Registration Dossier
Registration Dossier
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EC number: 226-241-3 | CAS number: 5332-73-0
Developmental toxicity / teratogenicity
Administrative data
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 04 April 2018 - 04 June 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Cross-referenceopen allclose all
- Reason / purpose for cross-reference:
- reference to other study
Reference
- Endpoint:
- short-term repeated dose toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 05/01/2018- 05/09/2019
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- other: 14-day dose range finding study
- Principles of method if other than guideline:
- The study was run at a GLP compliant facility, however the study doesn't claim GLP compliance
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: batch TE767F05G
- Purity, including information on contaminants, isomers, etc.: 99.7%
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: The stability and homogeneity of the test item formulations were determined by Covance CRS Research Limited, Shardlow, UK Analytical Services within Covance Study Number: WD30CK. Results showed formulations to be stable for four hours.
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: - Species:
- rat
- Strain:
- Wistar
- Remarks:
- Wistar Han™:RccHan™:WIST
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Envigo RMS (UK) Limited, Oxon, UK
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: six weeks
- Weight at study initiation: males weighed 177 to 213g, the females weighed 145 to 164g
- Fasting period before study: no
- Housing: housed in groups of three by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 6 days
DETAILS OF FOOD AND WATER QUALITY: The diet, drinking water, bedding and environmental enrichment was considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 50 ± 20%
- Air changes (per hr): 15/h
- Photoperiod (hrs dark / hrs light): 12h/12h
IN-LIFE DATES: From: 05/01/2018 To: 21/02/2018 - Route of administration:
- oral: gavage
- Vehicle:
- arachis oil
- Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:
VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle: 0; 7.5; 18.75; 37.5mg/mL
- Amount of vehicle (if gavage): 4mL/kg
- Lot/batch no. (if required):
- Purity: - Analytical verification of doses or concentrations:
- no
- Remarks:
- Results of previous study showed formulations to be stable for four hours. Formulations for this study were prepared daily and were dosed within 2 hours of preparation. No analysis was conducted to determine the concentration of the test item formulation.
- Duration of treatment / exposure:
- 14 days
- Frequency of treatment:
- daily
- Dose / conc.:
- 0 mg/kg bw/day (nominal)
- Dose / conc.:
- 30 mg/kg bw/day (nominal)
- Dose / conc.:
- 75 mg/kg bw/day (nominal)
- Dose / conc.:
- 150 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- 3
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: preliminary toxicity work was undertaken, treating one male and one female rat at 300 mg/kg bw/day for four days. Treatment-related effects consisted of increased salivation, hunched posture, pilo-erection and noisy respiration and body weight losses in both animals. At necropsy both animals had dark lungs and thickening/sloughing on the non-glandular region of the stomach. The male also had a dark area on the non-glandular region of the stomach.
- Rationale for animal assignment (if not random): random - Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity, ill health or behavioral change immediately before dosing, up to thirty minutes after dosing and one hour after dosing.
Additionally observations were also made four hours following dosing (not at weekends).
All observations were recorded.
DETAILED CLINICAL OBSERVATIONS: No
BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on Days 1, 4, 8, 11 and 15.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Food consumption was recorded for each cage group for Days 1 to 4, 4 to 8, 8 to 11 and 11 to
15. Food conversion efficiency was calculated retrospectively.
FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes
Food conversion efficiency was calculated retrospectively.
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Water intake was measured and recorded daily for each cage group.
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: No
CLINICAL CHEMISTRY: No
PLASMA/SERUM HORMONES/LIPIDS: No
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No
IMMUNOLOGY: No - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: No - Statistics:
- Data were processed to give summary incidence or group mean and standard deviation values
where appropriate. - Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- One male and two females treated with 150 mg/kg bw/day showed an isolated incidence of
increased salivation post dosing on Day 14.
No such effects were evident in animals of either sex treated with 75 or 30 mg/kg bw/day. - Mortality:
- no mortality observed
- Body weight and weight changes:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Males treated with 150 mg/kg bw/day showed a slight reduction in body weight gain between
Days 4 and 8, however, recovery was evident thereafter.
No such effects were evident in females treated with 150 mg/kg bw/day or in animals of
either sex treated with 75 or 30 mg/kg bw/day. - Food consumption and compound intake (if feeding study):
- effects observed, non-treatment-related
- Description (incidence and severity):
- Males treated with 150 mg/kg bw/day showed a slight reduction in food consumption
between Days 4 and 8, however, recovery was evident thereafter.
No such effects were evident in females treated with 150 mg/kg bw/day or in animals of
either sex treated with 75 or 30 mg/kg bw/day. - Food efficiency:
- no effects observed
- Water consumption and compound intake (if drinking water study):
- effects observed, non-treatment-related
- Description (incidence and severity):
- Females from all treatment groups showed an increase in overall water consumption; however, a true dose related response was not evident. No such effects were evident in treated males.
- Gross pathological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- All males and two females treated with 150 mg/kg bw/day showed ulcerated stomachs at necropsy.
No such effects were evident in animals of either sex treated with 75 or 30 mg/kg bw/day. - Conclusions:
- Dose levels of 0 (Control), 10, 30 and 100 mg/kg bw/day are recommended for use in the planned Ninety Day Repeated Dose Oral (Gavage) Toxicity Study in the Rat
- Executive summary:
In this 14-day dose range finding study treatment at 150 mg/kg bw/day showed an incidence of reduced body weight gain and food consumption in males only (Days 4-8) and although recovery was evident thereafter, adverse macroscopic abnormalities (ulcerated stomachs) were evident in all males and two females from this treatment group. In view of these findings, 150 mg/kg bw/day is considered unsuitable for a longer administration period on future studies. Dose levels of 0 (Control), 10, 30 and 100 mg/kg bw/day are therefore recommended for use in the planned Ninety Day Repeated Dose Oral (Gavage) Toxicity Study in the Rat
- Reason / purpose for cross-reference:
- reference to other study
Reference
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 07 March 2018 - 9 August 2019
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 414 (Prenatal Developmental Toxicity Study)
- Version / remarks:
- dose range finding study
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- the analysis of the test item formulation was not performed
- Limit test:
- no
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: batch TE767F05G
- Purity, including information on contaminants, isomers, etc.: purity 99.7%
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: The stability and homogeneity of the test item formulations were determined by Covance CRS Research Limited, Shardlow, UK Analytical Services within Covance CRS Study Number: WD30CK. - Species:
- rat
- Strain:
- Sprague-Dawley
- Remarks:
- Sprague-Dawley Crl:CD (SD) IGS BR
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent.
- Age at study initiation: adult
- Weight at study initiation: 188 to 267g
- Fasting period before study: no
- Housing: housed individually in solid-floor polypropylene cages with stainless steel mesh lids furnished with softwood flakes
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: no
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 ºC
- Humidity (%): 50 ± 20%
- Air changes (per hr): 15/h
- Photoperiod (hrs dark / hrs light): 12h/12h
IN-LIFE DATES: From: 09/03/2018 To: 26/03/2018 - 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):
- Concentration in vehicle: 0; 7.5; 18.75; 37.5 mg/mL
- Amount of vehicle (if gavage): 4mL/kg - Analytical verification of doses or concentrations:
- no
- Remarks:
- Results of a previous study showed formulations to be stable for four hours. Formulations were therefore prepared daily and dosed within four hours of preparation. No analysis was conducted to determine the concentration of the test item formulations.
- Details on mating procedure:
- - Impregnation procedure: purchased timed pregnant
- Proof of pregnancy: positive evidence of mating referred to as day 0 of pregnancy - Duration of treatment / exposure:
- GD 3- GD19
- Frequency of treatment:
- daily
- Duration of test:
- 16 days
- Dose / conc.:
- 0 mg/kg bw/day (nominal)
- Dose / conc.:
- 30 mg/kg bw/day (nominal)
- Dose / conc.:
- 75 mg/kg bw/day (nominal)
- Dose / conc.:
- 150 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- 8
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: In a preliminary study one male and one (non-pregnant) female rat were treated with 300 mg/kg bw/day for four consecutive days. Both animals were terminated on Day 5 due to clinical signs of toxicity and body weight losses and at necropsy, macroscopic stomach and lung findings were evident in both animals. In the 14-day dose range finding study (Allt J., 2018) in non-pregnant animals, treatment at 150 mg/kg bw/day showed an incidence of reduced body weight gain and food consumption in males only (Days 4-8) and although recovery was evident thereafter, adverse macroscopic abnormalities (ulcerated stomachs) were evident in all males and two females from this treatment group. In view of these findings, and the fact that doses of 300mg/kg bw/d were not tolerated by non-pregnant animals, 150 mg/kg bw/day was used as the high dose in this preliminary development toxicity study.
- Rationale for animal assignment (if not random): random - Maternal examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: during the dosing period, observations were recorded immediately before dosing, up to thirty minutes after dosing and one hour after dosing. Additionally, observations were also performed four hours following dosing (not at weekends). All observations were recorded.
DETAILED CLINICAL OBSERVATIONS: No
BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on Day 3 (before the start of treatment) and on Days 4, 5, 8, 11, 14 and 17 of gestation. Body weights were also recorded for animals at terminal kill (Day 20).
FOOD CONSUMPTION: Yes
Food consumption was recorded for each individual animal for the periods Days 3 to 5, Days
5 to 8, 8 to 11, 11 to 14, 14 to 17 and 17 to 20.
WATER CONSUMPTION: Yes
- Time schedule for examinations: Water intake was observed daily by visual inspection of the water bottles for any overt changes.
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: All animals were subjected to a full external and internal examination and any macroscopic abnormalities were recorded. The ovaries and uteri of pregnant females were removed and examined. - 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: placental weight - Fetal examinations:
- - External examinations: Yes all per litter
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No
- Anogenital distance of all live rodent pups: no
- Fetal sex was examined
-Fetal weight was recorded - Indices:
- Percentage pre-implantation loss was calculated as:
(number of corpora lutea - number of implantations)/number of corpora lutea x100
Percentage post-implantation loss was calculated as:
(number of implantations - number of live fetuses)/number of implantations x 100
Sex ratio was calculated for each litter value using the following formula:
% male fetuses (sex ratio) = Number of male fetuses/Total number of fetuses x 100
- Clinical signs:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No test item effects were noted in clinical observations.
Isolated incidences of increased salivation was evident in one female treated with 75 mg/kg
bw/day on Day 9 and in two females treated with 150 mg/kg bw/day on Days 17 and 19.
No such effects were evident in females treated with 30 mg/kg bw/day. - Mortality:
- no mortality observed
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Females treated with 150 mg/kg bw/day showed reduced body weight gains between Days 3 and 5, with two females showing actual body weight losses at some point during this period. Recovery was evident thereafter. However, as a consequence of the initial reduced gains the overall body weight gain was slightly lower than controls. Gravid uterus weight for these females was comparable to controls. However, body weight gain for these females when adjusted for gravid uterus weight was reduced when compared to controls. Females treated with 75 mg/kg bw/day showed a reduction in body weight gain between Days 4 and 5, however, this was considered to be the result of two females showing actual body weight losses during this period. Recovery was evident thereafter and subsequent overall body weight gains for these females were comparable to controls. Gravid uterus weight and body weight gain when adjusted for gravid uterus weight for these females were comparable to controls. No such effects were evident in females treated with 30 mg/kg bw/day.
- Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- Females treated with 150 mg/kg bw/day showed lower food intake when compared to controls between Days 3 and 5. Slight reductions in food consumption for these females were also evident between Days 5 and 14. Food consumption for these females was comparable to controls between Days 14 and 17, however slightly reduced intake was again observed between Days 17 and 20. No such effects were evident in females treated with 75 or 30 mg/kg bw/day.
- Water consumption and compound intake (if drinking water study):
- no effects observed
- Gross pathological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Three females treated with 150 mg/kg bw/day and one female treated with 75 mg/kg bw/day had white patches on the non-glandular region of the stomach. A further female treated with 75 mg/kg bw/day had raised white ridges on the non-glandular region of the stomach. No such effects were detected in females treated with 30 mg/kg bw/day.
- Pre- and post-implantation loss:
- no effects observed
- Description (incidence and severity):
- There was no obvious effect of maternal treatment on litter data as assessed by numbers of
implantations, in-utero offspring survival (as assessed by the mean numbers of early or late
resorptions) and pre and post-implantation losses at 30, 75 or 150 mg/kg bw/day. - Early or late resorptions:
- no effects observed
- Other effects:
- no effects observed
- Description (incidence and severity):
- There was no difference in placenta weights among the groups.
- Fetal body weight changes:
- no effects observed
- Description (incidence and severity):
- There was no obvious effect of maternal treatment on live litter size or sex ratio at 30, 75 or 150 mg/kg bw/day.
Intergroup differences for mean fetal or litter weights did not indicate any obvious effects of maternal treatment at 30, 75 or 150 mg/kg bw/day. - Changes in sex ratio:
- no effects observed
- Changes in litter size and weights:
- no effects observed
- External malformations:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Neither the type, incidence nor distribution of external finding apparent for fetuses at Day 20 of gestation indicated an effect of maternal treatment on fetal development at 30, 75 or 150 mg/kg bw/day.
- Conclusions:
- The dose levels of 0 (Control), 30, 75 and 150 mg/kg bw/day are recommended for use in the main prenatal developmental toxicity study.
- Executive summary:
The oral administration of test item to pregnant rats by oral gavage during gestation at dose levels of 30, 75 and 150 mg/kg bw/day resulted in initial reductions in body weight gain at 150 and 75 mg/kg bw/day, slightly reduced food consumption at 150 mg/kg bw/day and macroscopic findings at 150 and 75 mg/kg bw/day which are likely due to the corrosive nature of the test item. Therefore, dose levels of 0 (Control), 30, 75 and 150 mg/kg bw/day are recommended for use in future prenatal developmental toxicity study.
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 019
- Report date:
- 2019
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 414 (Prenatal Developmental Toxicity Study)
- Version / remarks:
- adopted 22 January 2001
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
- Version / remarks:
- August 1998
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: Japanese Ministry of Agriculture, Forestry and Fisheries Testing guidelines for Toxicology studies, 12 NohSan No 8147
- Version / remarks:
- 24 November 2000
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: Commission Regulation (EC) No 440/2008 of 30 May 2008 test methods pursuant to Regulations (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
Test material
- Reference substance name:
- 3-methoxypropylamine
- EC Number:
- 226-241-3
- EC Name:
- 3-methoxypropylamine
- Cas Number:
- 5332-73-0
- Molecular formula:
- C4H11NO
- IUPAC Name:
- 3-methoxypropan-1-amine
- Test material form:
- liquid
Constituent 1
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: TE767F05G
- Expiration date of the lot/batch: 05 June 2019
- Purity: 99.7%
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark
- Solubility and stability of the test substance in the solvent/vehicle: stable for 4 hours
- Samples were taken of test item formulations and were analyzed on two occasions for
concentration of Methoxypropylamine (MOPA) at Envigo Analytical Laboratory, Shardlow. The results indicate that the prepared formulations were within 7% of the nominal concentration.
OTHER SPECIFICS: No correction for purity was made.
Test animals
- Species:
- rat
- Strain:
- Sprague-Dawley
- Remarks:
- Crl:CD (SD) IGS BR
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent
- Age at study initiation: no data
- Weight at study initiation: 199 to 285g
- Fasting period before study: no data
- Housing: The animals were housed in a single air-conditioned room within the Envigo Research Limited, Shardlow, UK Barrier Maintained Rodent Facility.
- Diet: ad libitum; A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK)
- Water: ad libitum; Mains drinking water was supplied from polycarbonate bottles attached to the cage.
- Acclimation period: no data
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 ºC
- Humidity (%): 50 ± 20%
- Air changes (per hr): at least fifteen air changes per hour
- Photoperiod (hrs dark / hrs light): twelve hours continuous light and twelve hours darkness.
IN-LIFE DATES: From: 07 April 2018 (first day of treatment) and 26 April 2018 (final day of necropsy).
Administration / exposure
- Route of administration:
- oral: gavage
- Vehicle:
- arachis oil
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS: Formulations were shown to be stable for four hours. Formulations were therefore prepared daily and dosed within four hours of preparation.
VEHICLE
- Concentration in vehicle: 7.5, 18.75 and 37.5 mg/mL
- Treatment volume: 4 mL/kg - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- The test item concentration in the test samples was determined by gas chromatography (GC) using an external standard technique. The test item gave a chromatographic profile consisting of a single peak.
The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, linearity of detector response, method accuracy and precision.
The homogeneity and stability was confirmed for test item in Arachis Oil BP formulations at nominal concentrations of 2.5 mg/mL and 100 mg/mL when stored for 4 hours during study number WD30CK.
The mean concentrations of test item in test formulations analyzed for the study were within ± 10% of nominal concentrations, confirming accurate formulation. - Details on mating procedure:
- Animals were delivered in two batches containing females prior to Day 3 of gestation. The day that positive evidence of mating was observed was designated Day 0 of gestation.
- Duration of treatment / exposure:
- between Days 3 and 19 of gestation
- Frequency of treatment:
- daily
- Duration of test:
- from Day 3 to 19 of gestation
Doses / concentrationsopen allclose all
- Dose / conc.:
- 0 mg/kg bw/day (nominal)
- Remarks:
- Control group
- Dose / conc.:
- 30 mg/kg bw/day (nominal)
- Remarks:
- low treatment group
- Dose / conc.:
- 75 mg/kg bw/day (nominal)
- Remarks:
- Intermediate treatment group
- Dose / conc.:
- 150 mg/kg bw/day (nominal)
- Remarks:
- high treatment group
- No. of animals per sex per dose:
- 24 females per group
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: The dose levels were chosen in collaboration with the Sponsor Representative and were based on available toxicity data including a Preliminary Oral (Gavage) Pre-Natal Development Toxicity Study in the Rat (Covance CRS Study Number VF78GT) and a 90 Day Oral (Gavage) Toxicity Study in the Rat (Covance CRS Study Number WD30CK).
In a preliminary study one male and one (non-pregnant) female rat were treated with 300 mg/kg bw/day for four consecutive days. Both animals were terminated on Day 5 due to clinical signs of toxicity and body weight losses. At necropsy, macroscopic stomach and lung findings were evident in both animals. In the 14-day dose range finding study (Allt J., 2018) in non-pregnant animals, treatment at 150 mg/kg bw/day showed an incidence of reduced body weight gain and food consumption in males only (Days 4-8) and although recovery was evident thereafter, adverse macroscopic abnormalities (ulcerated stomachs) were evident in all males and two females from this treatment group.
In the preliminary pre-natal development study in pregnant rats (Allt J., 2019), the oral administration of the test item by oral gavage during gestation at dose levels of 30, 75 and 150 mg/kg bw/day resulted in initial reductions in body weight gain at 150 and 75 mg/kg bw/day, slightly reduced food consumption at 150 mg/kg bw/day and macroscopic findings (stomach) at 150 and 75 mg/kg bw/day. Given the fact that the test item was not tolerated at doses of 300mg/kg bw/d in non-pregnant animals, that it is corrosive and caused stomach ulcers at doses of 150mg/kg bw/d in a 14-day dose range finding study in non-pregnant animals and it caused stomach effects in the dose range finding study in pregnant animals, the dose levels of 0 (Control), 30, 75 and 150 mg/kg bw/day were recommended for use in this main prenatal developmental toxicity study.
- Rationale for animal assignment: The animals were randomly allocated to treatment groups using a randomization procedure based on stratified body weight to ensure similarity between the treatment groups.
Examinations
- Maternal examinations:
- DETAILED CLINICAL OBSERVATIONS: Yes
Following arrival, all animals were examined for overt signs of toxicity, ill-health or behavioral changes once daily during the gestation period. Additionally, during the dosing period, observations were recorded immediately before and soon after dosing and one hour post dosing. All observations were recorded.
BODY WEIGHT: Yes
Individual body weights were recorded on Day 3 (before the start of treatment) and on Days 4, 5, 8, 11, 14 and 17 of gestation. Body weights were also recorded for animals at terminal kill (Day 20).
FOOD CONSUMPTION: Yes
Food consumption was recorded for each individual animal at Day 3, 5, 8, 11, 14, 17 and 20 of gestation.
WATER CONSUMPTION: Yes
Water intake was observed daily by visual inspection of the water bottles for any overt changes.
POST-MORTEM EXAMINATIONS: Yes
All animals were killed by carbon dioxide asphyxiation followed by cervical dislocation on Day 20 of gestation. All animals were subjected to a full external and internal examination and any macroscopic abnormalities were recorded. - Ovaries and uterine content:
- The ovaries and uteri of pregnant females were removed, examined and the following data recorded:
ii) Number, position and type of intrauterine implantation
iii) Fetal sex
iv) External fetal appearance
v) Fetal weight
vi) Placental weight
vii) Gravid uterus weight
The uteri of any apparently non-pregnant females were immersed in 0.5% ammonium polysulphide solution to reveal evidence of implantation.
Implantation types were divided into:
Early Death: No visible distinction between placental/decidual tissue and embryonic tissue
Late Death: Separate embryonic/fetal and placental tissue visible
Dead Fetus: A fetus that had died shortly before necropsy. These were included as late deaths for reporting purposes.
All implantations and viable fetuses were numbered according to their intrauterine position as follows (as an example):
Left Horn Cervix Right Horn
L1 L2 L3 L4 L5 L6 L7 L8 R1 R2 R3 R4 R5 R6 R7 R8
V1 V2 V3 V4 V5 V6 V7 V8 V9 V10 V11 V12 V13 V14 V15 V16
V = viable fetus - Fetal examinations:
- The fetuses were killed by subcutaneous injection of a suitable barbiturate agent. Fetuses from each litter were divided into two groups and examined for skeletal alterations and soft tissue alterations. Alternate fetuses were identified using an indelible marker and placed in Bouin’s fixative. Fetuses were subsequently transferred to distilled water and examined for visceral anomalies under a low power binocular microscope and then stored in 10% Buffered Formalin. The remaining fetuses were identified using cardboard tags marked with chinagraph pencil and placed into 70% IMS in distilled water.
The fetuses were subsequently eviscerated, processed and the skeletons stained with alizarin red S before being transferred to 50% glycerol for examination of skeletal development and anomalies and storage. - Statistics:
- Data was assessed using the R Environment for Statistical Computing. Initially, the distribution of the data was assessed by the Shapiro-Wilk normality test, followed by assessment of the homogeneity of the data using Bartlett’s test. Where considered appropriate, parametric analysis of the data was applied incorporating analysis of variance (ANOVA), which if significant, was followed by pair-wise comparisons using Dunnett’s test. Where parametric analysis of the data was considered to be unsuitable, non-parametric analysis of the data was performed incorporating the Kruskal-Wallis test which if significant was followed by the Mann-Whitney "U" test. Dose response relationships were also investigated by linear regression.
Probability values (p) are presented as follows:
p<0.001 ***
p<0.01 **
p<0.05 *
p≥0.05 (not significant) - Indices:
- Percentage pre-implantation loss was calculated as:
[(number of corpora lutea-number of implantations)/number of corpora lutea] x 100
Percentage post-implantation loss was calculated as:
[(number of implantations - number of live fetuses)/number of implantations] x 100
Sex ratio was calculated as:
% male fetuses (sex ratio) = (number of male fetuses/total number of fetuses) x 100
Results and discussion
Results: maternal animals
General toxicity (maternal animals)
- Clinical signs:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Instances of increased salivation were evident in all females treated with 150 mg/kg bw/day between Days 13 and 19 and in two females treated with 75 mg/kg bw/day on Day 19 only.
No such effects were detected in females treated with 30 mg/kg bw/day.
The following observations were noted in a small number of control or treated females and in isolation, these were considered to be incidental and unrelated to the test item. One control female had scattered scabs on one occasion, another control female had red/brown staining around the eyes on one occasion, one female treated with 75 mg/kg bw/day had red/brown staining around the mouth on one occasion, and three females treated with 150 mg/kg bw/day had generalized fur loss between Days 16 and 20, with one of these females also having red/brown staining around the mouth on one occasion. - Dermal irritation (if dermal study):
- not examined
- Mortality:
- no mortality observed
- Description (incidence):
- There were no unscheduled deaths.
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- Body weight and body weight gain, including adjustment for the contribution of the gravid uterus, was unaffected by treatment at 30, 75 or 150 mg/kg bw/day.
Females from all treatment groups showed a statistically significant increase (p<0.05-0.001) in body weight gain between Days 3 and 4 of gestation. An increase in body weight gain is considered not to reflect an adverse effect of treatment and the increase was not dose related and was therefore considered not to be of toxicological significance. - Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- No adverse effect on food consumption was evident in females from any treatment group.
Females treated with 150 mg/kg bw/day exhibited a statistically significant reduction (p<0.01) in food consumption between Days 8 and 11 of gestation. As no such effects were evident before or after this period and all but two individual values for food consumption at this treatment group were within the historical control data range, this reduction was considered not to be of any toxicological significance. - Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- no effects observed
- Description (incidence and severity):
- Daily visual inspection of water bottles did not reveal any overt intergroup differences.
- 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, treatment-related
- Description (incidence and severity):
- Sixteen females treated with 150 mg/kg bw/day had ulceration on the non-glandular region of the stomach. One of these females also had a pale stomach. A further five females from this treatment group had ulceration on both the non-glandular and glandular regions of the stomach. Eleven females treated with 75 mg/kg bw/day had ulceration on the non-glandular region of the stomach and one female treated with 30 mg/kg bw/day had white ridges on the non-glandular region of the stomach.
One female treated with 150 mg/kg bw/day had a mass on the right side of the abdominal cavity. In isolation this was considered to be incidental and unrelated to treatment. - Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- not examined
- Histopathological findings: neoplastic:
- not examined
Maternal developmental toxicity
- Number of abortions:
- no effects observed
- Pre- and post-implantation loss:
- no effects observed
- Description (incidence and severity):
- There was no treatment-related effect observed in the pre- and post-implantation loss in all dose groups, when comparing to the control group:
- preimplantation loss: 15.6%, 18.1%, 17.1%, 18.7% at 0, 30, 75 and 150 mg/kg bw/day
- post-implantation loss: 2.3%, 1.3%, 1.5%, 2.7% at 0, 30, 75 and 150 mg/kg bw/day
All data was within the historical control range available for this species and strain. - Total litter losses by resorption:
- no effects observed
- Description (incidence and severity):
- There were no total litter losses by resorption observed.
- Early or late resorptions:
- no effects observed
- Description (incidence and severity):
- There were no treatment-related effects on early or late resorptions observed.
- Dead fetuses:
- no effects observed
- Description (incidence and severity):
- There were no treatment-related effects on the number of dead fetuses observed.
- Changes in pregnancy duration:
- no effects observed
- Description (incidence and severity):
- Cumulative Body Weight Changes were recorded until day 20 of pregnancy, suggesting a duration of 20 days for all females.
- Changes in number of pregnant:
- no effects observed
- Description (incidence and severity):
- There was no treatment-related, toxicologically relevant effect on the number of pregnant dams per dose group. The number of pregnant females per dose group were 24/24, 24/24, 24/24 and 24/24 at 0,25, 100 and 250 mg/kg bw/day, resp. No historical control data is available for this parameter.
- Details on maternal toxic effects:
- There was no obvious effect of maternal treatment on litter data as assessed by numbers of implantations, in-utero offspring survival (as assessed by the mean numbers of early or late resorptions), live litter size, sex ratio and pre and post-implantation losses at 30, 75 or 150 mg/kg bw/day.
Intergroup differences for mean fetal, litter or placental weights did not indicate any adverse effects of maternal treatment at 30, 75 or 150 mg/kg bw/day.
Effect levels (maternal animals)
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 150 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Basis for effect level:
- clinical signs
- gross pathology
- Remarks on result:
- other: at a lesser extent, also evident in females treated with 75 mg/kg bw/day
Maternal abnormalities
- Key result
- Abnormalities:
- no effects observed
Results (fetuses)
- Fetal body weight changes:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Intergroup differences for mean fetal, litter or placental weights did not indicate any adverse effects of maternal treatment at 30, 75 or 150 mg/kg bw/day.
Male fetal weight and combined fetal weights for litters from females treated with 30 mg/kg bw/day showed a statistically significant increase (p<0.05-0.01) when compared to controls. An increase in fetal weight is generally not considered to represent an adverse effect of treatment and in the absence of a similar effect at the higher dosages, the intergroup differences are considered not to be of toxicological significance. - Reduction in number of live offspring:
- no effects observed
- Description (incidence and severity):
- There is no treatment-related effect on the number of live offspring.
- Changes in sex ratio:
- no effects observed
- Description (incidence and severity):
- There was no treatment-related effect observed on sex ratio.
- Changes in litter size and weights:
- no effects observed
- Description (incidence and severity):
- There was no treatment-related effects observed on litter weight or litter size.
- Changes in postnatal survival:
- no effects observed
- Description (incidence and severity):
- There was no treatment-related effects observed on postnatal survival.
- External malformations:
- no effects observed
- Description (incidence and severity):
- Neither the type, incidence nor distribution of external finding apparent for fetuses at Day 20 of gestation indicated an adverse effect of maternal treatment on fetal development at 30, 75 or 150 mg/kg bw/day.
A statistically significant increase (p<0.05) in the number of fetuses/litters showing a large placenta was evident at 150 mg/kg bw/day. The group mean value (4.7%) was slightly above the historical control range (0.0 - 4.2%), however, this was considered to be the result of one litter which had five fetuses that had this finding. With this litter excluded, the group mean value was within the historical control range. In the absence of any associated effects, the intergroup difference was considered not to be of toxicological significance. - Skeletal malformations:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Skeletal examinations of fetuses on Day 20 of gestation did not indicate any obvious effect of maternal treatment on fetal development at 30, 75 or 150 mg/kg bw/day.
Statistically significant increases (p<0.01) in the number of fetuses/litters showing a ossification centre associated with the first lumbar vertebra, incomplete ossification of the squamosal of the skull and zygomatic process of maxilla of the skull were noted in females treated with 150 mg/kg bw/day. The observations of one variant at a higher incidence compared with controls is not significant when evaluated in isolation. In the absence of a particular pattern of abnormal skeletal development of skeletal structures affecting treated fetuses, the observation of one affected skeletal structure can be considered unlikely to represent true developmental abnormality. This also takes account of these findings being seen regularly on this study type amongst control group fetuses.
A statistically significant increase (p<0.05) in the number of fetuses/litter showing incomplete ossification of the sternebra was evident at 75 mg/kg bw/day. Statistically significant reductions (p<0.05-0.01) in incomplete ossification of the cervical (neural) arch and incomplete ossification of the occipital (supra-occipital) was evident at 30 mg/kg bw/day. In the absence of similar findings at higher dosages or any particular pattern of abnormal skeletal development of skeletal structures affecting treated fetuses, the observation of one affected skeletal structure can be considered unlikely to represent true developmental abnormality. - Visceral malformations:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Visceral examinations of fetuses on Day 20 of gestation did not indicate any obvious effect of maternal treatment on fetal development at 30, 75 or 150 mg/kg bw/day.
A statistically significant increase (p<0.05) in the number of fetuses/litter showing a nonuniform patterning of the rugae was evident at 75 and 30 mg/kg bw/day. In the absence of a similar finding at the highest dosage, the intergroup differences were considered unrelated to treatment. - Details on embryotoxic / teratogenic effects:
- No treatment-related changes were detected in the offspring parameters measured or on embryofetal development. The ‘No Observed Effect Level’ (NOEL) for developmental toxicity was therefore considered to be 150 mg/kg bw/day.
Effect levels (fetuses)
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 150 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: developmental toxicity
Fetal abnormalities
- Key result
- Abnormalities:
- no effects observed
Overall developmental toxicity
- Key result
- Developmental effects observed:
- no
Any other information on results incl. tables
Table 1 Summary of Female Performance
Category | Number of Females at Dose Level (mg/kg bw/day) | |||
0 (Control) | 30 | 75 | 150 | |
Initial Group Size | 24 | 24 | 24 | 24 |
Pregnant | 24 | 24 | 24 | 24 |
Table 2 Summary Incidence of Daily Clinical Observations
Dose Level (mg/kg bw/day) | Number of Animals | Clinical Observations | Number Showing Effect (Days post coitum affected) |
0 (Control) | 24 | No Abnormalities Detected Scattered Scabs | 22 (0-20) 1 (5) |
|
| Red/Brown Staining around the Eyes | 1 (9) |
30 | 24 | No Abnormalities Detected | 24 (0-20) |
75 | 24 | No Abnormalities Detected Increased Salivation | 21 (0-20) 2 (19) |
|
| Red/Brown Staining Around the Mouth | 1 (19) |
150 | 24 | No Abnormalities Detected Fur Loss (Hind Quarters) | 3 (0-20) 3 (16-20) |
|
| Red/Brown Staining Around the Mouth | 1 (13) |
|
| Increased Salivation | 20 (13-19) |
Table 3 Group Mean Body Weight Values
Dose Level (mg/kg bw/day) |
| 3 | 4 | Body Weight (g) on Day of Gestation 5 8 11 14 | 17 | 20 | |||
0 (Control) | mean sd | 260.7 21.6 | 261.6 20.9 | 267.0 20.2 | 280.6 22.9 | 300.8 23.1 | 320.0 25.0 | 347.4 27.4 | 395.8 33.6 |
| n | 24 | 24 | 24 | 24 | 24 | 24 | 24 | 24 |
30 | mean sd | 258.6 16.2 | 261.3 17.4 | 266.4 17.9 | 280.0 19.5 | 300.1 21.0 | 318.7 23.2 | 349.5 23.8 | 393.9 30.6 |
| n | 24 | 24 | 24 | 24 | 24 | 24 | 24 | 24 |
75 | mean sd | 256.8 15.8 | 261.7 15.5 | 264.7 16.2 | 276.6 16.5 | 295.9 17.3 | 314.8 18.6 | 342.8 22.1 | 388.9 28.7 |
| n | 24 | 24 | 24 | 24 | 24 | 24 | 24 | 24 |
150 | mean sd | 255.8 16.3 | 260.5 16.4 | 264.2 16.9 | 276.1 15.6 | 293.9 17.3 | 312.6 17.7 | 339.6 21.5 | 384.5 26.7 |
| n | 24 | 24 | 24 | 24 | 24 | 24 | 24 | 24 |
Table 4 Group Mean Body Weight Change Values
Dose Level
(mg/kg bw/day) | 3 to 4 |
| Body Weight Change (g) during Days of Gestation 4 to 5 5 to 8 8 to 11 11 to 14 14 to 17 | 17 to 20 | |||||
0 (Control) | mean sd | 0.9 5.6 |
| 5.4 3.7 | 13.6 5.3 | 20.2 4.8 | 19.2 6.1 | 27.4 5.3 | 48.4 8.1 |
| n | 24 |
| 24 | 24 | 24 | 24 | 24 | 24 |
30 | mean sd | 2.8* 3.8 |
| 5.1 3.9 | 13.6 4.6 | 20.1 4.4 | 18.6 6.5 | 30.8 5.3 | 44.4 9.2 |
| n | 24 |
| 24 | 24 | 24 | 24 | 24 | 24 |
75 | mean sd | 4.9*** 2.9 |
| 3.0 3.2 | 11.9 4.4 | 19.3 4.7 | 18.9 5.5 | 28.0 6.6 | 46.1 8.3 |
| n | 24 |
| 24 | 24 | 24 | 24 | 24 | 24 |
150 | mean sd | 4.7* 7.8 |
| 3.7 3.6 | 11.9 4.1 | 17.8 5.5 | 18.7 5.2 | 27.0 8.2 | 44.9 7.7 |
| n | 24 |
| 24 | 24 | 24 | 24 | 24 | 24 |
Dose Level (mg/kg bw/day) |
| 4 | Cumulative Body Weight Change (g) from Day 3 of Gestation 5 8 11 14 17 | 20 | ||||
0 (Control) | mean sd | 0.9 5.6 | 6.3 5.1 | 19.9 7.9 | 40.1 8.6 | 59.3 10.7 | 86.7 13.2 | 135.1 19.3 |
| n | 24 | 24 | 24 | 24 | 24 | 24 | 24 |
30 | mean sd | 2.8* 3.8 | 7.8 4.5 | 21.4 6.7 | 41.5 8.4 | 60.1 11.5 | 90.9 12.3 | 135.3 19.8 |
| n | 24 | 24 | 24 | 24 | 24 | 24 | 24 |
75 | mean sd | 4.9*** 2.9 | 7.9 3.2 | 19.8 5.6 | 39.1 6.2 | 58.0 7.9 | 86.0 10.1 | 132.1 16.6 |
| n | 24 | 24 | 24 | 24 | 24 | 24 | 24 |
150 | mean sd | 4.7* 7.8 | 8.4 8.1 | 20.3 9.1 | 38.1 10.9 | 56.8 12.2 | 83.8 15.8 | 128.7 21.4 |
| n | 24 | 24 | 24 | 24 | 24 | 24 | 24 |
Table 5 Group Mean Gravid Uterus Weight and Adjusted Body Weight and Body Weight Change Values
Dose Level (mg/kg bw/day) |
| Body Weight (g) on Days of Gestation 3 20 | Body Weight Change (g) during Days of Gestation 3-20 | Gravid Uterus Weight (g) | Adjusted Body Weight (g) Day 20 | Adjusted Body Weight Change (g) 3-20 | |
0 (Control) | mean sd | 260.7 21.6 | 395.8 33.6 | 135.1 19.3 | 80.985 13.696 | 314.8 27.1 | 54.1 11.5 |
| n | 24 | 24 | 24 | 24 | 24 | 24 |
30 | mean sd | 258.6 16.2 | 393.8 30.4 | 135.2 19.5 | 78.509 14.164 | 315.3 25.8 | 56.7 15.9 |
| n | 24 | 24 | 24 | 24 | 24 | 24 |
75 | mean sd | 256.8 15.8 | 388.9 28.7 | 132.1 16.6 | 80.375 12.871 | 308.5 23.0 | 51.8 11.4 |
| n | 24 | 24 | 24 | 24 | 24 | 24 |
150 | mean sd | 255.8 16.3 | 384.5 26.7 | 128.7 21.4 | 77.238 19.951 | 307.3 19.3 | 51.5 12.2 |
| n | 24 | 24 | 24 | 24 | 24 | 24 |
Table 6 Group Mean Food Consumption Values
Dose Level (mg/kg bw/day) |
| Food Consumption (g/rat/day) between Days of Gestation 3 - 5 5 - 8 8 - 11 11 - 14 14-17 17 - 20 | |||||
0 (Control) | mean sd | 21.4 1.8 | 23.0 2.5 | 24.2 2.2 | 24.3 2.4 | 25.3 4.8 | 27.3 4.5 |
| n | 24 | 23 | 24 | 24 | 24 | 24 |
30 | mean sd | 21.0 2.8 | 23.2 3.2 | 23.7 3.0 | 24.4 6.0 | 26.5 5.1 | 26.3 3.4 |
| n | 24 | 24 | 24 | 24 | 24 | 24 |
75 | mean sd | 21.5 3.3 | 24.2 10.0 | 24.1 6.4 | 23.2 2.7 | 26.0 4.9 | 26.2 3.6 |
| n | 24 | 24+ | 23# | 24 | 24 | 24 |
150 | mean sd | 20.6 4.6 | 21.2 2.0 | 21.7** 2.5 | 22.6 1.9 | 24.1 4.9 | 26.0 5.1 |
| n | 24 | 24 | 24 | 24 | 24 | 24 |
+ = Food consumption for Female 61 calculated for Days 5-7 only due to female being present in incorrect cage on Day 8.
# = Data unavailable for Female No. 63, due to erroneous value recorded for food allocated on Day 8.
Table 7 Summary Incidence of Necropsy Findings
|
| Dose Level (mg/kg bw/day) |
| |
0 (Control) | 30 | 75 | 150 | |
TERMINAL DEATH
Number of Animals Examined |
24 |
24 |
24 |
24 |
External: Generalized Fur Loss |
0 |
0 |
0 |
1 |
Mass on Right Side | 0 | 0 | 0 | 1 |
Internal: Stomach – Non-Glandular Region: White Ridges |
0 |
1 |
0 |
0 |
Stomach – Non-Glandular Region: Ulcerated | 0 | 0 | 11 | 16 |
Stomach: Ulcerated | 0 | 0 | 0 | 5 |
Stomach: Pale | 0 | 0 | 0 | 1 |
Outer Right Side of Abdominal Cavity: Mass | 0 | 0 | 0 | 1 |
No Abnormalities Detected
|
24
|
23 |
13
|
3
|
Table 8 Group Mean Litter Data Values
Dose Level (mg/kg bw/day) |
| Number of Corpora Lutea | Number of Implants | Number of Embryonic/Fetal Deaths | Implantation Loss % | Number of Live Implants | % Male Fetuses | Mean Male Fetal Weight (g) | Mean Female Fetal Weight (g) | Mean Fetal Weight (g) | Mean Placental Weight (g) | Litter Weight (g) | Total Placental Weight (g) | |||||
Early | Late | Total | Pre | Post | Male | Female | Total | |||||||||||
0 (Control) | mean sd | 16.3 2.3 | 13.8 2.6 | 0.2 0.5 | 0.1 0.3 | 0.3 0.6 | 15.6 12.3 | 2.3 3.8 | 6.7 2.2 | 6.7 2.0 | 13.4 2.5 | 50.3 13.0 | 3.917 0.315 | 3.742 0.306 | 3.831 0.286 | 0.537 0.065 | 51.054 9.067 | 7.137 1.459 |
| n | 23# | 24 | 24 | 24 | 24 | 23 | 24 | 24 | 24 | 24 | 24 | 24 | 24 | 24 | 23+ | 24 | 23+ |
30 | mean sd | 15.4 2.4 | 12.8 2.8 | 0.1 0.3 | 0.0 0.2 | 0.2 0.4 | 18.1 13.4 | 1.3 3.0 | 6.3 1.8 | 6.3 2.2 | 12.6 2.9 | 52.1 15.2 | 4.253** 0.546 | 3.906 0.271 | 4.140* 0.569 | 0.571 0.132 | 50.878 9.780 | 6.834 1.202 |
| n | 24 | 24 | 24 | 24 | 24 | 24 | 24 | 24 | 24 | 24 | 24 | 24 | 23 | 24 | 24+ | 24 | 24+ |
75 | mean sd | 16.2 2.3 | 13.5 2.5 | 0.2 0.4 | 0.0 0.2 | 0.2 0.4 | 17.1 7.4 | 1.5 3.0 | 7.1 2.7 | 6.2 2.1 | 13.3 2.5 | 53.0 17.2 | 3.961 0.291 | 3.771 0.305 | 3.877 0.279 | 0.540 0.045 | 51.140 8.766 | 7.138 1.334 |
| n | 24 | 24 | 24 | 24 | 24 | 24 | 24 | 24 | 24 | 24 | 24 | 24 | 24 | 24 | 24 | 24 | 24 |
150 | mean sd | 15.3 2.2 | 12.6 3.2 | 0.1 0.4 | 0.0 0.2 | 0.2 0.5 | 18.7 14.7 | 2.7 10.3 | 6.5 2.5 | 5.9 2.5 | 12.4 3.4 | 52.9 14.3 | 4.029 0.264 | 3.781 0.309 | 3.913 0.289 | 0.608 0.165 | 48.735 13.796 | 7.201 1.968 |
| n | 24 | 24 | 24 | 24 | 24 | 24 | 24 | 24 | 24 | 24 | 24 | 24 | 24 | 24 | 24 | 24 | 24 |
+ = Placental weights not recorded in error for Female No. 21’s litter and for fetus No. 8 from Female No. 25
# = Data unavailable for Female No. 16
Table 9 Summary Incidence of Fetal External Findings
| External Findings |
| Dose level (mg/kg bw/day) |
|
| ||||||||
0 (Control) | 30 | 75 |
| 150 | |||||||||
| Number of fetuses (litters) examined |
|
| ||||||||||
322 (24) | 303 (24) | 318 (24) |
| 298 (24) | |||||||||
NF | NL | %† | NF | NL | %† | NF | NL | %† | NF | NL | %† | ||
Total Number Affected |
| 4 | 1 | 1.3 | 8 | 5 | 5.9 | 7 | 6 | 2.2 | 15 | 6 | 6.6 |
Small Fetus |
| 4 | 1 | 1.3 | 6 | 4 | 1.8 | 5 | 4 | 1.6 | 8 | 4 | 2.8 |
Pale Fetus |
| 1 | 1 | 0.3 | 0 | 0 | 0.0 | 0 | 0 | 0.0 | 0 | 0 | 0.0 |
Large Fetus |
| 0 | 0 | 0.0 | 2 | 1 | 4.2 | 0 | 0 | 0.0 | 0 | 0 | 0.0 |
Large Placenta |
| 0 | 0 | 0.0 | 2 | 1 | 4.2 | 1 | 1 | 0.3 | 9 | 5 | 4.7* |
Small Placenta |
| 2 | 1 | 0.6 | 1 | 1 | 0.2 | 2 | 2 | 0.7 | 1 | 1 | 0.3 |
Table 10 Summary Incidence of Fetal Visceral Findings
Visceral Findings |
|
|
|
| Dose Level (mg/kg bw/day) |
|
|
|
| ||||
| 0 (Control) |
|
| 30 | 75 |
|
| 150 |
| ||||
|
|
|
| Number of Fetuses (litters) Examined |
|
|
|
| |||||
| 168 (24) |
|
| 156 (24) | 164 (24) |
|
| 157 (24) |
| ||||
NF |
| NL | %† | NF | NL | %† | NF | NL | %† | NF | NL | %† | |
External Hemorrhage |
0 |
|
0 |
0.0 |
0 |
0 |
0.0 |
1 |
1 |
0.5 |
0 |
0 |
0.0 |
Head Rugae - non-uniform patterning |
2 |
|
2 |
1.0 |
11 |
8 |
6.8* |
8 |
8 |
4.8* |
5 |
2 |
3.9 |
Abdomen Liver - additional lobe between right and left median |
2 |
|
2 |
1.2 |
2 |
1 |
1.0 |
0 |
0 |
0.0 |
0 |
0 |
0.0 |
Umbilical artery - left-sided | 0 |
| 0 | 0.0 | 0 | 0 | 0.0 | 1 | 1 | 0.8 | 2 | 2 | 1.2 |
Testis - partially undescended | 4 |
| 3 | 2.4 | 4 | 4 | 2.5 | 1 | 1 | 0.6 | 2 | 2 | 1.2 |
Testis - malrotated | 0 |
| 0 | 0.0 | 0 | 0 | 0.0 | 0 | 0 | 0.0 | 1 | 1 | 0.5 |
Ureter - kinked | 15 |
| 10 | 10.0 | 11 | 7 | 7.0 | 12 | 6 | 8.3 | 9 | 7 | 7.3 |
Ureter - dilated | 11 |
| 7 | 7.9 | 3 | 3 | 1.9 | 8 | 4 | 5.9 | 5 | 4 | 4.9 |
Kidney - malpositioned | 0 |
| 0 | 0.0 | 0 | 0 | 0.0 | 1 | 1 | 0.8 | 0 | 0 | 0.0 |
Renal pelvic cavitation - increased | 8 |
| 6 | 4.4 | 8 | 7 | 5.0 | 7 | 5 | 4.5 | 10 | 6 | 7.5 |
Renal papilla - absent | 1 |
| 1 | 0.5 | 2 | 2 | 1.3 | 3 | 2 | 2.3 | 1 | 1 | 1.4 |
Renal medulla - reduced in size | 1 |
| 1 | 0.5 | 0 | 0 | 0.0 | 0 | 0 | 0.0 | 0 | 0 | 0.0 |
Thorax Thymus - lobe partially undescended |
5 |
|
4 |
2.8 |
5 |
4 |
2.9 |
3 |
3 |
1.8 |
4 |
4 |
2.2 |
Atrium - enlarged | 4 |
| 4 | 2.3 | 1 | 1 | 0.7 | 2 | 2 | 1.1 | 3 | 3 | 1.6 |
Ventricle - reduced in size | 0 |
| 0 | 0.0 | 0 | 0 | 0.0 | 1 | 1 | 0.5 | 0 | 0 | 0.0 |
Ventricle - vacuole | 0 |
| 0 | 0.0 | 0 | 0 | 0.0 | 1 | 1 | 0.5 | 0 | 0 | 0.0 |
Total | 30 |
| 18 | 18.5 | 31 | 14 | 18.9 | 27 | 14 | 17.0 | 30 | 15 | 20.3 |
Table 11 Summary Incidence of Fetal Skeletal Findings
Skeletal Findings |
| Dose Level (mg/kg bw/day) |
|
| ||||||||
0 (Control) | 30 | 75 |
| 150 | ||||||||
| Number of Fetuses (litters) Examined |
|
| |||||||||
154 (24) | 147 (24) | 154 (24) |
| 141 (24) | ||||||||
NF | NL | %† | NF | NL | %† | NF | NL | %† | NF | NL | %† | |
Skull Fontanelle (anterior) - large |
2 |
1 |
1.4 |
2 |
2 |
1.1 |
9 |
4 |
5.2 |
1 |
1 |
0.6 |
Nasal - incomplete ossification | 10 | 5 | 6.2 | 6 | 5 | 3.9 | 18 | 9 | 10.5 | 19 | 9 | 12.5 |
Nasal/Frontal - sutural bone | 1 | 1 | 0.7 | 0 | 0 | 0.0 | 0 | 0 | 0.0 | 0 | 0 | 0.0 |
Frontal - incomplete ossification | 1 | 1 | 0.7 | 0 | 0 | 0.0 | 5 | 4 | 2.9 | 8 | 5 | 5.1 |
Frontal - unossified area | 1 | 1 | 0.7 | 3 | 2 | 1.9 | 5 | 3 | 3.5 | 3 | 2 | 1.6 |
Parietal - incomplete ossification | 6 | 4 | 3.7 | 3 | 2 | 2.1 | 8 | 6 | 5.0 | 20 | 10 | 14.2 |
Interparietal - incomplete ossification | 29 | 11 | 18.4 | 14 | 8 | 9.1 | 30 | 12 | 18.5 | 38 | 14 | 25.7 |
Occipital (Supra-occipital) - incomplete ossification | 17 | 12 | 11.1 | 6 | 4 | 4.1* | 23 | 10 | 14.2 | 26 | 14 | 18.4 |
Occipital (Supra-occipital) - unossified area(s) | 12 | 8 | 7.2 | 7 | 6 | 4.3 | 8 | 5 | 4.6 | 8 | 7 | 6.5 |
Squamosal - incomplete ossification | 7 | 5 | 4.4 | 8 | 4 | 5.4 | 14 | 7 | 8.1 | 30 | 14 | 20.3** |
Squamosal - unossified area(s) | 1 | 1 | 0.8 | 0 | 0 | 0.0 | 1 | 1 | 0.5 | 1 | 1 | 1.4 |
Jugal - incomplete ossification | 5 | 3 | 3.2 | 4 | 4 | 2.6 | 4 | 4 | 2.5 | 8 | 6 | 6.7 |
Zygomatic process of maxilla - incomplete ossification | 4 | 3 | 2.6 | 7 | 6 | 4.3 | 13 | 9 | 7.6 | 18 | 11 | 12.5** |
Zygomatic process of squamosal - incomplete ossification | 1 | 1 | 0.7 | 0 | 0 | 0.0 | 2 | 2 | 1.1 | 6 | 3 | 4.2 |
Zygomatic process of squamosal - fused to jugal | 1 | 1 | 0.7 | 0 | 0 | 0.0 | 0 | 0 | 0.0 | 0 | 0 | 0.0 |
Premaxilla - incomplete ossification | 0 | 0 | 0.0 | 0 | 0 | 0.0 | 3 | 3 | 1.8 | 2 | 1 | 1.4 |
Hyoid - incomplete ossification | 14 | 9 | 8.6 | 17 | 11 | 11.9 | 11 | 8 | 7.0 | 16 | 9 | 10.1 |
Hyoid - not ossified | 14 | 10 | 9.1 | 5 | 5 | 3.6 | 11 | 6 | 6.4 | 14 | 7 | 8.3 |
Presphenoid - incomplete ossification | 1 | 1 | 0.7 | 0 | 0 | 0.0 | 1 | 1 | 0.7 | 2 | 2 | 1.3 |
Presphenoid - not ossified | 0 | 0 | 0.0 | 1 | 1 | 0.7 | 2 | 2 | 1.3 | 0 | 0 | 0.0 |
NOTE: a fetus may appear in more than one category
Skeletal Findings | Dose Level (mg/kg bw/day) |
|
| |||||||||
0 (Control) | 30 | 75 |
| 150 |
| |||||||
Number of Fetuses (litters) Examined |
|
| ||||||||||
154 (24) | 147 (24) | 154 (24) |
| 141 (24) |
| |||||||
NF | NL | %† | NF | NL | %† | NF | NL | %† | NF | NL | %† | |
Vertebral Column Odontoid - ossification present |
0 |
0 |
0.0 |
1 |
1 |
4.2 |
0 |
0 |
0.0 |
1 |
1 |
0.7 |
Ventral arch of vertebra 1 - ossification present | 45 | 18 | 31.7 | 49 | 18 | 36.3 | 37 | 16 | 23.9 | 34 | 11 | 22.2 |
Cervical (neural) arch - incomplete ossification | 8 | 7 | 5.1 | 0 | 0 | 0.0** | 10 | 7 | 6.3 | 16 | 9 | 10.1 |
Thoracic centrum - incomplete ossification | 12 | 9 | 8.1 | 3 | 2 | 1.8 | 10 | 5 | 6.3 | 7 | 6 | 5.0 |
Thoracic centrum - not ossified | 3 | 1 | 2.1 | 0 | 0 | 0.0 | 1 | 1 | 0.8 | 2 | 2 | 1.3 |
Thoracic centrum - bipartite ossification | 5 | 4 | 3.8 | 2 | 2 | 1.4 | 2 | 2 | 1.1 | 1 | 1 | 0.8 |
Thoracic centrum - dumb-bell-shaped | 20 | 10 | 13.9 | 19 | 11 | 16.2 | 19 | 13 | 11.7 | 21 | 14 | 18.5 |
Thoracic centrum - asymmetrically ossified | 4 | 4 | 3.0 | 1 | 1 | 0.6 | 2 | 2 | 1.1 | 3 | 3 | 2.2 |
Thoracic centrum - misaligned | 1 | 1 | 0.8 | 0 | 0 | 0.0 | 0 | 0 | 0.0 | 0 | 0 | 0.0 |
Thoracic centrum - hemicentric | 2 | 2 | 1.5 | 0 | 0 | 0.0 | 0 | 0 | 0.0 | 0 | 0 | 0.0 |
Thoracic vertebra - absent (arch and centrum) | 1 | 1 | 0.8 | 0 | 0 | 0.0 | 0 | 0 | 0.0 | 0 | 0 | 0.0 |
Lumbar centrum - incomplete ossification | 3 | 2 | 2.1 | 0 | 0 | 0.0 | 0 | 0 | 0.0 | 0 | 0 | 0.0 |
Lumbar centrum - bipartite ossification | 0 | 0 | 0.0 | 0 | 0 | 0.0 | 1 | 1 | 0.8 | 0 | 0 | 0.0 |
Lumbar centrum - dumb-bell-shaped | 0 | 0 | 0.0 | 0 | 0 | 0.0 | 1 | 1 | 0.8 | 0 | 0 | 0.0 |
Lumbar centrum - asymmetrically ossified | 0 | 0 | 0.0 | 0 | 0 | 0.0 | 1 | 1 | 0.8 | 0 | 0 | 0.0 |
Sacral centrum - incomplete ossification | 0 | 0 | 0.0 | 0 | 0 | 0.0 | 1 | 1 | 0.8 | 0 | 0 | 0.0 |
Sacral centrum - bipartite ossification | 0 | 0 | 0.0 | 0 | 0 | 0.0 | 1 | 1 | 0.8 | 0 | 0 | 0.0 |
Sacral (neural) arch - incomplete ossification | 25 | 11 | 15.9 | 22 | 9 | 15.3 | 31 | 13 | 19.9 | 39 | 14 | 26.5 |
Sacral (neural) arch - not ossified | 0 | 0 | 0.0 | 0 | 0 | 0.0 | 1 | 1 | 0.6 | 0 | 0 | 0.0 |
Caudal vertebrae - less than 4 ossified | 28 | 14 | 18.0 | 24 | 12 | 14.8 | 47 | 13 | 28.0 | 51 | 15 | 32.8 |
Number of pre-sacral vertebrae = 25/27 | 1 | 1 | 0.8 | 1 | 1 | 0.7 | 1 | 1 | 0.8 | 1 | 1 | 2.1 |
Skeletal Findings | Dose Level (mg/kg bw/day) |
| ||||||||||
0 (Control) | 30 | 75 |
| 150 | ||||||||
Number of Fetuses (litters) Examined |
| |||||||||||
154 (24) | 147 (24) | 154 (24) |
| 141 (24) | ||||||||
NF | NL | %† | NF | NL | %† | NF | NL | %† | NF | NL | %† | |
Ribs Ossification centre - associated with 7th cervical vertebra |
0 |
0 |
0.0 |
1 |
1 |
0.8 |
0 |
0 |
0.0 |
2 |
2 |
1.4 |
14th rib - extra - associated with 1st lumbar vertebra | 0 | 0 | 0.0 | 1 | 1 | 0.7 | 0 | 0 | 0.0 | 0 | 0 | 0.0 |
Ossification centre - associated with 1st lumbar vertebra | 2 | 2 | 1.5 | 8 | 4 | 5.5 | 4 | 3 | 2.5 | 18 | 11 | 14.1** |
One or more ribs - wavy | 0 | 0 | 0.0 | 0 | 0 | 0.0 | 0 | 0 | 0.0 | 2 | 1 | 1.0 |
One or more ribs - thickened | 1 | 1 | 0.6 | 0 | 0 | 0.0 | 0 | 0 | 0.0 | 2 | 1 | 1.0 |
Rib - short | 3 | 1 | 1.8 | 1 | 1 | 0.7 | 2 | 2 | 1.5 | 0 | 0 | 0.0 |
Rib - fused | 2 | 2 | 1.5 | 0 | 0 | 0.0 | 0 | 0 | 0.0 | 0 | 0 | 0.0 |
Rib - incomplete ossification | 0 | 0 | 0.0 | 0 | 0 | 0.0 | 0 | 0 | 0.0 | 1 | 1 | 0.5 |
Rib - interrupted ossification | 1 | 1 | 0.8 | 0 | 0 | 0.0 | 0 | 0 | 0.0 | 0 | 0 | 0.0 |
Rib - not ossified | 1 | 1 | 0.8 | 0 | 0 | 0.0 | 0 | 0 | 0.0 | 0 | 0 | 0.0 |
Rib - absent | 1 | 1 | 0.8 | 0 | 0 | 0.0 | 0 | 0 | 0.0 | 0 | 0 | 0.0 |
Rib - no articulation point | 1 | 1 | 0.8 | 0 | 0 | 0.0 | 1 | 1 | 0.5 | 0 | 0 | 0.0 |
Rib - bifurcated | 1 | 1 | 0.8 | 0 | 0 | 0.0 | 0 | 0 | 0.0 | 0 | 0 | 0.0 |
Costal cartilage - misaligned | 3 | 2 | 2.0 | 2 | 2 | 1.2 | 3 | 3 | 1.6 | 1 | 1 | 0.7 |
Costal cartilage - not fused to sternebra | 14 | 9 | 8.7 | 11 | 9 | 7.5 | 12 | 7 | 7.9 | 14 | 7 | 11.1 |
Sternebrae Sternebra - incomplete ossification |
4 |
2 |
2.7 |
0 |
0 |
0.0 |
11 |
8 |
6.2* |
4 |
3 |
2.7 |
Sternebra - not ossified | 3 | 1 | 2.1 | 0 | 0 | 0.0 | 1 | 1 | 0.7 | 0 | 0 | 0.0 |
Sternebra - bipartite ossification | 0 | 0 | 0.0 | 0 | 0 | 0.0 | 0 | 0 | 0.0 | 1 | 1 | 0.7 |
Sternebra - misaligned | 2 | 2 | 1.3 | 4 | 4 | 2.4 | 6 | 5 | 3.3 | 8 | 6 | 4.9 |
Sternum - split | 0 | 0 | 0.0 | 0 | 0 | 0.0 | 1 | 1 | 0.7 | 0 | 0 | 0.0 |
Skeletal Findings | Dose Level (mg/kg bw/day) |
|
| |||||||||
0 (Control) | 30 | 75 |
| 150 |
| |||||||
Number of Fetuses (litters) Examined |
|
| ||||||||||
154 (24) | 147 (24) | 154 (24) | 141 (24)
| |||||||||
NF | NL | %† | NF | NL | %† | NF | NL | %† | NF | NL | %† | |
Sternebrae (continued) Xiphoid cartilage - split |
0 |
0 |
0.0 |
0 |
0 |
0.0 |
1 |
1 |
0.7 |
0 |
0 |
0.0 |
Xiphoid cartilage - partially split | 14 | 11 | 9.9 | 9 | 8 | 5.7 | 8 | 8 | 5.1 | 14 | 11 | 10.1 |
Pectoral Girdle Scapula - misshapen |
1 |
1 |
0.6 |
5 |
5 |
6.6 |
2 |
2 |
1.0 |
4 |
2 |
2.6 |
Pelvic Girdle Ischium - not ossified |
0 |
0 |
0.0 |
0 |
0 |
0.0 |
1 |
1 |
0.7 |
0 |
0 |
0.0 |
Ischium - incomplete ossification | 1 | 1 | 0.7 | 1 | 1 | 0.7 | 4 | 2 | 2.6 | 2 | 2 | 1.2 |
Pubis - not ossified | 0 | 0 | 0.0 | 0 | 0 | 0.0 | 3 | 3 | 1.8 | 1 | 1 | 0.7 |
Pubis - incomplete ossification | 8 | 3 | 5.4 | 8 | 5 | 5.8 | 11 | 8 | 7.0 | 13 | 8 | 9.3 |
Forelimbs Metacarpal - not ossified |
51 |
20 |
31.8 |
41 |
15 |
27.0 |
65 |
19 |
42.2 |
57 |
17 |
37.7 |
Metacarpal - incomplete ossification | 0 | 0 | 0.0 | 1 | 1 | 0.7 | 4 | 3 | 2.9 | 3 | 3 | 1.9 |
Forepaw phalanges - 1 or more - ossified | 14 | 10 | 10.5 | 13 | 9 | 12.8 | 10 | 5 | 6.5 | 10 | 5 | 7.0 |
Humerus - incomplete ossification | 2 | 2 | 1.3 | 1 | 1 | 0.7 | 2 | 2 | 1.7 | 7 | 5 | 5.6 |
Humerus - hole | 1 | 1 | 0.6 | 0 | 0 | 0.0 | 1 | 1 | 0.6 | 0 | 0 | 0.0 |
Hindlimbs Metatarsal - 1st - ossified |
0 |
0 |
0.0 |
1 |
1 |
4.2 |
0 |
0 |
0.0 |
0 |
0 |
0.0 |
Metatarsal - not ossified | 0 | 0 | 0.0 | 0 | 0 | 0.0 | 1 | 1 | 0.7 | 0 | 0 | 0.0 |
Metatarsal - incomplete ossification | 3 | 2 | 2.2 | 0 | 0 | 0.0 | 5 | 4 | 2.9 | 2 | 2 | 1.3 |
Femur - incomplete ossification | 12 | 7 | 7.7 | 2 | 2 | 1.4 | 9 | 6 | 5.9 | 7 | 6 | 4.6 |
Total | 132 | 24 | 86.6 | 118 | 24 | 81.4 | 130 | 24 | 82.7 | 126 | 24 | 90.2 |
Applicant's summary and conclusion
- Conclusions:
- The oral administration of the test substance to pregnant rats by oral gavage during gestation at dose levels of 30, 75 and 150 mg/kg bw/day, resulted in treatment-related macroscopic abnormalities detected in females treated with 150 and 75 mg/kg bw/day. The macroscopic findings detected were considered to be the result of localized irritation from the test item formulations and are not considered to reflect systemic toxicity. The ‘No Observed Adverse Effect Level’ (NOAEL) for the pregnant female was therefore considered to be 150 mg/kg bw/day.
No treatment-related changes were detected in the offspring parameters measured or on embryofetal development. The ‘No Observed Effect Level’ (NOEL) for developmental toxicity was therefore considered to be 150 mg/kg bw/day.
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