-fetal study in the rabbit (Envigo Study No. TMR0110), in which the high dose of 150 mg/kg/day elicited extended periods of low food intake in some animals and resulted in one animal being killed for reasons of animal welfare, whereas the intermediate dose of 115 mg/kg/day elicited extended periods of low food consumption in some animals but did not result in premature death. A dose level of 150 mg/kg/day was therefore considered too high for subsequent investigation on a main embryo-fetal study, and therefore the dose level of 115 mg/kg/day was selected as the high dose for the current main embryo-fetal development study.
Intermediate and low dose levels of 50 and 15 mg/kg/day, respectively, were selected to evaluate the response at lower dose levels to identify any dose response. The lowest dose level of 15 mg/kg/day was expected to provide a clear No Observed Effect Level for extended periods of low maternal food intake.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: during aclimatization at least once per day, during gestation at least twice daily
- Animals were inspected visually for evidence of ill-health or reaction to treatment. Cages and cage-trays were inspected daily for evidence of animal ill-health amongst the occupant.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily during the treatment period at the following times during the day: 1 to 2 hours after completion of dosing, as late as possible in the working day. Detailed physical examination on days 0, 6, 12, 18, 24 and 29 after mating
- Detailed physical examination was performed on each animal to monitor general health.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly during acclimatization, on days 0, 3, 6 to 29 after mating.
- All adult animals

FOOD CONSUMPTION: Yes
- Time schedule: daily from day 1 after mating.
- All adult animals
- The weight of food supplied supplied to each animal, that remaining and an estimate of any spilled was recorded
- Consumption of hay and vegetables were monitored qualitatively but not quantitatively. No entry was made in the raw data for the provision of vegetables during the period of 26 Jan 16 – 01 February 2016 and thus during this period only, a twice weekly schedule cannot be verified from the raw data.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #: day 29 after mating
- Females that exhibit pregnancy loss were killed on the day of detection.
- All adult animals were killed by intravenous injection of sodium pentobarbitone
- Macroscopic pathology: a full macroscopic examination of the tissues was performed for each animal. 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.

OTHER:
MORTALITY:
- Time schedule: a viability check was performed near the start and end of each working day.
- Animals were killed for reasons of animal welfare where necessary or if they exhibited pregnancy loss.
Ovaries and uterine content:
For females surviving to term, the following was recorded:
- uterus: gravid uterine weight (including cervix and ovaries).

The following were recorded for all animals (including those prematurely sacrificed, where possible):
- For each ovary/uterine horn: number of corpora lutea, implantation sites, resorption sites (classified as early or late), fetuses (live and dead).
- Apparently non pregnant animals and for apparently empty uterine horns: the absence or number of uterine implantation sites was confirmed.
- Females exhibiting pregnancy loss: expelled uterine contents were identified and examined, as appropriate.
Fetal examinations:
- External examinations: Yes: [all viable fetuses per litter ]
- Soft tissue examinations: No
- Skeletal examinations: Yes [one third per litter ], fixed in Industrial Methylated Spirit and stained with Alizarin Red
- Head examinations: Yes: [appr. 50% of eviscerated fetuses ], fixed in Bouin's fluid
- Remaining fetuses will be eviscerated and fixed in Industrial Methylated Spirit.
- All live fetuses: Internal examination of neck and thoracic and abdominal cavities, sex recorded and individual identification within litter.
- Fetuses were killed by subcutaneous injection of sodium pentobarbitone.
Statistics:
Body weight, gravid uterus weight, food consumption, corpora lutea, implantations, live young, fetal, placental and litter weight data:
- A parametric analysis if Bartlett's test for variance homogeneity (Bartlett 1937) was not significant at the 1% level. For pretreatment data, analysis of variance was used to test for any group differences. Where this was significant (p<0.05) inter group comparisons using t-tests, with the error mean square from the one-way analysis of variance, were made. For all other comparisons the F1 approximate test was applied. If the F1 approximate test for monotonicity of dose-response was not significant at the 1% level, Williams' test for a monotonic trend was applied. If the F1 approximate test was significant, Dunnett's test (Dunnett 1955, 1964) was performed instead.
- A non-parametric analysis if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. For pretreatment data, Kruskal-Wallis’ test (Kruskal and Wallis 1952, 1953) was used to test for any group differences. Where this was significant (p<0.05) inter group comparisons using Wilcoxon rank sum tests (Wilcoxon 1945) were made. For all other comparisons the H1 approximate test, the non-parametric equivalent of the F1 test described above, was applied.If the H1 approximate test for monotonicity of dose-response was not significant at the 1% level, Shirley's test for a monotonic trend was applied. If the H1 approximate test was significant, Steel's test (Steel 1959) was performed instead.

Litter size and survival indices and fetal, placental and litter weight and gravid uterine weight data, if 75% of the data (across all groups) were the same value:
- Fisher’s exact tests (Fisher 1973).

Pre/post implantation loss and sex ratio: generalized mixed linear model with binomial errors, a logit link function and litter as a random effect (Lipsitz 1991).

Resorptions: exact Wilcoxon rank sum test (Wilcoxon 1945)
Indices:
- Pre-implantation loss (%) = (Number of corpora lutea – Number of implantations) / Number of corpora lutea x 100
Where the total number of implantations exceeds corpora lutea observed, pre-implantation loss was assumed to be 0.

- Post implantation loss (%) = (Number of implantations – Number of live fetuses) / Number of implantations x 100
Historical control data:
Historical control data will be routinely supplied for selected observations where this information is considered to assist interpretation of study data, and will normally include both fetal and litter incidences.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
- Female number 82 receiving 115 mg/kg/day was despatched to necropsy for reason of animal welfare on day 23 of gestation following a prolonged period of marked inappetance from day 9 of gestation and subsequent body weight loss of 590 g from day 6 of gestation. At necropsy this female showed 8 normal implantations, and there were no remarkable macroscopic observations. This prolonged period of reduced food intake is likely to be related to treatment.
- Female number 83 receiving 115 mg/kg/day was despatched to necropsy following evidence of abortion. This female had shown body weight loss of 310 g from day 10 of gestation, and food consumption had been very low from day 11 of gestation. Fetal material was observed in the cage under tray on day 20 of gestation. At necropsy this female showed a pale liver, and the animal was confirmed as pregnant with 12 implantations, 11 of which were live young and one was an aborted implant. Due to its isolated nature, a relationship between treatment and abortion is considered unproven.
- Female number 58 receiving 50 mg/kg/day was despatched to necropsy for reason of animal welfare following the observation of a small amount of clotted blood, and subsequently further blood, in the cage under tray, and signs of piloerection and dark eyes on day 15 of gestation. This female had progressively lost bodyweight (to a total of 260 g) with the most marked loss occurring from day 13 of gestation, and food consumption was very low during days 13 and 14. At necropsy there were no macroscopic changes, but the animal was confirmed as pregnant with 11 implantations, which were all noted to be early resorptions. This rabbit therefore showed total litter resorption. Our preliminary evaluation is that this is not related to treatment.
- Female number 65 receiving 50 mg/kg/day was despatched to necropsy for reason of animal welfare on day 23 of gestation following the observation of a swelling on the ventral abdomen (suspected hernia) on days 22-23 of gestation. This female also showed signs of little hay/water consumed, underactive behaviour, reduced body temperature (assessed by touch), deep and irregular breathing, thin build, few faeces, reduced urine output and dark eyes on day 23 of gestation. Body weight loss of 330 g was evident from day 15 of gestation, and food consumption was markedly low from day 14 of gestation. At necropsy the hernia was confirmed (jejunum protruded through the abdomen wall), stomach contents were dark and liquefied, the stomach was distended with dark areas on the glandular mucosa, the jejunum and duodenum were distended and the caecum had firm contents. This female was pregnant with 10 implantations, all of which were late resorptions. This rabbit therefore also showed total litter resorption. Our preliminary evaluation is that this is not related to treatment.
- In animals surviving to scheduled termination there was an increased incidence of females showing clinical signs indicative of inappetance at 115 mg/kg/day. These signs included thin build, little hay eaten, reduced urine output and few, small and pale faeces. Three females receiving 50 mg/kg/day were also noted to have thin build.
- There were no other test article related clinical signs, nor any dosing signs, observed.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
- Two females receiving 115 mg/kg/day, and two females receiving 50 mg/kg/day, were killed for reasons of animal welfare.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- At 115 mg/kg/day mean body weight gain during days 6-14 of gestation was similar to Controls, however, during days 14-28 of gestation mean bodyweight loss of approximately 120 g was evident, compared to body weight gain (ca. 90 g) in the Control group. When mean values of body weight and body weight gain were adjusted for the contribution of the gravid uterus, overall maternal mean body weight loss during days 6-29 of gestation was greater than Controls at 115 mg/kg/day, confirming that the maternal portion of gestational body weight gain was affected by treatment at this high dose level.
- Body weight performance at 15 or 50 mg/kg/day was unaffected by treatment with mean body weight gain during days 6-14 and 14-28 being similar to Control values. When adjusted for the contribution of the gravid uterus, adjusted body weight change values at 50 or 15 mg/kg/day were similar to Controls indicating that maternal weight gain was unaffected by treatment at these dose levels.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- Mean values of food consumption at 115 mg/kg/day were lower than Controls from day 15 of gestation to termination on day 29 of gestation. Resulting overall mean food consumption values for the treatment period (days 6-28) were 79 % of Controls.
- Food consumption at 50 or 15 mg/kg/day was similar to Controls throughout gestation and unaffected by treatment.
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:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
- There were no maternal findings observed at macroscopic examination that were considered to relate to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not specified
Number of abortions:
not examined
Pre- and post-implantation loss:
not examined
Total litter losses by resorption:
not examined
Early or late resorptions:
not examined
Dead fetuses:
not examined
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
not examined
Other effects:
not specified
Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Key result
Abnormalities:
no effects observed
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Litter data:
- In addition to the premature deaths observed, there was also one female in each treatment group that was observed to be not pregnant at necropsy on day 29 after mating. The following assessment of litter data is therefore based on a total of 22, 21, 19 and 19 pregnant females reaching full term in Groups 1-4, respectively.
- Mean post-implantation loss at 115 mg/kg/day was marginally higher than Control values, resulting in a slight increase in the numbers of early and late, and therefore total, resorptions; and subsequently slightly fewer live young when compared with Control values.
- Embryo-fetal survival was unaffected by treatment at 15 or 50 mg/kg/day with mean numbers of implantations, resorptions, live young and percentages of sex ratio and pre and post-implantation loss being similar to Control values.

Placental, litter and fetal weight
- Mean placental, litter and fetal weights at 115 mg/kg/day were marginally lower than Controls.
- Mean placental, litter, and male, female and overall fetal weights at 15 or 50 mg/kg/day were similar to Controls and unaffected by treatment.

Macropathology:
- At 115 mg/kg/day there was an increased fetal and litter incidence of the minor abnormalities of large anterior fontanelle, delayed/incomplete ossification/ unossified sternebrae, pubes, epiphyses, talus, metacarpals/metatarsals/phalanges compared to the concurrent Control group, and which were outside of Historical Control Data (HCD) ranges (See Annex 3). This delay in ossification is a transient stage in fetal development that may be attributed to the slight decrease in mean fetal weight observed and as such this is not considered to be adverse.
- In addition, at 115 mg/kg/day there was also a slightly increased incidence of the minor abnormality of fused/partially fused/misaligned sternebral hemicentres, with associated misaligned costal cartilage when compared to concurrent Control, and these were also slightly outside of HCD ranges. As these incidences were small (max 4 fetuses from 3 litters) a relationship to treatment is considered not proven.
- At 15 and 50 mg/kg/day there was a high incidence of the minor abnormality of delayed/incomplete ossification/ unossified sternebrae compared to concurrent control and outside of HCD ranges. As at 115 mg/kg/day this delay in ossification is a transient stage in fetal development and as such this is not considered to be adverse.
Key result
Dose descriptor:
NOAEL
Effect level:
115 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: fetotoxicity
Abnormalities:
not specified
Key result
Developmental effects observed:
no

Formulation analysis:

The mean concentrations of the test substance in test formulations were within applied limits +/- 10%, confirming the accuracy of formulation. Difference from mean values were within 2% confirming precise analysis.

Conclusions:
Based on the results of this prenatal developmental toxicity study, it was concluded that the No-Adverse-Effect-Level (NOAEL) of the test substance for maternal toxicity was 50 mg/kg/day as the higher dose of 115 mg/kg/day elicited low food consumption that directly resulted in a single mortality, and elicited clinical signs relating to inappetance and overall body weight loss during gestation compared with body weight gain in all other groups.
The NOAEL for embryo-fetal survival, development and growth is set at 115 mg/kg/day when the test substance is administered during organogenesis in the rabbit.
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-10-13 to 2016-01-05
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
other: OECD guideline 407
Principles of method if other than guideline:
The study design was based on OECD guideline 407.
GLP compliance:
no
Remarks:
the work performed generally followed GLP principles
Limit test:
no
Specific details on test material used for the study:
- Name of test material (as cited in study report): POPDA
- Substance type: yellow liquid
- Physical state: liquid
- Analytical purity: 100%
- Impurities (identity and concentrations): no data
- Purity test date: no data
- Lot/batch No.: DR66700414
- Expiration date of the lot/batch:2016-12-05
- Stability under test conditions: refrigerated (nominally 4 – 8°C) or ambient (nominally 21°C) storage for 15 days
- Storage condition of test material: at ambient temperature in the dark
- Action of test substance: reaction products of propane-1,2-diol, propoxylated by amination of the terminal hydroxyl groups
- purity/weighing factor: none
- total correction factor: none
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: 36 female (sexually mature, virgin) New Zealand White rabbits, supplied by Charles River (France)
- Age at start of the study: 18-19 weeks (pilot phase); 24 to 25 weeks (preliminary phase)
- Weight range at start of the study: 3.15 - 4.26 kg (pilot phase); 3.87 - 4.54 kg (preliminary phase)
- Fasting period before study: no data
- Housing: individual housing during acclimatization and gestation, one stock male and one female during mating; suspended cages fitted with perforated floor panels and mounted in batteries. Undertrays lined with absorbent paper were changed at least 3 times a week. Cages were also fitted with a plastic resting platform. The cages constituting each group were blocked by group and mounted in batteries.
- Diet (e.g. ad libitum): restricted, initially 150 g/rabbit/day during acclimatisation up to one week prior to the onset of mating and 200 g/animal/day thereafter; Harlan Teklad 2930, pelleted diet. The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent. Should an individual show a significant non-treatment related reduced food consumption, moistened diet (50g pelleted diet moistened with 50 ml of water) was offered, the consumption was recorded. In addition, a small supplement of autoclaved hay was given on a daily basis to promote gastric mobility and a small amount of chopped fresh vegetables were given twice weekly.
- Water (e.g. ad libitum): ad libitum, potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals. Water bowls were also provided.
- Acclimation period: 15 days (pilot phase), 55 days (preliminary phase)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16-20 °C
- Humidity (%): 40-70%
- Although conditions were occasionally outside the indicated ranges, these deviations were minor and/or of short duration and were considered not to have influenced the health of the animals and/or the outcome of the study.
- Air changes (per hr): filtered fresh air which was passed to atmosphere and not recirculated
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
- Pilot phase: from 2015-11-03 to: 2015-11-26
- Preliminary phase: from 2015-12-13 to 2016-01-05
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
purified
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- The required amount of test item for the highest concentration was mixed with sufficient vehicle to form a solution and mixed with a magnetic stirrer. The solution was made up to the required volume with vehicle and stirred again with a magnetic stirrer. The remaining group was formulated by serial dilution with further quantities of vehicle.
- Frequency of preparation: weekly, batches may be prepared in advance but will be used within documented stability limits.
- Storage of formulations: at ambient temperature (21°C)
- Detailed records of compound usage were maintained. The amount of test item necessary to prepare the formulations and the amount actually used were determined on each occasion. The difference between these amounts was checked before the formulations were dispensed.
- Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.

VEHICLE
- Concentration in vehicle:
- pilot phase: 15 mg/ml (75 mg/kg) and 30 mg/ml (150 mg/kg);
- preliminary phase: 23 mg/ml (115 mg/kg) and 30 mg/ml (150 mg/kg)
- Amount of vehicle (if gavage): 5 ml/kg
Analytical verification of doses or concentrations:
no
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused: no data
- M/F ratio per cage: 1:1 using identified stock New Zealand White bucks
- Length of cohabitation: no data
- Further matings after two unsuccessful attempts: no data
- Verification of same strain and source of both sexes: yes, a colony of stock males of the same strain was maintained specifically for the purpose of mating; these animals are not part of the study and were maintained as stock animals.
- Proof of pregnancy: natural mating observed referred to as day 0 of pregnancy
- checks: natural mating observed
- after mating: each female was injected intravenously with 25 i.u. luteinising hormone
- day 0 of gestation = day of mating
Duration of treatment / exposure:
days 6 to 28, after mating
Frequency of treatment:
once daily, at approximately the same time each day
Duration of test:
22 days (day 6 to day 28 after mating)
No. of animals per sex per dose:
6 females per dose (pilot and preliminary phase), except control group (3 in pilot phase and 3 in preliminary phase)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Pilot phase: the dose levels for the Pilot phase were selected in conjunction with the Sponsor based on a previous Preliminary Toxicity study in the New Zealand White Rabbit (Study number TMR0109). In that study, a single dose of 600 mg/kg/day was not tolerated, and was associated with marked macroscopic findings relating to depressions or perforation on the stomach, and other gastro-intestinal findings including dark areas on the jejunum and thickened omental adipose tissue.
Repeat daily dosing at a lower dose of 300 mg/kg/day was initially tolerated for a period of 11 days, but did elicit a marked reduction in food consumption for two out of three females. After receiving 11 doses one female was found dead and at macroscopic examination showed multiple depressions on the stomach. Repeat daily dosing at 300 mg/kg/day was subsequently suspended due to the similarity of the stomach findings in the decedent to those observed at the higher dose of 600 mg/kg/day. At necropsy a total of two out of three females receiving a daily dose of 300 mg/kg/day showed a marked reduction in food intake and macroscopic findings in the stomach and GI tract whilst the third animal was not similarly affected.
It is considered that daily dosing at 300 mg/kg/day for a period of 11 days or more is clearly unsuitable for any further investigation. Due to the marked nature of the findings observed in the stomach and GI tract the starting high dose level for the pilot phase of this preliminary embryo-fetal study in the New Zealand White Rabbit is set at 150 mg/kg/day, with the low dose set at 75 mg/kg/day.
Preliminary phase: Results from the pilot phase of the current study suggested potential effects in individual animals in terms of food consumption, and the possibility of marginally low fetal weights, at 150 mg/kg/day. The high dose level of 150 mg/kg/day was therefore repeated on the preliminary phase in order to substantiate these findings. A lower dose level of 115 mg/kg/day was also implemented on the preliminary phase to investigate the response between 75 and 150 mg/kg/day.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily (at least once per day during the acclimatisation period)
- Animals visually inspected for evidence of reaction to treatment or ill-health. Cages and cage-trays were inspected daily for evidence of animal ill-health amongst the occupant.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily during the treatment period at the following times in relation to dose administration: pre-dose observation, 1 to 2 hours after dosing, as late as possible in the working day; detailed physical examination was performed on days 0, 6, 12, 18, 23 and 29 after mating to monitor general health.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly during acclimatisation, on day of mating (day 0) and days 3 and 6-29 after mating
- all adult animals

FOOD CONSUMPTION: Yes
- Time schedule: daily from day 1 after mating
- all adult animals
- The weight of food supplied to each animal, that remaining and an estimate of any spilled was recorded.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS:
- Necropsy: all adult animals were subject to a detailed necropsy. Animals surviving until the end of the scheduled study period were killed at day 29 after mating; All adult animals were killed by intravenous injection of sodium pentobarbitone. Fetuses were killed by subcutaneous injection of sodium pentobarbitone.
- Macroscopic pathology: 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.

MORTALITY:
- A viability check was performed near the start and end of each working day. Animals were killed for reasons of animal welfare where necessary.
Ovaries and uterine content:
- The uterine content was examined after termination only for females exhibiting pregnancy loss.
- For females surviving to full term (day 29 after mating), the following was recorded: gravid uterine weight (including cervix and ovaries)
- The following was recorded for all animals (including those prematurely sacrificed, where possible): for each ovary/uterine horn: number of: corpora lutea, implantation sites, intrauterine deaths (assessed as early or late resorptions), fetuses (live and dead); apparently non-pregnant animals and for apparently empty uterine horns: the absence or number of uterine implantation sites was confirmed.
- Any photographs of unusual findings were taken at the discretion of the necropsy supervisor.
- Fetuses and placentae were dissected from the uterus and weighed individually.
Fetal examinations:
- External examinations: Yes, each placenta and fetus was externally examined.
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No
- Internal examination of neck and thoracic and abdominal cavities was performed and any abnormalities were recorded, sampled as appropriate and retained in appropriate fixative.
- The sex of each fetus was recorded.
- Grossly normal fetuses were discarded.
Statistics:
No statistical analysis performed.
Indices:
- Prenatal losses are seperated into pre- and post-implantation phases. Pre-implantation loss was considered to reflect losses due to non-fertilization of ova and failure to implant.
Pre-implantation loss (%) = (Number corpora lutea – Number implantations)/Number corpora lutea x 100
Where the total number of implantations exceeds corpora lutea observed, preimplantation loss was assumed to be zero (i.e. no pre-implantation loss was considered to have occurred).
- Post implantation loss (%) = (Number implantations – Number live fetuses)/Number implantations x 100
- All group values and SD (as appropriate) were calculated from the individual litter values.
Historical control data:
no data
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Mortality and clinical signs:
One female (number 15) receiving 150 mg/kg/day in the pilot phase was despatched to necropsy for reasons of animal welfare following an extended period of inappetance and clinical signs indicative of inappetance (little diet/water consumed, small and reduced faeces) and underactive behaviour. Upon veterinary examination it was deemed necessary to despatch this female to necropsy. Macroscopic examination revealed no findings, in particular no findings related to abnormalities of the gastro-intestinal tract. This mortality was considered to be related to inappetance, which is likely to be caused by the test material.There were no premature deaths at 150 mg/kg/day in the prelim phase.
There were no premature mortalities at 115 or 75 mg/kg/day.
In animals surviving to scheduled termination there were no consistent clinical signs, nor were there any dosing signs, observed that were considered to be related to treatment at any of the dose levels investigated.
Signs considered to be related to treatment related effects upon food consumption included two animals at 115 mg/kg/day, and one at 150 mg/kg/day, on the prelim phase that were observed to be thin, and pale and few faeces, which were observed in 4 animals at 115 mg/kg/day and few faeces in two animals receiving 150 mg/kg/day.

Body weight and gravid uterine weight:
Body weight was not clearly affected by treatment.
On the pilot phase, group mean overall body weight gain during Days 6-29 of gestation was lower than controls amongst females receiving 75 or 150 mg/kg/day; however, when mean values of body weight and body weight gain were adjusted for the contribution of the gravid uterus, overall maternal mean body weight loss during Days 6-29 of gestation was similar to controls at both dose levels investigated. This suggested that the reduced gravid uterine weight observed was the influencing factor in treated animals not gaining as much weight as controls and that the maternal portion of gestational body weight gain was unaffected by treatment.
On the prelim phase, slight group mean overall bodyweight loss was evident at 115 mg/kg/day and overall bodyweight gain lower than that of Controls was evident at 150 mg/kg/day. When mean values of body weight and body weight gain were adjusted for the contribution of the gravid uterus, overall maternal mean body weight loss during Days 6-29 of gestation was slightly greater than controls at both dose levels.
At 115 mg/kg/day overall body weight change was heavily influenced by two females (numbers 23 and 24) showing marked weight loss of 0.40 – 0.41 kg, however, all other females at this dose level showed lower weight gain than in controls.
At 150 mg/kg/day on the prelim phase overall body weight change was heavily influenced by a single female (number 28) that showed weight loss of 0.20 kg, however, all other females at this dose level (i.e. 5/6 females) showed overall weight gain that was similar to controls.
Individual values of weight change following adjustment for the contribution of the gravid uterus indicated 4 / 6 females at 115 mg/kg/day, and two females at 150 mg/kg/day, with greater adjusted body weight loss than controls. Control values were also influenced by one female (number 6) showing overall body weight gain after adjustment for the contribution of the gravid uterus, due to a very low litter size (3 implantations).

Food consumption:
At 150 mg/kg/day, four out of a total of 12 animals at this dose level showed periods of low food intake. Females 15 (pilot phase) and 28 (prelim phase) showed low food from day 15 after mating, female 16 showed low food during days 15-20 after mating and female 18 showed low food intake from day 22 after mating.
At 115 mg/kg/day three out of six females showed periods of low food intake. Female 23 showed low food from day 13 after mating, female 24 showed low food intake from day 14 after mating and female 19 showed low food from day 24 after mating.
These incidences were compared to no similar cases in the control or 75 mg/kg/day groups.

Macropathology
There were no maternal findings observed at macroscopic examination of adult females receiving 75, 115 or 150 mg/kg/day that were considered to relate to treatment.
Key result
Dose descriptor:
dose level: other
Remarks:
no remarks
Effect level:
ca. 115 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks:
no remarks
Basis for effect level:
mortality
Remarks on result:
other: 115 mg/kg considered appropriate high dose in main OECD414 study due to mortality observed at 150 mg/kg as a direct result of sustained period of very low food intake.
Abnormalities:
not specified
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Litter data:
All females on the pilot phase were pregnant. One Group 3 female receiving 150 mg/kg/day was killed for welfare reasons therefore the assessment of litter data is based on a total of 6, 6 and 5 pregnant females reaching full term in Groups 1-3, respectively.
One female on the preliminary phase was found to be not pregnant at necropsy, female 27 receiving 150 mg/kg/day. The assessment of litter data on the preliminary phase is therefore based on a total of 6, 6 and 5 pregnant females reaching full term in Groups 1, 4 and 5, respectively.
Embryo-fetal survival was unaffected by treatment at 75, 115 or 150 mg/kg/day with mean numbers of implantations, resorptions, live young and percentages of sex ratio and pre- and post-implantation loss being similar to control values.

Placenta, litter and fetal weight:
Mean placental weight at 150 mg/kg/day was marginally lower than controls in the pilot phase but similar to Controls in the preliminary phase, therefore considered unrelated to treatment.
On the pilot phase mean litter weight was lower than controls in both treated groups, despite mean litter size being considered essentially similar across all groups. In the preliminary phase, mean litter weight was similar to Controls at 150 mg/kg/day but marginally lower than controls at 115 mg/kg/day.
Mean male, female and overall fetal weights at 75, 115 or 150 mg/kg/day were marginally lower than controls, with evidence of a slight dose response apparent. This was replicated on both the pilot and prelim phases at 150 mg/kg/day.

Macropathology:
There were no consistent fetal abnormalities observed at macroscopic examination of adult females receiving 75, 115 or 150 mg/kg/day.
There was one fetus in a litter of an adult female receiving 75 mg/kg/day that showed cardiovascular abnormalities including dilated aortic arch, narrow pulmonary trunk and ventricular septal defect.
At 115 mg/kg/day there were three fetuses from a litter of an adult female receiving 115 mg/kg/day with dark thyroids, two of these fetuses also showed enlarged thyroids.
There was one fetus in a litter of an adult female receiving 150 mg/kg/day on the pilot phase that showed the cardiovascular abnormality of retroesophageal subclavian artery, and further findings of small lungs and bilateral forepaw flexure, and a single fetus at 150 mg/kg/day on the preliminary phase that showed unilateral forepaw extension and flexure.
In isolation these findings are currently of uncertain relationship to treatment.
Key result
Dose descriptor:
dose level:
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
fetal/pup body weight changes
changes in litter size and weights
Remarks on result:
not determinable
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
Based on the results of this study it is concluded that a dose level of 150 mg/kg/day would be too high for investigation on a main embryo-fetal study in the pregnant New Zealand White Rabbit, due to the mortality observed as a direct result of a sustained period of very low food intake.
A dose level of 115 mg/kg/day would therefore be considered appropriate for use as the high dose on a main embryo-fetal study in the pregnant New Zealand White Rabbit. This dose level could be expected to elicit slight maternal toxicity in terms of prolonged periods of lower food intake in some females, but did not result in any premature mortality on this preliminary study.


Endpoint:
developmental toxicity
Study period:
defined by ECHA decision
Data waiving:
other justification
Justification for data waiving:
other:
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Species:
rat
Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
115 mg/kg bw/day
Study duration:
subacute
Species:
rabbit
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

Screening study via topical application

No adverse effects on development were demonstrated in a reproductive/developmental toxicity screening study (OECD 421), with the test substance administered up to 30 mg/kg/day via dermal application.

Prenatal development toxicity study in rabbit

A combined pilot and preliminary embryo-fetal development study was performed in rabbits with daily administration from day 6 to 28 after mating of the test substance at 75 or 150 mg/kg/day (pilot phase) or 115 or 150 mg/kg/day (preliminary phase) (Renaut, 2016; Klimisch 1, OECD guideline 414). Mortality was observed as a direct result of a sustained period of very low food intake, hence 150 mg/kg/day is considered too high to investigate in the main embryo-fetal development study. The dose level of 115 mg/kg/day is considered appropriate as high dose, as no premature mortality in this study is observed.

Three groups of 22 females received the test substance at doses of 15, 50 or 115 mg/kg/day by oral gavage administration, from Day 6 to 28 after mating. A similarly constituted Control group received the vehicle only, purified water, at the same volume dose as treated groups. Animals were killed on Day 29 after mating for reproductive assessment and fetal examination.

Clinical observations, body weight, and food consumption are recorded. Adult females were examined microscopically at necropsy on Day 29 after mating; the gravid uterine weight was recorded, and all fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination of the head or skeletal examination.

Treatment of pregnant female New Zealand White rabbits with the test substance daily from Day 6 to Day 28 after mating, inclusive, at 115 mg/kg/day was associated with a prolonged period of low food intake in one female to an exten that necessitated premature euthanasia for welfare reasons. There was also one female killed for welfare reasons following abortion at 115 mg/kg/day; however, in isolation this was considered unlikely to be a result of treatment.

At 50 mg/kg/day there were no mortalities that were considered to be related to treatment.

At 115 mg/kg/day there was an increased incidence, when compared to Controls, of females showing clinical signs indicative of inappetence including thin build, little hay eaten, reduced urine output and few, small and pale faeces. These signs were not observed at the same magnitude at 15 or 50 mg/kg/day.

Mean maternal body weight loss during Days 14 -29 of gestation, and markedly low food intake during Days 15 -28 of gestation, were observed at 115 mg/kg/day only. Body weight and food consumption at 15 or 50 mg/kg/day was similar to the control group and unaffected by treatment.

There were no maternal findings observed at macroscopic examination of adult females receiving 15, 50 or 115 mg/kg/day that were considered to relate to treatment.

Embryo-fetal survival was considered unaffected by treatment at dose levels up to 115 mg/kg/day.

There were no effects of treatment upon embryo-fetal growth or development at any of the dose levels investigated that were considered to be adverse. At 115 mg/kg/day mean fetal weight was slightly lower than in the control group and this, and the higher incidence of a number of minor fetal skeletal abnormalities (primarily delayed ossification), may reflect the reduced maternal food consumption observed at this dose level. At 15 or 50 mg/kg/day there was a higher incidence of delayed ossification of the sternebrae but this was considered no to represent an adverse effect of treatment.

Based on the results of this prenatal development toxicity study, it was concluded that the No-Adverse-Effect-Level (NOAEL) of the test substance for maternal toxicity was 50 mg/kg/day as the higher dose of 115 mg/kg/day elicited low food consumption that directly resulted in a single mortality, and elicited clinical signs relating to inappetence and overall body weight loss during gestation compared with body weight gain in all other groups. The NOAEL for embryo-fetal survival, development and growth is set at 115 mg/kg/day when the test substance is administered during organogenesis in the rabbit.

Prenatal development toxicity study in rat

A developmental toxicity study in a second species (the rat) is being performed, per ECHA decision.

Justification for classification or non-classification

Based on the available data and according to the criteria of the CLP Regulation, the substance should not be classified for toxicity to reproduction.

Additional information