Registration Dossier

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

Based on the results of the read across study, the NOAEL for reproductive toxicity was considered to be 200 mg/kg bw/day.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From 12 Jan, 2010 to 19 May, 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
KL2 due to RA
Justification for type of information:
Refer to section 13 of IUCLID for details on the read-across justification.
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:CD(SD)]
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC, USA
- Age at study initiation: ca. 63 d
- Housing: stainless steel wire mesh cages suspended above cage board
- Diet (e.g. ad libitum): PMI Nutrition International, LLC Certified Rodent LabDiet® 5002
- Water (e.g. ad libitum): yes
- Acclimation period: 17 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.8 - 22.7°C
- Humidity (%): 38.2 - 45.1%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 29 January 2010 To: 19 May 2010
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): corn oil
- Concentration in vehicle: 0, 5, 15 or 40 mg/ml
- Amount of vehicle (if gavage): 5ml/kg bw
- Lot/batch no. (if required): YF0793, YR1134, and YJ0917
Details on mating procedure:
The animals were paired on a 1:1 basis within each treatment group following 14 d of treatment for the males and females. A breeding record containing the male and female identification numbers and the start date of cohabitation was maintained. Each female was housed in the home cage of the male. Positive evidence of mating was confirmed by the presence of a vaginal copulatory plug or the presence of sperm following a vaginal lavage and verified by a second biologist. Each mating pair was examined daily. The day when evidence of mating was identified was termed gestation day 0. If evidence of copulation was not detected after 14 days of pairing, any females that had not shown evidence of mating were placed in plastic maternity cages.

For the purpose of calculating pre-coital intervals, rats paired over a 12-hour dark cycle were considered to have been paired for 1 day.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Fifteen-day room temperature resuspension homogeneity and stability of the test substance formulated in the vehicle at concentrations of 10 and 200 mg/mL were established in a previous study (Stump, Draft, WIL-738003). Therefore, resuspension homogeneity and stability were not assessed for the 15 and 40 mg/mL formulations prepared for the current study.

Prior to the initiation of dose administration, quadruplicate samples for homogeneity and concentration determination were collected from the top, middle, and bottom strata of the 5 mg/mL non-dosing formulation. However, because the analytical results of the initial samples as well as the back-up samples did not meet the WIL Research Laboratories, LLC’s SOP requirements, a new 5 mg/mL non-dosing formulation was prepared. Quadruplicate samples for homogeneity and concentration determination were collected from the top, middle, and bottom strata of the new 5 mg/mL non-dosing formulation. In addition, quadruplicate samples for resuspension homogeneity and stability determinations were collected from aliquots prepared from this same non-dosing formulation following room temperature storage for 5 and 13 days; the aliquots were stirred for at least 60 minutes prior to sampling. Quadruplicate samples for homogeneity and concentration analyses were collected from the top, middle, and bottom of the test substance formulations prepared for the first week of dose administration; samples were also collected from the middle stratum of the vehicle control formulation. Additionally, quadruplicate samples for concentration analysis were collected from the middle stratum of each dosing formulation and vehicle control formulation prepared for the remainder of the study. One set of duplicate samples from each collection was subjected to the appropriate analyses. The remaining set of duplicate samples was stored frozen (approximately -70°C ± 5°C) as back-up. All analyses were conducted by the Analytical Chemistry Department at WIL Research Laboratories, LLC using a validated high performance liquid chromatography method with ultraviolet absorbance detection.
Duration of treatment / exposure:
The males were dosed once daily during study Days 0-27 (14 days prior to pairing through 1 day prior to scheduled euthanasia) for a total of 28 doses. The females were dosed once daily during study Days 0 through the day prior to euthanasia (14 days prior to pairing through lactation day 4) for a total of 40-47 doses. Females that failed to deliver were dosed through the day prior to euthanasia (post-mating Day 25) for a total of 41 doses.
Frequency of treatment:
Daily
Dose / conc.:
25 mg/kg bw/day (nominal)
Dose / conc.:
75 mg/kg bw/day (nominal)
Dose / conc.:
200 mg/kg bw/day (nominal)
No. of animals per sex per dose:
12 males and 12 females
Control animals:
yes
Positive control:
None
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All rats were observed twice daily, once in the morning and once in the afternoon, for moribundity and mortality. A detailed physical examination was conducted weekly on each animal beginning approximately 1 week prior to the initiation of dose administration, including on the day of necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual male body weights were recorded beginning approximately 1 week prior to the initiation of dose administration, on the first day of dose administration and weekly thereafter until the day prior to scheduled euthanasia. Individual female body weights were recorded beginning approximately 1 week prior to the initiation of dose administration, on the first day of dose administration and weekly thereafter until the day evidence of copulation was observed.

FOOD CONSUMPTION:
- Individual food consumption was recorded on the corresponding weekly body weight days until pairing. Food intake was not recorded during the mating period. Once evidence of mating was observed, female food consumption was recorded on gestation days 0, 4, 7, 11, 14, 17, and 20 and on lactation days 1 and 4. Following mating, food consumption for the female with no evidence of mating was measured on a weekly basis until parturition. When food consumption could not be determined for an animal during a given interval (due to an unscheduled death, as females enter gestation/lactation, weighing error, food spillage, obvious erroneous value, etc.), group mean values were calculated for that interval using the available data.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at necropsy (Study Day 28 for males and Lactation Day 5 for females)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: 6 animals/sex/group

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at necropsy (Study Day 28 for males and Lactation Day 5 for females)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: 6 animals/sex/group

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- FOB assessments were recorded for 6 animals/sex/group prior to dose administration and fasting for clinical pathology sampling on study day 27 (males) and lactation day 4 (females).
- Locomotor activity counts were recorded for 6 animals/sex/group prior to dose administration on study day 27 (males) and on lactation day 4 (females); the same animals evaluated for FOB were selected for locomotor activity assessment.

GROSS PATHOLOGY

HISTOPATHOLOGY
Litter observations:
LITTER VIABILITY AND DEATHS
Each litter was examined daily for survival, and all deaths were recorded. All pups were individually identified by application of tattoo markings on the digits following completion of parturition. A daily record of litter size was maintained. Intact offspring dying were necropsied using a fresh dissection technique, which included examination of the heart and major vessels (Stuckhardt and Poppe, 1984). The carcass of each pup was then discarded.

LITTER CLINICAL OBSERVATIONS
Litters were examined daily for survival and any adverse changes in appearance or behavior. Each pup received a detailed physical examination on PND 1 and 4. Any abnormalities in nursing behavior were recorded.

LITTER BODYWEIGHTS
Pups were individually weighed on PND 1 and 4. Mean pup weights were presented by sex for each litter and by dose group. When body weights could not be determined for a pup during a given interval (due to an unscheduled death, weighing error, etc.), group mean values were calculated for that interval using the available data. The time periods a given pup was not weighed were left blank or designated as “NA” on the individual report tables.

LITTER SEX DETERMINATION
Pups were individually sexed on PND 0 (if possible), 1, and 4.
Clinical signs:
effects observed, treatment-related
Dermal irritation (if dermal study):
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Description (incidence):
Test substance-related mortality and/or moribundity occured at 75 and 200 mg/kg bw/day.Test substance-related mortality and/or moribundity occured at 75 and 200 mg/kg bw/day.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In the 200 mg/kg bw/day group males, lower mean body weight gains were noted during the overall pre-mating (Days 0-13) and treatment (Days 0-27) periods, with correspondingly lower mean food consumption during the pre-mating period. As a result, mean male body weight in this group was 8.4% lower than controls on Day 27. For the 75 mg/kg bw/day group males, slightly lower mean body weight gains were noted throughout the treatment period, with the most severe reduction occurring during Days 21-27. However, the magnitude of these differences was not sufficient to affect mean body weights when compared to controls and mean food consumption was similar to that of controls during the pre-mating period (Days 0-13). The effects were therefore not considered to be treatment-related. Mean body weights, body weight gains and food consumption were unaffected by test substance administration in the 25 mg/kg bw/day group males throughout the study and in the 25, 75, and 200 mg/kg bw/day group females during the pre-mating, gestation and lactation periods.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
See above
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
At the scheduled necropsy, there were higher mean absolute neutrophil counts at 75 and 200 mg/kg bw/day, consistent with the test substance-related ulceration and associated inflammation in the non-glandular stomach.
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related histopathologic alterations included ulceration, epithelial hyperplasia and hyperkeratosis, and/or chronic-active inflammation in the non-glandular stomach at 75 and 200 mg/kg bw/day.
Hypertrophy of the zona fasciculate of the adrenal cortex and lymphoid depletion was also noted in the 200 mg/kg bw/day group males and females, respectively. Mean numbers of corpora lutea, unaccounted-for sites and implantation were similar to control values.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Male and female mating and fertility, male copulation and female conception indices, mean number of days between pairing and coitus, gestation length, and the process of parturition were unaffected by test substance administration at all dosage levels. Mean numbers of pups born, live litter size, percentage of males at birth, mean pup body weights, and pup body weight gains were unaffected by parental test substance administration. No test substance-related clinical findings or macroscopic findings for pups that were found dead were noted at any dosage level.
Key result
Dose descriptor:
NOAEL
Effect level:
> 200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive function (sperm measures)
reproductive performance
Key result
Dose descriptor:
NOAEL
Effect level:
75 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
Other effects:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Mean numbers of pups born, live litter size, percentage of males at birth, mean pup body weights, and pup body weight gains were unaffected by parental test substance administration. No test substance-related clinical findings or macroscopic findings for pups that were found dead were noted at any dosage level.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
> 200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
body weight and weight gain
Key result
Reproductive effects observed:
no

None

Conclusions:
Based on the results of the read across study, the test substance NOAEL for reproductive toxicity and neonatal toxicity is considered to be >200 mg/kg bw/day (highest tested dose).
Executive summary:

A study was conducted to determine the repeated dose and reproduction/developmental toxicity of the read across substance, PETIA to rat in a screening study conducted according to OECD Guideline 422 and EPA Guideline OPPTS 870.3650, in compliance with GLP. The read across substance was administered orally in corn oil by gavage once daily to 3 groups of 12 male and 12 female Crl:CD(SD) rats. Dosage levels were 25, 75 and 200 mg/kg bw/day, administered at 5 mL/kg. Read across substance-related mortality and/or moribundity occured at 75 and 200 mg/kg bw/day. Clinical findings were recorded at all dose levels and included salivation or evidence thereof, yellow and red material primarily around the mouth and wiping of mouth in the bedding (females only). Incidences of rales were also noted sporadically at 75 and 200 mg/kg bw/day. All findings were primarily noted at the time of dose administration or shortly thereafter and were attributed to the irritating properties of the substance. In the 200 mg/kg bw/day group males, lower mean body weight gains were noted during the overall pre-mating (Days 0-13) and treatment (Days 0-27) periods, with correspondingly lower mean food consumption during the pre-mating period. As a result, mean male body weight in this group was 8.4% lower than controls on Day 27. For the 75 mg/kg bw/day group males, slightly lower mean body weight gains were noted throughout the treatment period, with the most severe reduction occurring during Days 21-27. However, the magnitude of these differences was not sufficient to affect mean body weights when compared to controls and mean food consumption was similar to that of controls during the pre-mating period (Days 0-13). The effects were therefore not considered to be treatment-related. Mean body weights, body weight gains and food consumption were unaffected by test substance administration in the 25 mg/kg bw/day group males throughout the study and in the 25, 75, and 200 mg/kg bw/day group females during the pre-mating, gestation and lactation periods. No substance-related effects were noted during the functional observation battery (FOB) or locomotor activity evaluations at any dosage level. At the scheduled necropsy, there were higher mean absolute neutrophil counts at 75 and 200 mg/kg bw/day, consistent with the substance-related ulceration and associated inflammation in the non-glandular stomach. Gross observations consisted of thickened non-glandular stomach in the 75 and 200 mg/kg bw/day group males and females and adhesions to the liver or spleen in males at 200 mg/kg bw/day. Substance-related histopathologic alterations included ulceration, epithelial hyperplasia and hyperkeratosis, and/or chronic-active inflammation in the non-glandular stomach at 75 and 200 mg/kg bw/day. Organ weight changes included higher mean adrenal gland weights in the 75 mg/kg bw/day group males and 200 mg/kg bw/day group males and females and lower mean thymus weight in the 200 mg/kg bw/day group females. Hypertrophy of the zona fasciculata of the adrenal cortex and lymphoid depletion was also noted in the 200 mg/kg bw/day group males and females, respectively. Mean numbers of corpora lutea, unaccounted-for sites and implantation were similar to control values. Male and female mating and fertility, male copulation and female conception indices, mean number of days between pairing and coitus, gestation length and the process of parturition were unaffected by read across substance administration at all dosage levels. Mean numbers of pups born, live litter size, percentage of males at birth, mean pup body weights and pup body weight gains were unaffected by parental substance administration. No substance-related clinical findings or macroscopic findings for pups that were found dead were noted at any dosage level (Stump, 2011). Based on the results of the read across study, the test substance NOAEL for reproductive toxicity and neonatal toxicity is considered to be >200 mg/kg bw/day (highest tested dose).

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
200 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The information requirements for this tonnage band is sufficiently met with the available data.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

A study was conducted to determine the repeated dose and reproduction/developmental toxicity of the read across substance, PETIA to rat in a screening study conducted according to OECD Guideline 422 and EPA Guideline OPPTS 870.3650, in compliance with GLP. The read across substance was administered orally in corn oil by gavage once daily to 3 groups of 12 male and 12 female Crl:CD(SD) rats. Dosage levels were 25, 75 and 200 mg/kg bw/day, administered at 5 mL/kg. Read across substance-related mortality and/or moribundity occurred at 75 and 200 mg/kg bw/day. Clinical findings were recorded at all dose levels and included salivation or evidence thereof, yellow and red material primarily around the mouth and wiping of mouth in the bedding (females only). Incidences of rales were also noted sporadically at 75 and 200 mg/kg bw/day. All findings were primarily noted at the time of dose administration or shortly thereafter and were attributed to the irritating properties of the substance. In the 200 mg/kg bw/day group males, lower mean body weight gains were noted during the overall pre-mating (Days 0-13) and treatment (Days 0-27) periods, with correspondingly lower mean food consumption during the pre-mating period. As a result, mean male body weight in this group was 8.4% lower than controls on Day 27. For the 75 mg/kg bw/day group males, slightly lower mean body weight gains were noted throughout the treatment period, with the most severe reduction occurring during Days 21-27. However, the magnitude of these differences was not sufficient to affect mean body weights when compared to controls and mean food consumption was similar to that of controls during the pre-mating period (Days 0-13). The effects were therefore not considered to be treatment-related. Mean body weights, body weight gains and food consumption were unaffected by test substance administration in the 25 mg/kg bw/day group males throughout the study and in the 25, 75, and 200 mg/kg bw/day group females during the pre-mating, gestation and lactation periods. No substance-related effects were noted during the functional observation battery (FOB) or locomotor activity evaluations at any dosage level. At the scheduled necropsy, there were higher mean absolute neutrophil counts at 75 and 200 mg/kg bw/day, consistent with the substance-related ulceration and associated inflammation in the non-glandular stomach. Gross observations consisted of thickened non-glandular stomach in the 75 and 200 mg/kg bw/day group males and females and adhesions to the liver or spleen in males at 200 mg/kg bw/day. Substance-related histopathologic alterations included ulceration, epithelial hyperplasia and hyperkeratosis, and/or chronic-active inflammation in the non-glandular stomach at 75 and 200 mg/kg bw/day. Organ weight changes included higher mean adrenal gland weights in the 75 mg/kg bw/day group males and 200 mg/kg bw/day group males and females and lower mean thymus weight in the 200 mg/kg bw/day group females. Hypertrophy of the zona fasciculata of the adrenal cortex and lymphoid depletion was also noted in the 200 mg/kg bw/day group males and females, respectively. Mean numbers of corpora lutea, unaccounted-for sites and implantation were similar to control values. Male and female mating and fertility, male copulation and female conception indices, mean number of days between pairing and coitus, gestation length and the process of parturition were unaffected by read across substance administration at all dosage levels. Mean numbers of pups born, live litter size, percentage of males at birth, mean pup body weights and pup body weight gains were unaffected by parental substance administration. No substance-related clinical findings or macroscopic findings for pups that were found dead were noted at any dosage level (Stump, 2011). Based on the results of the read across study, the test substance NOAEL for reproductive toxicity and neonatal toxicity is considered to be >200 mg/kg bw/day (highest tested dose).

Effects on developmental toxicity

Description of key information

Based on the results of the read across study in rabbits, the NOAELs for maternal and developmental toxicity are both considered to be 75 mg/kg bw/day (highest tested dose).

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From May 20, 2014 to September 02, 2014
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
KL2 due to RA
Justification for type of information:
Refer to section 13 of IUCLID for details on the read-across justification.
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories France, L'Arbresle Cedex, France.
- Age at study initiation: 17-19 weeks
- Housing: individually housed in labelled cages with perforated floors (Ebeco, Germany, dimensions 67 x 62 x 55 cm) and shelters (Ebeco, Germany, dimensions 40 x 32 x 23 cm).
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days prior to pairing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24
- Humidity (%): 40-70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12-h light/12-h dark cycle
Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): based on trial formulations performed at WIL Research Europe
- Amount of vehicle (if gavage): 1 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on a single occasion during the treatment phase (10 July 2014). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 h at room temperature under protection from light was also determined (highest and lowest concentration).

Duplicate samples (approximately 500 mg), which were taken from the formulations using a pipette, were accurately weighed into volumetric flasks of 20 mL. For determination of accuracy, samples were taken at middle position (50% height) or at top, middle and bottom position (90%, 50% and 10% height). The samples taken at 90%, 50% and 10% height were also used for the determination of the homogeneity of the formulations. For determination of stability, additional samples were taken at 50% height and stored at room temperature protected from light for 6 h. The volumetric flasks were filled up to the mark with acetonitrile. The solutions were further diluted to obtain an end solution of 25/75 (v/v) acetonitrile/water and concentrations within the calibration range. All solutions containing the test substance were protected from light.

Analytical conditions:
- Instrument: Acquity UPLC system (Waters, Milford, MA, USA)
- Detector: Acquity UPLC TUV detector (Waters)
- Column: Acquity UPLC BEH Shield RP-18, 100 mm × 2.1 mm i.d., dp = 1.7 μm (Waters)
- Column temperature: 40°C±1°C
- Injection volume: 10 μL
- Mobile phase: 30/70 (v/v) acetonitrile/water
- Flow 0.45 mL/min
- UV detection: 225 nm


The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
Details on mating procedure:
One female was placed on a one-to-one-basis in the cage of a male rabbit. The time of mating was established by visual observation of mating. This day was designated Day 0 post-coitum.
Duration of treatment / exposure:
From Day 6 to Day 28 post-coitum, inclusive.
As a misgavage was suspected, female no. 45 (Group 3) was not dosed on Days 13 and 14 post-coitum. This animal died on Day 16 post-coitum.
Frequency of treatment:
Once daily for 7 days/week, approximately the same time each day
dose.
Duration of test:
From Day 6 to Day 28 post-coitum, inclusive
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
25 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
75 mg/kg bw/day (nominal)
No. of animals per sex per dose:
24, 22, 26 and 24 females in groups 1, 2, 3 and 4, respectively; group 1 being control
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Dose levels were selected based on the results of a dose range finding study. In this study, six mated rabbits per groups were dosed at 0, 25, 50 or 75 mg/kg bw/day for Days 6 to 28 postcoitum,inclusive, by oral gavage. At 75 mg/kg bw/day, toxicity consisted of reduced faeces production, reduced body weight gain (with a body weight loss on Days 6 to 9 post-coitum) and reduced food consumption on Days 6 to 9 post-coitum. At 75 mg/kg bw/day, one female had dark red fluid in the uterus. This animal had 5 live fetuses and 6 early resorptions, which resulted in a slightly increased post-implantation loss at this dose. At all dose levels, fetal body weights were slightly lower compared to controls, but this was not statistically significant and did not show a dose relationship.
Maternal examinations:
MORTALITY/VIABILITY:
At least twice daily. Animals showing pain, distress or discomfort, which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ (2000)7). The circumstance of any death was recorded in detail.

CLINICAL SIGNS:
At least once daily from Day 0 post-coitum onwards up to the day prior to necropsy. The time of onset, grade and duration of any observed signs were recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) were scored. Cage debris was examined in case of abortions.

BODY WEIGHTS:
Days 0, 3, 6, 9, 13, 16, 20, 23, 26, 29 post-coitum.

FOOD CONSUMPTION:
Days 0-3, 3-6, 6-9, 9-13, 13-16, 16-20, 20-23, 23-26 and 26-29 post-coitum.

WATER CONSUMPTION:
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

POST-MORTEM EXAMINATIONS:
All animals surviving to the end of the observation period, all moribund animals and all animals showing abortion were euthanized and subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs. All macroscopic abnormalities were recorded, collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution). Females which had abortion or spontaneous death, were necropsied within 24 h of abortion.
Ovaries and uterine content:
Each ovary and uterine horn of animals surviving to planned necropsy was dissected and examined as quickly as possible to determine:

- The number of corpora lutea.
- The weight of the (gravid) uterus.
- The number and distribution of live and dead fetuses.
- The number and distribution of embryo-fetal deaths.
- The weight of each fetus.
- The sex of each fetus.
- Externally visible macroscopic fetal abnormalities.

Animals found dead or sacrificed before planned necropsy, were subjected to relevant examinations of the ovaries and uterine horns.
Fetal examinations:
External, visceral, and skeletal findings were recorded as developmental variations or malformations.

External:
Each viable fetus was examined in detail and weighed. All live fetuses were euthanized. The nonviable fetus (the degree of autolysis was minimal or absent) was examined, crown-rump length measured and weighed. The crown-rump length of late resorptions was measured and a gross external examination performed. Late resorptions with malformations were fixed in 10% buffered formalin.

Visceral (Internal):
All fetuses were examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities were opened and dissected. This examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development. The sex of all fetuses was determined by internal examination. The heads were removed from approximately one-half of the fetuses in each litter and placed in Bouin's solution. Tissues were then transferred to a 70% aqueous ethanol for subsequent processing and soft-tissue examination of all groups using the Wilson sectioning technique. After examination, the tissues were stored in 10% formalin. All carcasses, including the carcasses without heads, were eviscerated, skinned and fixed in identified containers containing 96% aqueous ethanol for subsequent examination of skeletons.

Skeletal:
The eviscerated fetuses from Groups 1 and 4, following fixation in 96% aqueous ethanol, were macerated in potassium hydroxide and stained with Alizarin Red S. Subsequently, the skeletal examination was done on all fetuses from Groups 1 and 4. Since no treatment related effects in the high dose group were seen, skeletal examination was not extended to the fetuses from the low and mid dose group. All specimens were archived in glycerin with bronopol as preservative. A few bones were not available for skeletal examination because they were accidentally damaged or lost during processing. Evaluation by the fetal pathologist and study director determined there was no influence on the outcome of the individual or overall skeletal examinations, or on the integrity of the study as a whole.
Clinical signs:
no effects observed
Description (incidence and severity):
No toxicologically relevant clinical signs were noted up to 75 mg/kg bw/day. Incidental findings that were noted included alopecia, scars, scales, scabs, lean appearance, hunched posture, laboured respiration, rales, ulcer, diarrhoea, a wound, and salivation. These findings occurred within the range of background findings to be expected for rabbits of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological relevance.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No toxicologically relevant mortalities occurred up to 75 mg/kg bw/day.
Ten animals died preterm
-Five animals showed an abortion and had to be terminated early. Macroscopic examination showed abnormalities of the heart (enlarged, pale or soft), stomach (many black foci on glandular mucosa, many black-brown or dark red foci, irregular surface of the forestomach or gelatinous contents), gallbladder (enlarged or many greenish foci), oviducts (several watery-clear cysts), pancreatic lymph nodes (enlarged and dark red), alopecia, thoracic cavity (containing watery-clear fluid), lungs (many, black foci), liver (pale), general pale discolouration, or watery-clear contents in the caecum.
- Five animals died due to complications of the gavage procedure. Macroscopic examination of these animals revealed abnormalities in the trachea (contents: reddish/dark red foamy or hemorrhagic/clotted blood), lungs (reddish foamy contents, many dark red foci, dark red discolouration or isolated tan focus), or advanced autolysis.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 50 and 75 mg/kg bw/day, overall a body weight loss was noted on Day 9 post-coitum and reduced body weight gain was noted on Day 13 post-coitum. These changes were statistically significant but did not show a dose response. During the remainder of treatment, mean body weights recovered to normal. Corrected (for uterus) body weight gain was unaffected at these dose levels. At 25 mg/kg bw/day, body weights and (corrected) body weight gain remained in the same range as controls over the treatment period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Absolute and relative food consumption were statistically significantly lower on Day 6-13 post-coitum at 75 mg/kg bw/day and on Day 6-9 post-coitum at 50 mg/kg bw/day. Food consumption was slightly lower on Day 13-16 post coitum (not statistically significant). Subsequently, mean food consumption recovered to normal. Food consumption before or after allowance for body weight of treated animals at 25 mg/kg bw/day remained in the same range as controls.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
During the observation period several animals in all groups (vehicle control and test item treated) showed reduced water intake on one or more days. In the absence of a clear treatment-related effect, no quantitative measurement of water consumption was introduced during this study.
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:
no effects observed
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment. The incidence of incidental findings among control and treated animals was within the background range of findings that are encountered among rabbits of this age and strain, and did not show a dose-related trend. These necropsy findings were therefore considered to be of no toxicological relevance.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
not examined
Early or late resorptions:
no effects observed
Dead fetuses:
not examined
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
not examined
Other effects:
not examined
Description (incidence and severity):
MATERNAL PREGNANCY DATA
For the control, low, mid and high-dose groups, there were respectively 22, 21, 21 and 19 litters with viable fetuses available on Day 29 post-coitum. In the control group, out of 24 mated animals two died preterm; all rabbits were pregnant. At 25 mg/kg bw/day, out of 22 mated animals one had an abortion. At 50 mg/kg bw/day, out of 26 mated animals two aborted, two died preterm (both pregnant) and one was not pregnant. At 75 mg/kg bw/day, out of 24 mated animals two aborted, one died preterm (pregnant) and two were not pregnant.
Key result
Dose descriptor:
NOAEL
Effect level:
75 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
clinical signs
dead fetuses
early or late resorptions
gross pathology
mortality
number of abortions
organ weights and organ / body weight ratios
pre and post implantation loss
total litter losses by resorption
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Fetal body weight was unaffected by treatment up to 75 mg/kg bw/day. Mean fetal body weights (both sexes combined) were 36.3, 37.8, 35.5 and 37.5 grams in the control, 25, 50 and 75 mg/kg bw/day group, respectively.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
There was no effect on fetal viability up to 75 mg/kg bw/day. Litter proportions of viable or dead fetuses and early resorptions were all within the normal range of biological variation.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Sex ratio was unaffected by treatment up to 75 mg/kg bw/day.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
Litter sizes were not affected by treatment up to 75 mg/kg bw/day. Mean litter sizes were 9.5, 8.6, 9.2 and 8.4 viable fetuses per group for the control, 25, 50 and 75 mg/kg bw/day group, respectively.






Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
There were no external developmental malformations or variations up to 75 mg/kg bw/day for fetuses at planned necropsy. Two fetuses of one litter from 50 mg/kg bw/day group showed hyperextension of the right hind limb. As this only affected one litter and this malformation was noted in the historical control data, it was not considered toxicologically relevant.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
At 75 mg/kg bw/day, there was a slight increase in skeletal developmental variations. This included three parameters indicating a developmental delay: unossified metacarpals and/or metatarsals (11.7% at 75 mg/kg bw/day versus 7.7% in the control group), slightly or moderately mal-aligned sternebrae (6.0% at 75 mg/kg bw/day versus 1.8% in the control group) and unossified tarsals (2.9% at 75 mg/kg bw/day versus 1.1% in the control group). As these increases were only slight and not statistically significant, these were not considered toxicologically significant. Additionally, the number of fetuses with caudal shift of the pelvic girdle was increased at 75 mg/kg bw/day (31.7% per litter; 52 fetuses in 11 litters) compared to the control group (17.1% per litter; 36 fetuses in 13 litters). Without any corroborative findings and as the number of affected litters was even less than controls, this finding was not considered toxicologically relevant. All malformations and remaining skeletal variations recorded in this study were not considered to be treatment related as they occurred infrequently, at lower levels in the high dose group, occurred at frequencies that were within the range of available historical control data, or were noted in control fetuses only.
Visceral malformations:
no effects observed
Description (incidence and severity):
There were no treatment related effects on visceral morphology following treatment up to 75 mg/kg bw/day. Retrocaval ureter was noted at 0.8% in the control group, 1.8% at 25 mg/kg bw/day, 1.1% at 50 mg/kg bw/day and 2.6% at 75 mg/kg bw/day. At 25 and 75 mg/kg bw/day, this was just outside the historical control maximum of 1.4%. Without a clear dose response relationship, these slight increases were not considered toxicologically significant. Any remaining visceral malformations and variations were not considered treatment related as they occurred infrequently, did not follow a dose-related trend, or occurred at frequencies that were within the range of available historical control data.
Other effects:
no effects observed
Description (incidence and severity):
The numbers of fetuses (litters) available for external and visceral morphological evaluation were 208 (22), 180 (21), 194 (21) and 160 (19) in control, 25, 50 and 75 mg/kg bw/day groups respectively. Soft tissue cephalic examination was done for approximately half of the fetuses of all groups. Skeletal examinations were performed for all fetuses of control and 75 mg/kg bw/day groups.
Key result
Dose descriptor:
NOAEL
Effect level:
75 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
reduction in number of live offspring
changes in sex ratio
fetal/pup body weight changes
external malformations
skeletal malformations
visceral malformations
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

None

Conclusions:
Based on the results of read across study, the test substance NOAELs for maternal and developmental toxicity is considered at 75 mg/kg bw/day (highest tested dose).
Executive summary:

An oral prenatal developmental toxicity study in rabbits was conducted with the read across substance PETIA according to OECD Guideline 414, EU method B.31 and EPA OPPTS 870.3700 in compliance with GLP. Ninety six mated female rabbits were assigned to four dose groups. The number of animals was 24, 22, 26 and 24 for Groups 1, 2, 3 and 4, respectively. The read across substance was administered once daily by oral gavage from Days 6 to 28 post-coitum, inclusive, at doses of 25, 50 and 75 mg/kg bw/day (Groups 2, 3 and 4, respectively). The rabbits of the control group received the vehicle, Arachid oil, alone. Females were checked daily for the presence of clinical signs. Food consumption and body weight were determined at periodic intervals. All animals surviving to Day 29 post-coitum were subjected to an examination post-mortem and external, thoracic and abdominal macroscopic findings were recorded. The uteri, placentae and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations and corpora lutea were recorded. The fetuses were weighed, sexed and examined for external, visceral and skeletal malformations and developmental variations. No maternal or developmental toxicity was observed in any of the groups (Beekhuijzen, 2014). Based on the results of read across study, the test substance NOAELs for maternal and developmental toxicity is considered at 75 mg/kg bw/day (highest tested dose).

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
75 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

An oral prenatal developmental toxicity study in rabbits was conducted with the read across substance PETIA according to OECD Guideline 414, EU method B.31 and EPA OPPTS 870.3700 in compliance with GLP. Ninety six mated female rabbits were assigned to four dose groups. The number of animals was 24, 22, 26 and 24 for Groups 1, 2, 3 and 4, respectively. The read across substance was administered once daily by oral gavage from Days 6 to 28 post-coitum, inclusive, at doses of 25, 50 and 75 mg/kg bw/day (Groups 2, 3 and 4, respectively). The rabbits of the control group received the vehicle, Arachid oil, alone. Females were checked daily for the presence of clinical signs. Food consumption and body weight were determined at periodic intervals. All animals surviving to Day 29 post-coitum were subjected to an examination post-mortem and external, thoracic and abdominal macroscopic findings were recorded. The uteri, placentae and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations and corpora lutea were recorded. The fetuses were weighed, sexed and examined for external, visceral and skeletal malformations and developmental variations. No maternal or developmental toxicity was observed in any of the groups (Beekhuijzen, 2014). Based on the results of the read across study, the NOAELs for both maternal and developmental toxicity are considered to be 75 mg/kg bw/day (highest tested dose).


 


The available guideline compliant developmental toxicity and teratology screening studies for the read-across substance PETIA in rabbits and rats support the conclusion that the substance does not represent a developmental toxicity hazard in humans. Indeed, the recent OECD Guideline 414 developmental toxicity study in rabbits as well as the OECD Guideline 422 reproductive/developmental screening study in rats did not reveal any evidence of developmental toxicity. Based on this evidence, DPHA is also not expected to present a development toxicity hazard.

Justification for classification or non-classification

Based on the lack of toxicity to fertility in parental animals or to development of fetuses as observed in the combined repeated dose toxicity and reproductive/developmental screening toxicity study and a prenatal developmental toxicity study conducted with the read-across substance PETIA, DPHA is not considered to warrant reproductive/developmental toxicity classification according to CLP (EC 1272/2008) criteria.

Additional information