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Effects on fertility

Description of key information

No adverse effects were observed, either in the adult animals or in the offspring, at the highest dose (200 mg/kg bw) used in an OECD 422 study (28-day exposure) via the oral route.

Link to relevant study records
Reference
Endpoint:
toxicity to reproduction
Remarks:
other: OECD Guideline 422Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From January to September 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to standard guidelines in compliance with GLP.
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
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 days 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
Remarks:
Doses / Concentrations:
0, 25, 75 or 200 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
12 males and 12 females
Control animals:
yes
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
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
effects observed, treatment-related
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.
Dose descriptor:
NOAEL
Remarks:
reproduction
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No substance-related effects on reproductive performance at any dose
Dose descriptor:
NOAEL
Remarks:
maternal toxicity
Effect level:
75 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
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.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed
Reproductive effects observed:
not specified

None

Conclusions:
Under the conditions of this screening study, no test substance-related effects were observed on reproductive performance at any dosage level. As such, a dosage level of 200 mg/kg bw/day was considered to be the no-observed-adverse-effect level (NOAEL) for reproductive toxicity of PETIA when administered orally by gavage to Crl:CD(SD) rats. 
Executive summary:

A study was conducted to determine the reproductive toxicity of PETIA to rats after oral exposure according to OECD Guideline 422.

Under the conditions of this screening study, no test substance-related effects were observed on reproductive performance at any dosage level. As such, a dosage level of 200 mg/kg bw/day was considered to be the no-observed-adverse-effect level (NOAEL) for reproductive toxicity of PETIA when administered orally by gavage to Crl:CD(SD) rats. 

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
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

According to the Regulation EC 1907/2006, a two-generation reproductive toxicity study (one species, male and female, most appropriate route of administration, having regard to the likely route of human exposure) must be proposed in the REACH dossier (Annex IX) if the 28-day or 90-day study indicates adverse effects on reproductive organs or tissues. An OECD 422 study (28-day exposure) via the oral route is available which has not shown any adverse effects, either in the adult animals or in the offspring at the highest dose (200 mg/kg bw) used. Dosage levels were selected based on the results of previous studies and were provided by the Sponsor after consultation with the Study Director. In a previous 14-day toxicity study in Crl:CD(SD) rats (Stump, Draft, WIL-738003), the maximum tolerated dose was exceeded at 1000 mg/kg/day. At 300 mg/kg/day, lower mean body weight gains, reduced food consumption, adverse clinical signs, and/or changes in mean organ weights were noted for males and females. Based on these results, dosage levels of 25, 75, and 200 mg/kg/day were selected to be evaluated in the current study.


Justification for selection of Effect on fertility via oral route:
The study used for this end-point is an OECD 422 study with a supporting substance (read-across). The study has been conducted under GLP. The substance used for read-across is a structural analogue with a similar functional group and comparable phys-chem properties as the substance to be registered.

Effects on developmental toxicity

Description of key information

No adverse effects were observed, either in the adult animals or in the offspring, at the highest dose (200 mg/kg bw) used in an OECD 422 study (28-day exposure) via the oral route.

In an OECD 414 study in rabbits, no developmental toxicity was observed at the highest dose of 75mg/kg.

No developmental toxicity was also observed in rats in an OECD 414 equivalent study, but the maximum dose tested was only 10mg/kg.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to standard guidelines in compliance with GLP.
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
other: OECD 422
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:CD(SD)]
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
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.
Details on mating procedure:
The animals were paired on a 1:1 basis within each treatment group following 14 days 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.
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
Remarks:
Doses / Concentrations:
0, 25, 75 and 200 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
12 males and 12 females
Control animals:
yes
Maternal 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
Fetal examinations:
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.
Dose descriptor:
NOAEL
Effect level:
75 mg/kg bw (total dose)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: no adverse effects observed
Abnormalities:
not specified
Developmental effects observed:
not specified

None

Conclusions:
Under the conditions of this screening study, no test substance-related effects were observed on the general physical condition of F1 pups at any dosage level. As such, a dosage level of 200 mg/kg bw/day was considered to be the no-observed-adverse-effect level (NOAEL) for neonatal toxicity of PETIA when administered orally by gavage to Crl:CD(SD) rats. 
Executive summary:

A study was conducted to determine the neonatal toxicity of PETIA to rats after oral exposure according to OECD Guideline 422.

Under the conditions of this screening study, no test substance-related effects were observed on the general physical condition of F1 pups at any dosage level. As such, a dosage level of 200 mg/kg bw/day was considered to be the no-observed-adverse-effect level (NOAEL) for neonatal toxicity of PETIA when administered orally by gavage to Crl:CD(SD) rats. 

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline stud
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. QA statement)
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: Females were approximately 17 - 19 weeks (1) (1) Female no. 22 (Group 1) was 27 weeks old at mating; the other females were 18-21 weeks old at mating
- Housing: Females were individually housed
- Diet (e.g. ad libitum): ad libitum (Global Diet 2030 from Harlan Teklad®, Mucedola, Milanese, Italy)
- Water (e.g. ad libitum): ad libitum (tap-water)
- Acclimation period: At least 5 days prior to pairing

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 24°C
- Humidity (%): 40 - 70%
- Air changes (per hr): 12/12
- Photoperiod (hrs dark / hrs light): 10

Route of administration:
oral: gavage
Vehicle:
arachis oil
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 per week
Duration of test:
29 days
No. of animals per sex per dose:
25 mg/kg: 22 females
50 mg/kg: 26 females
75 mg/kg: 24 females
control group: 24 females
Control animals:
yes, concurrent vehicle
Details on study design:
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. At 75 mg/kg, 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, one female (no. 21) 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.

The animals were allocated to the groups by computer-generated random algorithm according to body weight, with all animals within ± 25% of the mean. Upon observation of mating (Day 0 post-coitum), the females were distributed in a random sequence over the test groups. Females which were mated on the same day were classified in the same subgroup.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from Day 0 post-coitum onwards up to the day prior to necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: Days 0, 3, 6, 9, 13, 16, 20, 23, 26, 29 post-coitum

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Days 0-3, 3-6, 6-9, 9-13, 13-16, 16-20, 20-23, 23-26 and 26-29 postcoitum

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29

Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: externally visiable macroscopic fetal abnormalities, weight and sex of each fetus
Fetal examinations:
All fetuses were weighed, numbered, sexed, tagged and examined for esternal malformation
- External examinations: Yes
- Soft tissue examinations: Yes
- Head examinations: Yes
Statistics:
The following statistical methods were used to analyze the data:
Dunnett-test, Steel-test, Fisher Exact-test, Mann Whitney test, ANOVA test, Dunn’s test
Historical control data:
yes
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
No toxicologically relevant mortalities occurred up to 75 mg/kg.
Ten animals died preterm.
Five animals showed an abortion and had to be terminated early. One animal (no. 41) at 25 mg/kg aborted on Day 26 post-coitum, two animals (nos. 52 and 65) at 50 mg/kg aborted on Day 28 and 23 post-coitum, respectively, and two animals (nos. 68 and 84) at 75 mg/kg aborted on Day 28 postcoitum.
Five animals died due to complications of the gavage procedure, which was in part due to the high viscosity of the vehicle (i.e. arachnid oil). Two animals (nos. 4 and 19) of the control group died immediately after dosing on Day 10 post-coitum. At 50 mg/kg, one animal (no. 45) was found dead on Day 16 post-coitum, and one animal (no. 94) was killed in extremis on Day 28 post-coitum. One animal (no. 96) at 75 mg/kg was found dead on Day 28 post-coitum.
For these ten animals, clinical signs consisted of alopecia, reduced faeces production, laboured respiration, lean or pale appearance, scabs, lethargy, slow breathing, rales, a wound, diarrhoea, flat posture, hypotonia, piloerection, and/or hypothermia.
The five animals which had an abortion, showed reduced food consumption with concurrent body weight loss. Reduced water consumption was noted for eight animals (nos. 41, 45, 52, 65, 94, 68, 84 and 96).
Macroscopic examination of the animals that died due to complications of the gavage procedure 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. For the animals that had an abortion, 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.

Clinical signs:
- Reduced faeces production: control group: 15 animals; 25 mg/kg: 12 animals; 50 mg/kg: 19 animals; 75 mg/kg: 21 animals This was due to the large variation in food consumption, which is regularly seen for rabbits of this age and strain housed and treated under the conditions in this study. Without a clear dose response relationship, these signs were considered signs of no toxicological relevance.
- 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.

Body weight:
- At 50 and 75 mg/kg, overall a body weight loss was noted on Day 9 post-coitum and reduced body weight gain was noted on Day 13 post-coitum.
- At 25 mg/kg, body weights and (corrected) body weight gain remained in the same range as controls over the treatment period.
At 50 and 75 mg/kg, a clear reduction in food consumption was noted during the first days of treatment. This resulted in a body weight loss for most of the animals, which recovered during the remainder of the treatment period as food consumption returned to normal levels. As these effects were transient, they were not considered toxicologically relevant.

Food consumption:
- Absolute and relative food consumption were statistically significantly lower on Days 6-13 post-coitum at 75 mg/kg and on Days 6-9 post-coitum at 50 mg/kg. Food consumption was slightly lower on Days 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 remained in the same range as controls.

Water consumption:
- During the observation period several animals in all groups (vehicle control and test item treated) showed reduced water intake on one or more days.

Macroscopic examination:
- Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment.

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, out of 22 mated animals one had an abortion. At 50 mg/kg, out of 26 mated animals two aborted, two died preterm (both pregnant) and one was not pregnant. At 75 mg/kg, out of 24 mated animals two aborted, one died preterm (pregnant) and two were not pregnant.










Dose descriptor:
NOAEL
Effect level:
75 mg/kg bw/day
Basis for effect level:
other: no adverse effects observed
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Litter size:
Litter sizes were not affected by treatment up to 75 mg/kg.
There was no effect on fetal viability up to 75 mg/kg. Litter proportions of viable or dead fetuses and early resorptions were all within the normal range of biological variation.
The percentage of late resorptions at 25 mg/kg was slightly outside the historical control data (4.0% versus a maximum of 3.5% in the historical control data). As no dose response was noted, this slight increase was not considered toxicologically relevant.

Sex ration:
Sex ratio was unaffected by treatment up to 75 mg/kg.

Fetal body weight:
Fetal body weights were unaffected by treatment up to 75 mg/kg.

Fetal morphological examinations:
The numbers of fetuses (litters) available for external and visceral morphological evaluation were 208(22), 180(21), 194(21) and 160(19) in Groups 1, 2, 3, and 4, respectively. Soft tissue cephalic examination was done for approximately half of the fetuses of all groups. Skeletal examinations were performed for all fetuses of Groups 1 and 4.

External malformations and variations:
There were no external developmental malformations or variations up to 75 mg/kg for fetuses at planned necropsy.
Two fetuses of one litter (no. 94) of which the dam was killed in extremis on Day 28 post-coitum, 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.

Visceral malformations and variations:
There were no treatment related effects on visceral morphology following treatment up to 75 mg/kg.
Retrocaval ureter was noted at 0.8% in the control group, 1.8% at 25 mg/kg, 1.1% at 50 mg/kg and 2.6% at 75 mg/kg. At 25 and 75 mg/kg, 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.

Skeletal malformations and variations:
At 75 mg/kg, 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 versus 7.7% in the control group), slightly or moderately malaligned sternebrae (6.0% at 75 mg/kg versus 1.8% in the control group) and unossified tarsals (2.9% at 75 mg/kg 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 (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.




Dose descriptor:
NOAEL
Effect level:
75 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: no adverse effects observed
Abnormalities:
not specified
Developmental effects observed:
not specified

Accuracy, homogeneity and stability of formulations were demonstrated by analyses.

Maternal findings

At 50 and 75 mg/kg, a clear reduction in food consumption was noted during the first days of treatment. This resulted in a body weight loss for most of the animals, which recovered during the remainder of the treatment period as food consumption returned to normal levels. As these effects were transient, they were not considered toxicologically relevant.

No toxicologically significant changes were noted in any of the remaining maternal parameters investigated in this study (i.e. mortality, clinical appearance and macroscopic examination).

No maternal toxicity was noted at 25 mg/kg.

Developmental findings

No developmental toxicity was observed in the 25, 50 and 75 mg/kg groups.

In conclusion, based on the results in this prenatal developmental toxicity study, the No Observed Adverse Effect Level (NOAEL) for maternal and developmental toxicity for PETIA was established as being at least 75 mg/kg bw, since no adverse effect was observed.

The NOEL is 25 mg/kg bw based on the transient reduction in food consumption with corresponding body weight loss observed at 50 and 75 mg/kg bw. No higher dose levels could be tested in pregnant rabbits.

The test item, PETIA, did not elicit any teratogenic potential in rabbits.

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

No information is available for the registered substance. Read across has been performed to the structurally related substance PETIA, CAS 1245638 -61 -2, which has the same functional groups and is, due to the missing ethoxy elements expected to be more reactive than the registered substance.

Justification for classification or non-classification

No test item related, or adverse effects were noted at evaluation of the reproductive parameters during mating and gestation, delivery and post-partum/lactation period, under the conditions of this study.There were no adverse effects ascribed to test item administration on the F1 offspring viability, clinical signs, development or at observations following euthanasia. There were no test item-related changes observed in organ weights, at necropsy or at histopathology for the adult animals of either sex. The NOEL for effects in the offspring (F1) is considered to be 200 mg/kg bw/day.

In OECD 414 study in rabbits, there was also no indication of a developmental effect. This is supported by the results of a teratogenicity study in rats.

Therefore, there is no need to classify the substance for fertility and developmental toxicity, according to the Regulation EC 1272/2008 and the Directive 67/584/EEC.

Additional information