Registration Dossier
Registration Dossier
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EC number: 233-215-5 | CAS number: 10081-67-1
Endpoint summary
Administrative data
Key value for chemical safety assessment
Effects on fertility
Description of key information
Screening for reproductive / developmental toxicity
The results of this study indicated that per os application of the test article Dusnatox 86 didn’t show any significant changes in all used doses in comparison with the control animals:in clinical observation, bodyweight of animals, food consumption, length of pregnancy, number of pups and weight of litter, number of corpora lutea and implantation sites, relative weight of organs or macroscopical and histological changes.
Link to relevant study records
- Endpoint:
- screening for reproductive / developmental toxicity
- Remarks:
- based on test type (migrated information)
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 17 December 2008 to 13 July 2009
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Study performed in accordance with OECD test guideline in compliance with GLP and reported with a valid GLP certificate.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- yes
- Species:
- rat
- Strain:
- Wistar
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- Test Animals: 48 adult males and 48 females of Wistar rats from Velaz Praha, Czech Republic were used. The animals were acclimatised for 20 days before the study in the condition identical to the condition during the experiment. The acclimatisation was made according to SOPB-00188-AH, Animals quarantine. The mean bodyweight of animals at the start of experiment was in males 269.8g, in females 185g. Twelve males and twelve females were used in the control and in all dose groups. Husbandry: All animals were kept in cages with bedding in the experimental animal house with standard conditions. The sanitation was made according to standard operation procedure. The temperature was 22± 2°C, relative humidity 45 to 65%. They were followed by a room thermometer and hydrometer placed in the room. The room is equipped with central air-conditioner. The light regime was artificial 12-hour light / 12-hour dark cycle. The keeping of animals was organised to timetable:1. 4 animals in the cage (males and female separately)2. 1 male and 1 female in the cage (mating)3. 1 pregnant female individually4. 1 female and offspring individuallyFood and water: For feeding conventional laboratory diet (Top Dovo, PD Horné Dubové – Naháč) was used with an unlimited supply of drinking water.
- Route of administration:
- oral: gavage
- Vehicle:
- olive oil
- Details on exposure:
- Control article/VehicleIdentification: Olive oil (Olivae oleum raffinatum), product by G. HeessLot number: L 809182Purity: Quality of control article was declared with Certificate.Storage condition: The control article was stored under laboratory conditions in dark container. Justification for choice of vehicle: Dusantox 86 is not soluble in water, but olive oil was used as vehicle. Method of application: The test article was administered per os with the metal stomachic tube everyday at 8 a.m. Doses were calculated on the current bodyweight. The animals were weighed weekly. The fixed application volume of 0.5mL/100g bodyweight was used. Dusantox 86 is not soluble in water; the olive oil was used as a vehicle. The test substance was soluble in olive oil as vehicle every day. The vehicle was applied to control animals in volume 0.5mL/100g bodyweight.
- Details on mating procedure:
- Normally, 1:1 (1 male to 1 female) mating was used in this study. The female was place with the same male until pregnancy occurs or 14 days have elapsed. Every morning during the mating period the females were examined for the presence if sperm or vaginal plugs. Day 0 of pregnancy was defined as the day a vaginal plug or sperm are found. The pregnant female was kept individually up to 4th day post-partum.
- Analytical verification of doses or concentrations:
- not specified
- Details on analytical verification of doses or concentrations:
- Not specified.
- Duration of treatment / exposure:
- Males - 28 daysFemales - up to four days after birth (54 days)
- Frequency of treatment:
- Daily
- Details on study schedule:
- Daily dosing of both sexes began 14 days before mating. Dosing was continued in both sexes during mating period (max. 14 days – 2 complete oestrus cycles). Males were dosed after the mating period at least until the minimum total dosing period of 28 days. They were sacrificed by the overdose of anaesthetic (dietylether). Daily dosing of females were continued throughout pregnancy and up to the 4th day after birth. Female showing no evidence of copulation were dosed 29 days after the last day of mating period. They were sacrificed by overdose of anaesthetic. Females with offpring were sacrificed on day 4 post-partum.
- Remarks:
- Doses / Concentrations:0, 5, 25 & 50 mg/kg bw/dayBasis:actual ingested
- No. of animals per sex per dose:
- 12 animals per sex, per dose. (Total of 48 rats)
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- Groups Designation and Dose Levels: Three test groups and control group (vehicle) were used. Twelve animals of both sexes were used in every level group and in the control group.The animals were marked by serial number 1 – 48. All cages were marked with the number of the group, the dose and the serial number of the animals. The animals in the cage were marked by line I – IIII on the tail. Males were coloured with black and females with red. Selection of doses was estimated from available information about the test article. The low dose was estimated for 5mg.kg-1. The high dose level – 50mg.kg-1 represents 10-fold of the low dose level, medium dose level – 25mg.kg-1 represents 5-fold of the low dose level. Males (black)Females (red)ControlOlive oil(12) 1 -12(12) 1 – 12Low dose group5mg.kg-1(12) 13 – 24(12) 13 – 24Medium dose group25mg.kg-1(12) 25 – 36(12) 25 – 36High dose group50mg.kg-1(12) 37 – 48(12) 37 – 48
- Positive control:
- Not utilised in this study.
- Parental animals: Observations and examinations:
- General clinical observations were made once a day 1 hour after the test application. The health condition of the animals, reaction of the animals to the applied substance, their condition were monitored every day and recorded. The number of pregnant females was recorded.Bodyweight and Food Consumption: Individual weighing of the adult animals was performed once a week. The bodyweights of animals were recorded.
- Oestrous cyclicity (parental animals):
- Not measured
- Sperm parameters (parental animals):
- Not measured
- Litter observations:
- The number of live pups, weight of litters and sex of pups were observed.
- Postmortem examinations (parental animals):
- The animals were anesthetized with diethylether and necropsied. Males: at the end of the mating period (after 28 days of treatment period) and pregnant females after 4 days post-partum and non-pregnant females 28 days after mating period. Kidneys, testes, epididymides, uterus and ovaries were weighed during the necropsy. The samples from these organs with prostate and glands vesicles were processed for microscopic examination. Those tissue samples were fixed in bouin solution and prepared in the paraffin technical. The tissue segments were sectioned at 10μm by a Leica micrptome and stained with hematoxylin and eosin. The histological section was examined under a light microscope. Macroscopic Evaluation: At the time of death the animals were examined macroscopically for any abnormalities or pathological changes. Special attention was given to the organs of the reproductive system and the kidneys as target organ. The number of corpora lutea and count of implantation sites was monitored. Histopathological Examination: Histopathological evaluations have a prominent role in reproductive toxicity assessment. Detailed histological examination was performed on the uterus, ovaries, testes, epididydimes and kidneys (the target organ) of the animals of control and highest (50mg.kg-1) dose groups. The histopathological examination was made by standard operation procedures.
- Postmortem examinations (offspring):
- Postmorten exmainations not performed on offspring.
- Statistics:
- The results obtained during the study were statistically evaluated by statistical programme Statgraphics. Statistical evaluated was made separately for males, females and litters. For identification homogeneity groups were used the Bartllet’s test. In the case of homogeneity One-Way Analysis of variance with consecutive Multiple Rangers tests was accomplished. In case non homogeneity Kruskal-Wallis One Way Analysis by Ranks was applied.
- Reproductive indices:
- After sacrificed of all females were calculated reproduction indices Number of females with implantsFertility Index = -------------------------------- x 100 Number of females mating Number of females delivering live youngGestation (Pregnancy) Index = --------------------------------------------- x 100 Number of females with evidence of pregnancy Number of live offspringLive Birth Index = --------------------------------- x 100 Number of offspring delivered
- Offspring viability indices:
- Number of live offspring at lactation day 4Viability (Survival) Index = --------------------------------------------- x 100Number of live offspring delivered
- Clinical signs:
- no effects observed
- Body weight and weight changes:
- no effects observed
- Food consumption and compound intake (if feeding study):
- no effects observed
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Histopathological findings: non-neoplastic:
- no effects observed
- Other effects:
- not examined
- Reproductive function: oestrous cycle:
- no effects observed
- Reproductive function: sperm measures:
- no effects observed
- Reproductive performance:
- no effects observed
- Dose descriptor:
- NOEC
- Effect level:
- 50 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: clinical observation, bodyweight of animals, food consumption, length of pregnancy, number of pups and weight of litter, number of corpora lutea and implantation sites, relative weight of organs or macroscopical and histological changes.
- 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
- Dose descriptor:
- NOEC
- Generation:
- F1
- Effect level:
- 50 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: clinical observation, bodyweight of animals, food consumption, length of pregnancy, number of pups and weight of litter, number of corpora lutea and implantation sites, relative weight of organs or macroscopical and histological changes.
- Reproductive effects observed:
- not specified
- Conclusions:
- The results of this study indicated that per os application of the test article Dusnatox 86 didn’t show any significant changes in all used doses in comparison with the control animals:in clinical observation, bodyweight of animals, food consumption, length of pregnancy, number of pups and weight of litter, number of corpora lutea and implantation sites, relative weight of organs or macroscopical and histological changes.
- Executive summary:
This study was used to provide initial information on possible effects on reproduction and/or development, which may be evoked by Dusantox 86. The Wistar rats, 48 adult males and 48 adult females were divided into four groups. Three groups received the test article Dusantox 86 in graduated doses. The control group received the vehicle. Dusantox 86 is not soluble in water; olive oil was used as a vehicle. The dose of 5mg.kg-1was estimated as the low dose, the dose of 25mg.kg-1as the medium dose level and as the high dose level was selected 50mg.kg-1. Doses were calculated on the current bodyweight. The test article and vehicle were administered per os by metal stomachic tube every day. The males were applied 28 days – 14 days per-mating, 14 days mating. This dosing period (28 days) is considered sufficient to enable detection of the majority of effects on male fertility and spermatogenesis. The females were applicated 54 days – 14 days pre-mating, 14 days mating (with the objective of covering at least two complete oestrus cycles), 22 days gestation, and 4 days lactation. The application volume 0.5mL/100g weight was administered. All animals were monitored for signs of toxicity during application of the test article. All animals were weighed weekly. The bodyweight, food consumption and clinical observation were observed during the study. After finishing the test all animals were sacrificed by overdosing of anaesthetic and moved to macroscopical and histological evaluation. The organs of the reproductive system (the uterus, ovaries, testes, epididydimes, and accessory sex organs) and the kidneys as target organ were monitored. The number of implantation sites and corpora lutea was recorded. The results were elaborated in for of tables. The statistical programme Statgraphics was used for statistically evaluation of the results.
There were no test article-related deaths of animal during the study. There were not any visible signs of intoxication in all animals during clinical observation. The incidence of smooth stool in one animal of dose group 50mg.kg-1males and diarrhoea in one male of dose group 25mg.kg-1were observed. The incidence of alopecia in one female dose group of 50mg.kg-1was observed. These incidences were temporary and may not be indicate relation to the test application. The bodyweight of all animals moderately increased during the study. There were no significant differences between all dose groups and the control groups.
The food consumption was adequate. In control (olive oil) and highest (50mg.kg-1) dose groups 12 females were pregnant. In dose group 5mg.kg-19 females were pregnant and in dose group 25mg.kg-111 females were pregnant. In female dose group 5mg.kg-1three animals and in dose group 25 mg.kg-1one animal were not pregnant. The number of alive pups in all females of the control and all dose groups was stable. The relative weight of kidneys and reproduction organs in both sexes were without statistical differences. Number of corpora lutea and count of implantation sites in females of all dose groups and in the control group were without statistical differences. Detailed histological examination was performed on reproduction organs and kidneys (target organs) of the males and female of the control and the highest dose group. Macroscopical and histopathological findings showed that Dusantox 86 did not cause pathological lesions in organs of treated animals. The sporadic presence of lesions in the animals of the high dose groups was similar to control groups.
CONCLUSION
The results of this study indicated that per os application of the test article Dusnatox 86 didn’t show any significant changes in all used doses in comparison with the control animals:
in clinical observation, bodyweight of animals, food consumption, length of pregnancy, number of pups and weight of litter, number of corpora lutea and implantation sites, relative weight of organs or macroscopical and histological changes.
Reference
Table 1. Dusantox 86. Reproduction/Developmental Toxicity Screening Test (OECD 421) – Bodyweight, Males
Dose level | N | Bodyweight (g) / Day | ||||
1 | 7 | 14 | 21 | 28 | ||
Control
| 12/12 | 272.50± 12.88 | 286.17± 15.05 | 301.67± 20.38 | 321.67± 21.67 | 330.00± 26.63 |
Low 5mg.kg-1 | 12/12 | 265.83± 16.21 | 280.83± 16.21 | 295.00± 15.08 | 319.17± 18.32 | 325.83± 18.81 |
Medium 25mg.kg-1 | 12/12 | 270.00± 19.54 | 287.50± 23.79 | 299.17± 25.75 | 326.67± 29.65 | 331.67± 30.40 |
High 50mg.kg-1 | 12/12 | 270.83± 15.64 | 281.67± 19.92 | 299.17± 27.12 | 322.50± 31.66 | 329.17± 31.18 |
Table 2. Dusantox 86. Reproduction/Developmental Toxicity Screening Test (OECD 421) – Bodyweight, Pre-Mating Period, Females
Dose level | N | Bodyweight (g) / Day | ||
1 | 7 | 14 | ||
Control | 12/12 | 185.83± 9.96 | 186.67± 10.73 | 190.83± 9.00 |
Low 5mg.kg-1 | 12/12 | 186.67± 9.85 | 185.83± 10.84 | 190.00± 11.28 |
Medium 25mg.kg-1 | 12/12 | 183.33± 11.55 | 185.00± 7.98 | 188.33± 10.30 |
High 50mg.kg-1 | 12/12 | 184.17± 7.93 | 186.67± 12.31 | 188.33± 10.30 |
Table 3. Dusantox 86. Reproduction/Developmental Toxicity Screening Test (OECD 421) – Bodyweight, Mating Period, Females
Dose level | N | Bodyweight (g) / Day | ||
1 | 7 | 14 | ||
Control | 12/12 | 198.33± 9.37 | 215.83± 12.40 | 235.00± 11.68 |
Low 5mg.kg-1 | 12/12 | 199.17± 10.84 | 216.67± 16.14 | 232.50± 23.79 |
Medium 25mg.kg-1 | 12/12 | 198.33± 11.93 | 209.17± 13.11 | 230.83± 14.43 |
High 50mg.kg-1 | 12/12 | 200.83± 11.65 | 210.83± 12.40 | 230.83± 11.65 |
Table 4. Dusantox 86. Reproduction/Developmental Toxicity Screening Test (OECD 421) – Bodyweight, Pregnancy, Females
Dose level | N | Bodyweight (g) / Day | ||
1 | 14 | 21 | ||
Control | 12/12 | 200.00± 9.54 | 235.00± 11.68 | 281.67± 27.1 |
Low 5mg.kg-1 | 12/12 | 202.22± 9.72 | 240.00± 18.71 | 288.89± 28.92 |
Medium 25mg.kg-1 | 12/12 | 208.18± 13.28 | 232.73± 14.89 | 280.00± 24.08 |
High 50mg.kg-1 | 12/12 | 210.83± 12.40 | 232.50± 14.22 | 283.33± 25.70 |
N-Number of animals, the results are presented as mean± SD
Table 5. Dusantox 86. Reproduction/Developmental Toxicity Screening Test (OECD 421) – Bodyweight, F1 generation (Offspring)
Dose level | Bodyweight (g) / Day | |||
N | 1 | N | 4 | |
Control | 102 | 53.33± 30.25 | 101 | 70.83± 38.49 |
Low 5mg.kg-1 | 78 | 51.67± 23.98 | 78 | 75.56± 34.68 |
Medium 25mg.kg-1 | 79 | 43.64± 23.99 | 77 | 61.36± 35.08 |
High 50mg.kg-1 | 112 | 58.75± 17.34 | 110 | 77.92± 25.89 |
N-Number of pups, the results are presented as mean± SD
Table 6. Dusantox 86. Reproduction/Developmental Toxicity Screening Test (OECD 421) – Mean Number of Corpora Lutea and Implantation Sites
Dose level | N | Corpora Lutea | Implantation Sites |
Control | 12/12 | 13.00± 2.13 | 11.08± 3.50 |
Low 5mg.kg-1 | 12/9 | 10.67± 1.41 | 10.33± 1.00 |
Medium 25mg.kg-1 | 12/11 | 11.73± 1.56 | 11.36± 1.21 |
High 50mg.kg-1 | 12/12 | 12.58± 2.39 | 11.83± 1.95 |
N-Number of females, the results are presented as mean± SD
Table 11. Dusantox 86. Reproduction/Developmental Toxicity Screening Test (OECD 421) – Relative Weight of Organs, Males
Dose level | N | Kidneys (g) | Testis (g) | Epididymis (g) | |||
Right | Left | Right | Left | Right | Left | ||
Control | 12/12 | 0.36± 0.03 | 0.36± 0.02 | 0.46± 0.05 | 0.48± 0.07 | 0.089± 0.013 | 0.090± 0.010 |
Low 5mg.kg-1 | 12/12 | 0.36± 0.03 | 0.35± 0.03 | 0.48± 0.04 | 0.47± 0.04 | 0.086± 0.011 | 0.089± 0.012 |
Medium 25mg.kg-1 | 12/12 | 0.36± 0.03 | 0.35± 0.03 | 0.46± 0.06 | 0.47± 0.06 | 0.088± 0.011 | 0.085± 0.010 |
High 50mg.kg-1 | 12/12 | 0.38± 0.04 | 0.37± 0.04 | 0.49± 0.05 | 0.51± 0.07 | 0.094± 0.011 | 0.097± 0.011 |
Table 12. Dusantox 86. Reproduction/Developmental Toxicity Screening Test (OECD 421) – Relative Weight of Organs, Females
Dose level | N | Kidneys (g) | Ovaries (g) | Uterus (g) | ||
Right | Left | Right | Left | |||
Control | 12/12 | 0.037± 0.03 | 0.37± 0.02 | 0.38± 0.06 | 0.36± 0.07 | 0.29± 0.09 |
Low 5mg.kg-1 | 12/12 | 0.37± 0.04 | 0.35± 0.04 | 0.40± 0.08 | 0.41± 0.10 | 0.27± 0.04 |
Medium 25mg.kg-1 | 12/12 | 0.37± 0.03 | 0.36± 0.03 | 0.38± 0.11 | 0.38± 0.09 | 0.29± 0.07 |
High 50mg.kg-1 | 12/12 | 0.38± 0.04 | 0.36± 0.04 | 0.34± 0.03 | 0.37± 0.05 | 0.27± 0.05 |
N-Number of animals, the results are presented as mean± SD
Table 13. Dusantox 86. Reproduction/Developmental Toxicity Screening Test (OECD 421) – Percentage Evaluation of Results in Females
Dose level | N | Sperm-positive (%) | Pregnant (%) | With Live Pups (%) |
Control | 12/12 | 100 | 100 | 83.33 |
Low 5 mg.kg-1 | 12/9 | 75 | 75 | 66.67 |
Medium 25mg.kg-1 | 12/11 | 91.67 | 91.67 | 83.33 |
High 50mg.kg-1 | 12/12 | 100 | 100 | 100 |
N-Number of females
Table 14. Dusantox 86. Reproduction/Developmental Toxicity Screening Test (OECD 421) – F1 Generation (Offspring)
Dose level | Number of Pups | Live Pups (%) | Death Pups (%) | |||
Total | Mean/1 Female | Live | Death | |||
Control | 109 | 9 | 102 | 7 | 93.58 | 6.42 |
Low 5mg.kg-1 | 82 | 9 | 78 | 4 | 95.12 | 4.88 |
Medium 25mg.kg-1 | 90 | 8 | 79 | 11 | 87.78 | 12.22 |
High 50mg.kg-1 | 113 | 9 | 112 | 1 | 99.12 | 0.89 |
Table 15. Dusantox 86. Reproduction/Developmental Toxicity Screening Test (OECD 421) – Indices of Reproductive Parameters (%)
Dose level | Fertility | Gestation | Live Birth | Viability |
Control | 100 | 83.33 | 93.58 | 98.80 |
Low 5mg.kg-1 | 75 | 88.89 | 95.12 | 100 |
Medium 25mg.kg-1 | 91.67 | 90.91 | 87.78 | 97.47 |
High 50mg.kg-1 | 100 | 100 | 99.12 | 98.21 |
Table 16. Dusantox 86. Reproduction/Developmental Toxicity Screening Test (OECD 421) – Sex Distribution of Pups
Dose level | Total Number | Males | % | Females | % |
Control | 100 | 53 | 53.00 | 47 | 47.00 |
Low 5mg.kg-1 | 78 | 37 | 47.44 | 41 | 52.56 |
Medium 25mg.kg-1 | 77 | 36 | 46.75 | 41 | 53.25 |
High 50 mg.kg-1 | 110 | 49 | 49.00 | 61 | 61.00 |
Sex distribution – 4thday post-partum
Effect on fertility: via oral route
- Endpoint conclusion:
- no adverse effect observed
- Dose descriptor:
- NOAEL
- 50 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
The results of the reproduction/developmental screening study indicated that per os application of the test article Dusantox 86 didn’t show any significant changes in all used doses in comparison with the control animals:
in clinical observation, bodyweight of animals, food consumption, length of pregnancy, number of pups and weight of litter, number of corpora lutea and implantation sites, relative weight of organs or macroscopical and histological changes.
Effects on developmental toxicity
Description of key information
Based on the results obtained in the study, the NOAEL (No Observed Adverse Effect Level) for maternal and developmental toxicity can be set at 1000 mg/kg/day.
Link to relevant study records
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 12 February 2021 to 15 April 2021
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 414 (Prenatal Developmental Toxicity Study)
- Version / remarks:
- OECD guideline no. 414 “Prenatal Developmental Toxicity Study” of 25 June 2018.
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- yes
- Specific details on test material used for the study:
- No further details specified in the study report.
- Species:
- rat
- Strain:
- Sprague-Dawley
- Details on test animals or test system and environmental conditions:
- Animal supply and acclimatisation
A total of 116 Sprague Dawley SD virgin female rats, 9 weeks old (200-225 g) were ordered from Charles River Italia S.p.A., Calco (Lecco), Italy. The male rats used were from the same supplier, and were at least 11 weeks old (at least 350 g). After arrival the weight range was determined and the females were uniquely identified by tattoo on the hind feet. A health check was then performed by a veterinarian. An acclimatisation period of approximately 2 weeks was allowed before the start of treatment, during which time the health status of the animals was assessed by thorough observations.
Animal husbandry
The rats were housed in a limited access rodent facility. Animal room controls were set to maintain temperature and relative humidity at 22°C ± 2°C and 55% ± 15% respectively; actual conditions were monitored, recorded and the records retained. No relevant deviations from these ranges were recorded during the study. There were approximately 15-20 air changes per hour and the rooms were lit by artificial light for 12 hours each day.
Before mating for all animals and after mating for males, the animals were housed no more than 5 of one sex to a cage in clear polysulfone cages measuring 59.5×38×20 cm (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese). During the mating period, one male rat was housed with one female rat in clear polysulfone cages measuring 42.5×26.6×18 cm (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese) with a stainless steel mesh lid and floor. Each cage tray held absorbent material which was inspected and changed daily. After mating, the mated females were housed in clear polysulfone cages measuring 42.5×26.6×18 cm (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese).
Nesting material was provided inside suitable bedding bags. In addition, suitable nesting material (Scobis 0Mucedola) was provided as necessary. Nesting material was changed at least 2 times a week.
Drinking water was supplied ad libitum to each cage via water bottles.
A commercially available laboratory rodent diet (4 RF 21,Mucedola S.r.l., Via G. Galilei 4, 20019 SettimoMilanese (MI), Italy) was offered ad libitum throughout the study.
There was no information available to indicate that any non-nutrient substance likely to influence the effect of the test item was present in the drinking water or the diet. Records of analyses of water and diet are kept on file at ERBC.
Dated and signed records of activities relating to the day to day running and maintenance of the study in the animal house were recorded.
Allocation to groups
On the day of allocation (Day 0 post coitum) all females were weighed and allocated to the groups by computerised stratified randomisation to give approximately equal initial group mean body weights. Each female was identified within the study by ear notch and housed no more than 5 per cage.
The cages were identified by a label recording the study number, animal numbers and details of treatment. The arrangement of cages in batteries was such that cages from each treatment group were evenly distributed across the battery to minimise possible environmental effects. - Route of administration:
- oral: gavage
- Vehicle:
- CMC (carboxymethyl cellulose)
- Remarks:
- 0.5%
- Details on exposure:
- The required amount of the test item was suspended in the vehicle to reach the required concentrations of 5, 25 and 100 mg/mL. The formulation was prepared up to weekly interval based on the stability data measurements. Concentrations were calculated and expressed in terms of test item as supplied.
- Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Analysis was performed in a separate study in order to validate the analytical method and the formulation procedure and to verify the stability of the formulations (ERBC Study no. A4101). A 28 hour stability at room temperature and a 8 day stability at +4°C were verified in the range from 5 to 100 mg/mL.
Samples of the formulations prepared during the current study (the first and the last week of treatment) were analysed to check the homogeneity and concentration. The results were within the acceptability limits stated in ERBC protocols for suspensions. - Details on mating procedure:
- The females were paired with male rats. Females were paired one to one in the home cage of the male and left overnight. Vaginal smears were taken daily in themorning from the day after pairing until a positive identification of mating was made. The day of mating, as judged by the presence of spermin the vaginal smear or by the presence of a copulation plug, was considered as Day 0 of gestation (or Day 0 post coitum). Full mating records were maintained.
On the day of allocation (Day 0 post coitum) all females were weighed and allocated to the groups by computerised stratified randomisation to give approximately equal initial group mean body weights. Each female was identified within the study by ear notch and housed no more than 5 per cage.
The cages were identified by a label recording the study number, animal numbers and details of treatment. The arrangement of cages in batteries was such that cages from each treatment group were evenly distributed across the battery to minimise possible environmental effects. - Duration of treatment / exposure:
- All animals were dosed from Day 3 through Day 19 post coitum.
- Frequency of treatment:
- All animals were dosed once a day.
- Duration of test:
- 20 days
- Dose / conc.:
- 0 mg/kg bw/day (nominal)
- Remarks:
- Control Level
- Dose / conc.:
- 50 mg/kg bw/day (nominal)
- Remarks:
- Low Level
- Dose / conc.:
- 250 mg/kg bw/day (nominal)
- Remarks:
- Medium Level
- Dose / conc.:
- 1 000 mg/kg bw/day (nominal)
- Remarks:
- High Level
- No. of animals per sex per dose:
- Each group comprised 24 mated female rats.
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- Choice of the species, administration route and dose levels
The Sprague Dawley SD rat was the species and strain of choice because it is accepted by many regulatory authorities and there are ample experience and background data on this species and strain.
The oral route was selected as it is a possible route of exposure of the test item in man.
Dose levels were selected by the Sponsor.
Administration of test item
The test item was administered orally by gavage at a dose volume of 10 mL/kg body weight.
Control animals received the vehicle alone at the same dose volume. The dose was administered to each animal on the basis of the most recently recorded body weight and the volume administered was recorded for each animal. - Maternal examinations:
- Mortality
Throughout the study, all animals were checked early in each working day and again in the afternoon. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day.
Clinical signs
All clinical signs were recorded for individual animals. Each animal was observed daily and any clinical signs recorded starting from allocation until sacrifice.
Body weight
All animals were weighed on Days 0, 3, 6, 9, 12, 15, 18 and 20 post coitum.
Food consumption
Food consumption was measured on Days 3, 6, 9, 12, 15, 18 and 20 post coitum starting from Day 0 post coitum.
Terminal studies
Euthanasia
All animals at termination were killed by carbon dioxide inhalation on Day 20 post coitum and necropsied as detailed below. All foetuses were sacrificed by intraperitoneal injection of Sodium Thiopental followed by hypothermia.
Necropsy
The clinical history of the animal was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices). Changes were noted and the abnormalities preserved in 10% neutral buffered formalin.
Organ weight, fixation and preservation
From all females completing the scheduled test period, the thyroid and the brain were weighed, fixed and preserved in 10% neutral buffered formalin. The thyroid weight was determined after fixation. The ratio of thyroid weight to brain weight was calculated for each animal.
Thyroid histopathological examination
After dehydration and embedding in paraffin wax, sections of the tissue were cut at 5 micrometre thickness and stained with haematoxylin and eosin. Sections were examined for evaluation of pathological changes. - Ovaries and uterine content:
- The ovaries and uteri were examined to determine:
– Gravid uterine weight (not obtained from animals found dead or killed during the study);
– number of corpora lutea;
– number of implantation sites;
– number, sex and weight of all live foetuses;
– number and sex of dead foetuses (foetuses at termwithout spontaneous movements and breathing);
– number of intra-uterine deaths;
– gross evaluation of placentae.
Intra-uterine deaths were classified as:
– early resorptions: only placental remnants visible.
– late resorptions: placental and foetal remnants visible.
Uteri or individual uterine horns without visible implantations were immersed in a 20% solution of ammonium sulphide to reveal evidence of embryonic death at very early stages of implantation. - Blood sampling:
- On Day 20 post coitum, blood samples for thyroid hormones determination (approximately 1 mL), were collected, randomizing (equalised) between treatment groups, from the sublingual vein of all females, under slight isoflurane anaesthesia. This procedure was performed within a short time frame (e.g. two hours, if possible) on the morning of the day of necropsy.
Samples were transferred into tubes containing no anticoagulant and centrifuged at room temperature. The serum obtained was divided in two aliquots (300 µL in the aliquot A, the remaining in the aliquot B, if possible) and stored at -20°C, pending analysis.
Immunoanalysis was carried out by the Test Site, BioVetim (Study Phase number: BIOVX1610),
according to immunoanalytical methods validated at the Test Site:
– RV-T3/R/S-BKM/RIA-002 for T3
– RV-T4/R/S-BKM/RIA-002 for T4
– RV-TSH/R/S-IZO/RIA-002 for TSH
Samples were assayed to determine the serum levels of Total triiodothyronine (total T3), Total thyroxine (total T4) and Thyroid stimulating hormone (TSH) by RadioImmunoAssay (RIA).
Total number of samples: 96 × 2 aliquots
The aliquots were maintained at -20°C until transfer to the Test Site indicated below, where the immunoanalysis was carried out:
BIOVETIM
VetAgro Sup, aile 1 niveau 3
1 avenue Bourgelat
69280Marcy l’Etoile
France
Prior to shipment, the Principal Investigator was notified by e-mail.
Samples were labelled as follows: study number, animal number, group and sex, sampling date. Samples were transported in dry ice, by a dedicated courier and were subject to the latter’s standard insurance policy. In the first instance, only the aliquots A were despatched to the Test Site. The backup samples (aliquots B) will disposed of after Study Director’s authorisation and following Sponsor’s approval.
A Final Delegated Phase Report with the results of these analyses was sent to ERBC for inclusion in the Final Study Report. The results were presented as individual data (mean of duplicates, where applicable), group mean and group standard deviation. Evaluation of the data and the statistical analysis was performed in ERBC. - Fetal examinations:
- Examination of foetuses
All live foetuses were examined externally. Approximately one-half of the foetuses (i.e., routinely, every second live foetus) in each litter was preserved in Bouin’s solution for subsequent fixed-visceral examination. The remaining foetuses were eviscerated after which the carcasses were fixed in 95% (v/v) ethanol for subsequent skeletal examination.
Skeletal and fixed-visceral examinationswere performed in all groups. Structural deviations were classified as follows:
Malformations
Major abnormalities that are rare and/or affect the survival or health of the species under investigation.
Anomalies
Minor abnormalities that are detected relatively frequently.
Variants
A change that occurs within the normal population under investigation and is unlikely to adversely affect survival or health. This might include a delay in growth or morphogenesis that would have otherwise followed a normal pattern of development.
Anogenital distance (AGD):
The AGD of each live foetuses was measured on Day 20 post coitum. The AGD was normalized to the cube root of foetal body weight collected on Day 20 post coitum. Individual and mean data was reported. - Statistics:
- For continuous variables the significance of the differences amongst group means was
assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data.
Statistical analysis of non-continuous variables was carried out by means of the Kruskal-
Wallis test and intergroup differences between the control and treated groups assessed by a
non-parametric version of theWilliams test. The criterion for statistical significance was
p<0.05. The mean values, standard deviations and statistical analysis were calculated from
the actual values in the computer without rounding off. - Indices:
- Pre-implantation loss was calculated as a percentage from the formula:
Pre impl. Loss % = [(no. of corpora lutea – no. of implantations) / no. of corpora lutea] x 100
Post-implantation loss was calculated as a percentage from the formula:
Post impl. Loss% = [(no. of implantations – no. of foetuses) / no. of implantations] x 100
Total implantation loss was calculated as a percentage from the formula:
Total impl. Loss % = [(no. of corpora lutea – no. of live foetuses) / no. of corpora lutea] x 100
Sex ratios of the foetuses were calculated as the percentage of males.
All derived values (e.g., means, percentages, ratios) first were calculated within the litter and the group values derived as a mean of individual litter values. Foetal structural deviations were expressed as the percentage of affected foetuses relative to all foetuses examined per group, as well as in terms of the mean litter percentage of affected litters. - Historical control data:
- No specified in the study report
- Clinical signs:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No treatment-related clinical signs were detected throughout the study. A palpable subcutaneous mass was recorded in one lowdose female (no. 73) during the last day of study. This formation did not show microscopically changes. Other signs like hairloss or scabs were occasionally recorded during the study in single animals, with no correlation with the dose.
Thus they were considered as minor clinical signs and not related to the administration with the test item. - Dermal irritation (if dermal study):
- not examined
- Mortality:
- no mortality observed
- Description (incidence):
- All females survived to the final sacrifice on gestation Day 20 and were found pregnant with the exception of two controls (no. 31, 33) and one low dose female (no. 81).
The final number of females with live foetuses on gestation day 20 was of:
– 22 in the control group (0 mg/kg/day)
– 23 in the low dose group (50 mg/kg/day)
– 24 in the mid-dose group (250 mg/kg/day)
– 24 in the high dose group (1000 mg/kg/day) - Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- No treatment-related effects were seen in maternal body weight/body weight gain throughout the study.
- Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- No treatment-related effects were seen in food consumption throughout the study.
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- not examined
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Immunological findings:
- no effects observed
- Description (incidence and severity):
- Serum levels of T3, T4 and TSH did not differ between treated and control groups.
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Description (incidence and severity):
- Body weight at termination, uterus weight and the absolute weight gain of females were unaffected by treatment.
No effects were seen in thyroid weight between treated and control groups. - Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- At post mortem examination, no treatment-related macroscopic observations were noted in treated females receiving test item at ≥ 50 mg/kg/day, when compared to controls. Any macroscopic observations were within the range of expected spontaneous findings in SD rats of the same age considered incidental and unrelated to the test item.
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- no effects observed
- Description (incidence and severity):
- No treatment-related changes were noted in thyroid gland of females receiving 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline at ≥ 50 mg/kg/day, when compared to controls. The microscopic observations in control and treated females were consistent with those known to occur spontaneously in untreated SD rats of the same age and were considered incidental and unrelated to treatment.
- Histopathological findings: neoplastic:
- not examined
- Number of abortions:
- not specified
- Pre- and post-implantation loss:
- no effects observed
- Description (incidence and severity):
- There were no differences in the number of implantations, corpora lutea and the intrauterine
deaths between control and treated groups. - Total litter losses by resorption:
- not examined
- Early or late resorptions:
- not examined
- Dead fetuses:
- no effects observed
- Description (incidence and severity):
- The number of live foetuses, the mean litter and body weights and the percentage of males respect to females did not differ between control and treated groups.
- Changes in pregnancy duration:
- not examined
- Changes in number of pregnant:
- no effects observed
- Description (incidence and severity):
- The number of live foetuses, the mean litter and body weights and the percentage of males respect to females did not differ between control and treated groups.
- Details on maternal toxic effects:
- Treatment with the test item did not cause changes in body weight or food consumption at all dose levels when compared to controls throughout the study. In addition, the animals did not showtreatment-related clinical signs. The serumlevels of TSH, T3 and T4 hormones were comparable between control and treated groups. No treatment-related changes were noted at necropsy of females or at thyroid weight. The microscopic analysis of thyroid did not reveal findings at any dose level.
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 1 000 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Basis for effect level:
- body weight and weight gain
- changes in number of pregnant
- clinical signs
- dead fetuses
- food consumption and compound intake
- gross pathology
- histopathology: neoplastic
- maternal abnormalities
- mortality
- necropsy findings
- organ weights and organ / body weight ratios
- pre and post implantation loss
- Fetal body weight changes:
- no effects observed
- Description (incidence and severity):
- The body weights did not differ between control and treated groups.
- Reduction in number of live offspring:
- no effects observed
- Description (incidence and severity):
- The number of live foetuses did not differ between control and treated groups.
- Changes in sex ratio:
- no effects observed
- Description (incidence and severity):
- The the percentage of males respect to females did not differ between control and treated groups.
- Changes in litter size and weights:
- no effects observed
- Description (incidence and severity):
- The mean litter and body weights did not differ between control and treated groups.
- Changes in postnatal survival:
- not specified
- External malformations:
- no effects observed
- Description (incidence and severity):
- No treatment-related findings were recorded at external examination of foetuses. One single small foetus (body weight below 2.7g) was found in Group 3 as abnormality.
- Skeletal malformations:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Minor abnormalities or variations occurred in all groups and included for example altered ossification (asymmetrical, incomplete or no ossification) of several bones of the skull, sternebrae, thoracic/sacral vertebrae and the presence supernumerary ribs (14th). The incidence of the affected litters in treated groups was similar or even lower than observed in the control group or without dose relation. Therefore, the findings were considered unrelated to treatment.
- Visceral malformations:
- effects observed, non-treatment-related
- Description (incidence and severity):
- A total of three major abnormalities were found in the study. Two were related to the heart (incomplete septum and/or persistent truncus arteriosus) and observed in one control foetus (foetus no. 10; Dam no. 9) and in one foetus of group 3 (foetus 10; Dam no. 125).
The other major abnormality was related to brain (extreme enlarged lateral ventricles) and it was found in one foetus of group 3 (Dam no. 139). These malformations occurred in single instances and in the absence of a dose dependency. Therefore, they were considered incidental and unrelated to treatment.
The incidences of foetuses or litters with minor abnormalities or variations did not suggest any test item effect.
The overall data confirm that there were no treatment-related findings at fixed visceral examination of foetuses. - Details on embryotoxic / teratogenic effects:
- The number of corpora lutea, implantations, viable foetuses, intrauterine deaths and sex ratios were comparable between control and treated groups. No treatment-related effects were seen in the mean foetal weight. The external examination of foetuses including the measurement of the anogenital distance did not show differences in treated animals at all dose levels when compared to the control group.
No treatment-related findings were noted at skeletal and visceral examinations. Indeed, no evidence of teratogenicity was noted up to and including the dose level of 1000 mg/kg/day. - Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 1 000 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- changes in sex ratio
- fetal/pup body weight changes
- changes in litter size and weights
- external malformations
- skeletal malformations
- visceral malformations
- Key result
- Developmental effects observed:
- no
- Lowest effective dose / conc.:
- 1 000 mg/kg bw/day (nominal)
- Treatment related:
- no
- Conclusions:
- Based on the results obtained in the study, the NOAEL (No Observed Adverse Effect Level) for maternal and developmental toxicity can be set at 1000 mg/kg/day.
- Executive summary:
Study design
The effects of 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline were investigated in female Sprague Dawley SD rats during pregnancy and embryo-foetal development according to OECD guideline no. 414 “Prenatal Developmental Toxicity Study”. Females were in-house mated with sexually mature males of the same strain and then assigned to 4 groups of 24 females each as indicated below:
Group Number
Treatment
(mg/kg/day)+
Level
Female Numbers
(odd only)
1
2
3
4
0
50
250
1000
Control
Low
Medium
High
1-47
49-95
97-143
145-191
+: in terms of test item supplied
The test item was suspended in 0.5 % methyl cellulose (0.5% MC) and administered by oral gavage at 50, 250 and 1000 mg/kg/day with the dose volume set at 10 mL/kg body weight.
The animals were treated throughout the gestation period from Day 3 up to Day 19 post coitum. Control females received the vehicle (0.5 % MC) at the same dose volume during the same treatment period.
Parameters assessed included clinical observations, body weight and food consumption during the in-life phase. Blood collection for hormones determination (T3, T4 and TSH) was performed on Day 20 post coitum before dispatch to necropsy.
All females were caesarean-sectioned on Day 20 post coitum and subjected to post mortem examination along with thyroid and brain weights. The ovaries were examined and the corpora lutea counted. Gravid uterus was weighed and the uterine content examined for the number of implantations, intrauterine deaths and live foetuses.
Live foetuses were weighed, sexed and observed for external abnormalities. In addition, the anogenital distance (AGD) was recorded in all live foetuses.
Approximately one half of the foetuses in each litter was examined for fixed-visceral and skeletal abnormalities.
Histopathology of thyroid was performed in all groups.
Fate of females
All females survived to the final sacrifice on gestation Day 20 and were found pregnant with the exception of two controls and one low dose female.
The final number of females with live foetuses on gestation Day 20 was as follows:
– 22 in the control group (0 mg/kg/day)
– 23 in the low dose group (50 mg/kg/day)
– 24 in the mid-dose group (250 mg/kg/day)
– 24 in the high dose group (1000 mg/kg/day)
Clinical signs
No treatment-related findings were recorded.
Maternal body weight and body weight gain
No treatment-related effects were seen in maternal body weight/body weight gain throughout the study.
Food consumption
No treatment-related effects were seen in food consumption throughout the study.
Thyroid hormone determination
Serum levels of T3, T4 and TSH did not differ between treated and control groups.
Terminal body weight, uterus weight and absolute weight gain
Body weight at termination, uterus weight and the absolute weight gain of females were unaffected by treatment.
Organ weight
No effects were seen in thyroid weight between treated and control groups.
Litter data and sex ratios
Litter data and sex ratios were unaffected by treatment.
Anogenital distance
No differences were seen in the anogenital distance between treated and control groups both in male and females foetuses.
Macroscopic and microscopic examinations
At post mortem examination, no treatment-related macroscopic observations were noted in treated females receiving test item at ≥ 50 mg/kg/day, when compared to controls. Any macroscopic observations were within the range of expected spontaneous findings in SD rats of the same age considered incidental and unrelated to the test item.
External examinations of foetuses
No treatment-related effects were seen at external examination of foetuses.
Skeletal examinations of foetuses
No treatment-related effects were seen at external examination of foetuses.
Visceral examination of foetuses
No treatment-related effects were seen at external examination of foetuses.
Conclusion
Based on the results obtained in the study, the NOAEL (No Observed Adverse Effect Level) for maternal and developmental toxicity can be set at 1000 mg/kg/day.
Reference
SUMMARY OF REPRODUCTION DATA – GROUP DATA
| Dosage Levels mg/kg/day | |||
Observations | 0 | 50 | 250 | 1000 |
No. Dams Inseminated No. Dams That Conceived Percent Dams Conceived No. Dams Died During Study No. Dams with Resorptions only No. of Litters Mean Absolute Weight Gain Total No. Corpora Lutea Mean No. Corpora Lutea/Pregnancy Total No. Implantation Mean No. Implants/Pregnancy Total No. Live Fetuses Mean No. Live Fetuses/Pregnancy Total No. Dead Fetuses Mean No. Dead Fetuses/Pregnancy No. Dams with Resorptions Total No. Resorptions Mean No. Resorptions/Pregnancy Preimplantation Loss (%) Post Implantation Loss (%) Mean Live Foetal Weight (g) | 24 22 91.7 0 0 22 66.9 326 14.8 320 14.5 317 14.4 0 0.0 3 3 0.1 2.1 1.0 3.97 | 24 23 95.8 0 0 23 63.6 345 15.0 339 14.7 333 14.5 0 0.0 5 6 0.3 1.7 1.8 3.96 | 24 24 100.0 0 0 24 80.5 349 14.5 347 14.5 336 14.0 0 0.0 4 11 0.5 0.8 3.2 4.03 | 24 24 100.0 0 0 24 63.2 368 15.3 362 15.1 357 14.9 0 0.0 4 5 0.2 1.4 1.3 3.98 |
CLINICAL SIGNS OF FEMALES – GROUP INCIDENCE
Interval: 0 – 20 Days Group Observation | 1 (24) | 2 (24) | 3 (24) | 4 (24) | ||||
| A | B | A | B | A | B | A | B |
APPERANCE Scab(s) Presence if palpable mass |
2 0 |
8.3 0.0 |
1 1 |
4.2 4.2 |
0 0 |
0.0 0.0 |
0 0 |
0.0 0.0 |
Key:
() = Number of animals alive at start of interval
A = Number of animals affected
B = Percent of animals with observations during interval
BODY WEIGHT (g) OF PREGNANT FEMALES – GROUP DATA
Group(s) |
| Day of Phase | |||||||
0! | 3” | 6 | 9 | 12 | 15 | 18 | 20 | ||
1 | (n) Mean SD | 22 274.20 19.12 | 22 291.48 19.07 | 22 301.78 20.33 | 22 317.60 21.07 | 22 336.15 22.98 | 22 355.70 23.20 | 22 399.69 28.91 | 22 436.66 31.84 |
2 | (n) Mean SD | 23 272.39 12.58 | 23 285.38 12.44 | 23 300.41 13.42 | 23 312.48 13.97 | 23 331.93 14.84 | 23 351.01 17.37 | 23 392.40 20.65 | 23 430.58 24.53 |
3 | (n) Mean SD | 24 272.90 16.51 | 24. 294.75 18.34 | 24 308.83 17.96 | 24 323.23 18.55 | 24 342.84 19.29 | 24 363.27 22.68 | 24 409.54 26.36 | 24 448.51 30.88 |
4 | (n) Mean SD | 24 273.76 12.25 | 24 292.71 15.88 | 24 303.76 14.66 | 24 316.81 14.18 | 24 334.43 17.86 | 24 355.05 18.86 | 24 397.54 24.80 | 24 434.91 30.01 |
Note: ! = Gestation phase; “ = Dosing/Gestation phase
* = mean value of group is significantly different from control at p < 0.05
** = mean value of group is statistically different from control at p < 0.01
Statistical analysis: Dunnett’s test if group variances are homogenous; Modified t test if group variances are inhomogenous ($)
BODY WEIGHT GAIN PER DAY° (g) OF PREGNANT FEMALES – GROUP MEAN DATA
Group(s) |
| Day of Phase | ||||||
3 | 6 | 9 | 12 | 15 | 18 | 20 | ||
1 | (n) Mean SD | 22 5.762 2.126 | 22 3.431 2.144 | 22 5.274 2.985 | 22 6.184 1.561 | 22 6.518 1.651 | 22 14.662 2.579 | 22 18.485 2.957 |
2 | (n) Mean SD | 23 4.333 2.875 | 23 5.008 2.997 | 23 4.024 1.611 | 23 6.484 1.554 | 23 6.359 1.548 | 23 13.798 2.233 | 23 19.091 3.190 |
3 | (n) Mean SD | 24 7.284 1.749 | 24 4.6914 2.049 | 24 4.801 1.795 | 24 6.539 1.735 | 24 6.810 2.109 | 24 15.422 2.629 | 24 19.485 3.184 |
4 | (n) Mean SD | 24 6.319 2.705 | 24 3.683 1.848 | 24 4.350 1.814 | 24 5.871 2.015 | 24 6.875 1.897 | 24 14.162 2.729 | 24 18.685 3.493 |
Note: Data for Dosing/Gestation phase
* = mean value of group is significantly different from control at p < 0.05
** = mean value of group is statistically different from control at p < 0.01
Statistical analysis: Dunnett’s test if group variances are homogenous; Modified t test if group variances are inhomogenous ($)
°= mean daily body weight gain over the previous period starting from gestation day 0
FOOD CONSUMPTION° (g/animal/day) OF FEMALES – GROUP MEAN CAGE DATA
Group(s) |
| Day of Phase | ||||||
3 | 6 | 9 | 12 | 15 | 18 | 20 | ||
1 | (n) Mean SD | 9 22.47 2.50 | 9 25.08 1.70 | 9 26.59 1.99 | 9 26.85 1.89 | 9 27.61 2.53 | 9 29.97 3.20 | 9 30.58 2.99 |
2 | (n) Mean SD | 9 20.57 4.56 | 9 25.43 1.92 | 9 26.39 1.64 | 9 28.04 2.14 | 9 27.84 3.29 | 9 30.09 2.49 | 9 30.54 3.98 |
3 | (n) Mean SD | 9 23.96 1.37 | 9 25.99 1.70 | 9 27.30 1.28 | 9 27.94 1.38 | 9 29.05 1.79 | 9 31.70 2.06 | 9 32.29 2.58 |
4 | (n) Mean SD | 9 23.66 1.19 | 9 25.28 1.33 | 9 26.54 1.65 | 9 27.28 2.56 | 9 28.30 1.89 | 9 30.65 2.80 | 9 31.29 2.03 |
Note: Data for Dosing/Gestation phase
* = mean value of group is significantly different from control at p < 0.05
** = mean value of group is statistically different from control at p < 0.01
Statistical analysis: Dunnett’s test if group variances are homogenous; Modified t test if group variances are inhomogenous ($)
°= food consumed over the previous period starting from Day 0 post coitum
TERMINAL BODY WEIGHT, GRAVID UTERUS WEIGHT AND ABSOLUTE WEIGHT GAIN (g) OF PREGNANT FEMALES – GROUP MEAN DATA
Group |
| Terminal body weight (g) | Gravid uterus weight (g) | Absolute weight gain# (g) |
1 | Mean SD N | 427.77 32.18 22 | 86.72 15.79 22 | 66.85 18.32 22 |
2 | Mean SD N | 423.44 25.60 23 | 87.50 13.16 23 | 63.56 24.12 23 |
3 | Mean SD N | 439.63 30.86 24 | 86.28 20.79 24 | 80.45* 16.53 24 |
4 | Mean SD N | 426.77 30.71 24 | 89.84 17.13 24 | 63.18 19.95 24 |
# = Body weight at necropsy minus gravid uterus weight, minus body weight at Day 0 of pregnancy
* = mean value of group is significantly different from control
Statistical analysis: Kruskall Wallis test; Williams test if group means are different from control at p < 0.05
THYROID HORMONE DETERMINATION ON DAY 20 POST COITUM – GROUP MEAN DATA
Parameter/units | Group | 1 | 2 | 3 | 4 |
Triiodothyronine nmol/L | Mean SD n | 1.310 0.185 24 | 1.298 0.190 24 | 1.276 0.174 24 | 1.287 0.205 24 |
Thyroxine nmol/L | Mean SD | 20.4 4.2 24 | 19.8 4.1 24 | 19.0 1.9 24 | 18.5 1.9 24 |
THYROID STIMULATING HORMONE ng/ml | Mean SD n | 7.93 1.79 24 | 7.49 1.31 24 | 7.58 1.81 24 | 7.12 1.25 24 |
Controls from group(s): 1 Subgroup(s): 1
* = mean value of group is significantly different from control at p < 0.05
+ = mean value of group is significantly different from control at p < 0.01
LITTER DATA AND SEX RATIO OF FEMALES – GROUP MEAN DATA
Group |
| Corpora Lutea | Implan-tations | Uterine Deaths | Viable Young | % males | Implantation loss (%) | Litter Weight (g) | Mean Foetal Weight (g) | ||||||||
By sex | Combined | ||||||||||||||||
Early | Late | Total | Total | M | F | Pre | Post | Total | M | F | |||||||
1 | Mean SD (n) | 14.82 2.40 22 | 14.55 2.67 22 | 0.14 0.35 22 | 0.00 0.00 22 | 0.14 0.35 22 | 14.41 2.70 22 | 7.18 2.28 22 | 7.23 1.93 22 | 49.43 12.29 22 | 2.06 5.38 22 | 0.99 2.59 22 | 3.04 5.60 22 | 57.27 11.74 22 | 4.09 0.21 22 | 3.85 0.23 22 | 3.97 0.21 22 |
2 | Mean SD (n) | 15.00 2.30 23 | 14.74 2.30 23 | 0.22 0.52 23 | 0.04 0.21 23 | 0.26 0.54 23 | 14.48 2.33 23 | 6.87 2.01 23 | 7.61 1.90 23 | 47.26 11.23 23 | 1.67 3.92 23 | 1.76 3.59 23 | 3.41 5.10 23 | 57.30 9.46 23 | 4.05 0.29 23 | 3.89 0.22 23 | 3.96 0.24 23 |
3 | Mean SD (n) | 14.54 2.92 24 | 14.46 3.05 24 | 0.46 1.64 24 | 0.00 0.00 24 | 0.46 1.64 24 | 14.00 3.44 24 | 7.67 2.87 24 | 6.33 2.41 24 | 54.50 13.51 24 | 0.81 2.77 24 | 3.16 10.98 24 | 3.97 11.10 24 | 56.25 14.09 24 | 4.11 0.23 24 | 3.93 0.26 24 | 4.03 0.24 24 |
4 | Mean SD (n) | 15.33 3.13 24 | 15.08 2.95 24 | 0.21 0.51 24 | 0.00 0.00 24 | 0.21 0.51 24 | 14.88 2.91 24 | 7.71 2.61 24 | 7.17 2.51 24 | 51.94 13.65 24 | 1.41 3.32 24 | 1.31 3.21 24 | 2.70 4.48 24 | 58.96 10.91 24 | 4.07 0.21 24 | 3.90 0.20 24 | 3.98 0.19 24 |
* = mean value of group is significantly different from control
Statistical analysis: Kruskall Wallis test; William’s test of group means are different from control at p < 0.05
ANOGENITAL DISTANCE ON DAY 20 POST COITUM – GROUP MEAN DATA
MALES
Parameter/units | Group | 1 | 2 | 3 | 4 |
ANOGENITAL DISTANCE (mm) | Mean SD n | 4.30 0.26 22 | 4.24 0.30 23 | 4.36 0.28 24 | 4.37 0.25 24 |
Foetus weight (g) | Mean SD n | 4.10 0.21 22 | 4.05 0.29 23 | 4.11 0.23 24 | 4.07 0.21 24 |
ANOGENITAL DISTANCE ^ (NORMALISED) mm/g1/3 | Mean SD n | 2.69 0.16 22 | 2.66 0.18 23 | 2.72 0.16 24 | 2.74 0.16 24 |
^ = normalised for the cube root of the body weight collected on Day 20 post coitum (mm/g)
Statistical analysis: Kruskall Wallis test; T test if group mean differences are different from control at p < 0.05
* = mean value of group is significantly different from control
ANOGENITAL DISTANCE ON DAY 20 POST COITUM – GROUP MEAN DATA
FEMALES
Parameter/units | Group | 1 | 2 | 3 | 4 |
ANOGENITAL DISTANCE (mm) | Mean SD n | 2.54 0.53 22 | 2.48 0.60 23 | 2.53 0.55 24 | 2.69 0.50 24 |
Foetus weight (g) | Mean SD n | 3.85 0.23 22 | 3.89 0.22 23 | 3.93 0.26 24 | 3.90 0.20 24 |
ANOGENITAL DISTANCE ^ (NORMALISED) mm/g1/3 | Mean SD n | 1.62 0.35 22 | 1.58 0.38 23 | 1.60 0.34 24 | 1.71 0.32 24 |
^ = normalised for the cube root of the body weight collected on Day 20 post coitum (mm/g)
Statistical analysis: Kruskall Wallis test; T test if group mean differences are different from control at p < 0.05
* = mean value of group is significantly different from control
ABSOLUTE ORGAN WEIGHTS (g) – GROUP MEAN DATA
FEMALES
Organ: Brain | Controls from group: 1 | Data homogenous by Bartlett’s test (Dunnett’s test) | ||
Group Number/group Mean Standard deviation Group diff. at p > 0.05 Group diff. at p < 0.01 | Control 24 1.969 0.105 | 2 24 1.944 0.083 0.065 0.081 | 3 24 1.976 0.104 0.065 0.081 | 4 24 1.967 0.078 0.065 0.081 |
Analysis of variance: F ratio = 0.52 Df = 3/92 F probability = 0.677
Note: a * indicates group mean is significantly different from control at level of significance shown.
Organ: Thyroid | Controls from group: 1 | Data homogenous by Bartlett’s test (Dunnett’s test) | ||
Group Number/group Mean Standard deviation Group diff. at p > 0.05 Group diff. at p < 0.01 | Control 24 0.0241 0.0048 | 2 24 0.0241 0.0056 0.0031 0.0039 | 3 24 0.0222 0.0042 0.0031 0.0039 | 4 24 0.0215 0.0032 0.0031 0.0039 |
Analysis of variance: F ratio = 2.13 Df = 3/92 F probability = 0.100
Note: a * indicates group mean is significantly different from control at level of significance shown.
ORGAN WEIGHTS° TO BRAIN WEIGHT – GROUP MEAN DATA
FEMALES
Organ: Thyroid | Controls from group: 1 | Data homogenous by Bartlett’s test (Modified t test) | ||
Group Number/group Mean Standard deviation Group diff. at p > 0.05 Group diff. at p < 0.01 | Control 24 1.228 0.248 | 2 24 1.241 0.299 0.164 0.223 | 3 24 1.122 0.193 0.133 0.180 | 4 24 1.092 0.165 0.126* 0.171 |
Analysis of variance: F ratio = 2.49 Df = 3/92 F probability = 0.064
Note: a * indicates group mean is significantly different from control at level of significance shown.
° = expressed as % organ to brain weight ratio
MACROSCOPIC OBSERVATIONS OF FEMALES – FINAL SACRIFICE – GROUP INCIDENCE
| -- Females -- | |||
Group: Number in group: | 1 24 | 2 24 | 3 24 | 4 24 |
Skin Subcutaneous mass(es) |
0 |
1 |
0 |
0 |
Uterus Not pregnant |
2 |
1 |
0 |
0 |
Whole animal No abnormalities detected |
22 |
22 |
24 |
24 |
EXTERNAL EXAMINATION OF FOETUSES – GROUP INCIDENCE
Group | Organ | Cat | Observation(s) | No. Foetuses | No. Litters | ||||
Observed | Affected | % | Observed | Affected | % | ||||
1 | Whole foetus |
| No abnormalities detected | 317 | 317 | 100.00 | 22 | 22 | 100.00 |
2 | Whole foetus |
| No abnormalities detected | 333 | 333 | 100.00 | 23 | 23 | 100.00 |
3 | Whole foetus Whole foetus |
AN | No abnormalities detected Small | 336 336 | 335 1 | 99.70 0.30 | 24 24 | 23 1 | 95.83 4.17 |
4 | Whole foetus |
| No abnormalities detected | 357 | 357 | 100.00 | 24 | 24 | 100.00 |
SKELETAL EXAMINATION OF FOETUSES – GROUP INCIDENCE
Group | Organ | Cat | Observation(s) | No. Foetuses | No. Litters | ||||
Obs | Aff | % | Obs | Aff | % | ||||
1 | Forepaw(s) Forepaw(s) Pelvic girdle Ribs Ribs Ribs Sacral vertebrae Skull Skull Skull Skull Skull Skull Skull Skull Sternebrae Sternebrae Sternebrae Sternebrae Sternebrae Sternebrae Sternebrae Thoracic vertebrae Thoracic vertebrae Thoracic vertebrae | AN AN AN AN AN VA AN AN AN AN VA VA VA VA VA AN AN AN VA VA VA VA AN VA VA | Metacarpal(s) no ossification 4th Metacarpal(s) incomplete ossification Pubis incomplete ossification Short Rudimentary Short 14th Arch(es) incomplete ossification General incomplete ossification Hyoid no ossification Temporal incomplete ossification Nasal incomplete ossification Parietal incomplete ossification Supraoccipital incomplete ossification Hyoid incomplete ossification Interparietal incomplete ossification Asymmetrical ossification Fused No ossification 6th Incomplete ossification 6th Incomplete ossification No ossification 5th Incomplete ossification 5th Centrum asymmetrical ossification Centrum incomplete ossification Centrum dumb-bell shaped | 165 165 165 165 165 165 165 165 165 165 165 165 165 165 165 165 165 165 165 165 165 165 165 165 165 | 68 1 1 4 1 6 8 2 17 6 1 2 18 16 49 6 1 28 51 1 38 32 1 19 1 | 41.21 0.61 0.61 2.42 0.61 3.64 4.85 1.21 1.30 3.64 0.61 1.21 10.91 9.70 29.70 3.64 0.61 16.97 30.91 0.61 23.03 19.39 0.61 11.52 0.61 | 22 22 22 22 22 22 22 22 22 22 22 22 22 22 22 22 22 22 22 22 22 22 22 22 22 | 19 1 1 3 1 4 4 1 8 4 1 2 10 11 16 5 1 13 19 1 16 14 1 10 1 | 86.36 4.55 4.55 13.64 4.55 18.18 18.18 4.55 36.36 18.18 4.55 9.09 45.45 50.00 72.73 22.73 4.55 59.09 86.36 4.55 72.73 63.64 4.55 45.45 4.55 |
2 | Forepaw(s) Ribs Ribs Sacral vertebrae Skull Skull Skull Skull Skull Skull Skull Sternebrae Sternebrae Sternebrae Sternebrae Sternebrae Sternebrae Sternebrae Thoracic vertebrae Thoracic vertebrae Thoracic vertebrae Thoracic vertebrae | AN AN VA AN AN AN AN VA VA VA VA AN AN AN VA VA VA VA AN AN VA VA | Metacarpal(s) no ossification 4th Rudimentary Short 14th Arch(es) incomplete ossification Frontal incomplete ossification Hyoid no ossification Temporal incomplete ossification Interparietal incomplete ossification Parietal incomplete ossification Supraoccipital incomplete ossification Hyoid incomplete ossification Asymmetrical ossification No ossification 6th Bipartite 5th No ossification 5th Incomplete ossification 6th Incomplete ossification Incomplete ossification 5th Centrum no ossification Centrum bipartite Centrum incomplete ossification Centrum dumb-bell shaped | 171 171 171 171 171 171 171 171 171 171 171 171 171 171 171 171 171 171 171 171 171 171 | 58 3 3 5 1 15 1 33 1 13 9 11 17 2 43 41 3 53 1 2 15 1 | 33.92 1.75 1.75 2.92 0.58 8.77 0.58 19.30 0.58 7.60 5.26 6.43 9.94 1.17 25.15 23.98 1.75 30.99 0.58 1.17 8.77 0.58 | 23 23 23 23 23 23 23 23 23 23 23 23 23 23 23 23 23 23 23 23 23 23 | 20 3 3 5 1 9 1 16 1 6 6 10 8 1 16 18 2 19 1 2 9 1 | 86.96 13.04 13.04 21.74 4.35 39.13 4.35 69.57 4.35 26.09 26.09 43.48 34.78 4.35 69.57 78.26 8.70 82.61 4.35 8.70 39.13 4.35 |
3 | Forepaw(s) Ribs Ribs Ribs Sacral vertebrae Skull Skull Skull Skull Skull Skull Sternebrae Sternebrae Sternebrae Sternebrae Sternebrae Sternebrae Sternebrae Thoracic vertebrae Thoracic vertebrae | AN AN AN VA AN AN AN VA VA VA VA AN AN AN VA VA VA VA AN VA | Metacarpal(s) no ossification 4th Short Rudimentary Short 14th Arch(es) incomplete ossification Temporal incomplete ossification Hyoid no ossification Hyoid incomplete ossification Interparietal incomplete ossification Parietal incomplete ossification Supraoccipital incomplete ossification No ossification 6th No ossification Asymmetrical ossification No ossification 6th Incomplete ossification 6th Incomplete ossification 5th Incomplete ossification Centrum bipartite Centrum incomplete ossification | 174 174 174 174 174 174 174 174 174 174 174 174 174 174 174 174 174 174 174 174 | 60 1 1 1 6 1 7 7 38 2 9 13 1 9 32 49 46 1 3 21 | 34.48 0.57 0.57 0.57 3.45 0.57 4.02 4.02 21.84 1.15 5.17 7.47 0.57 4.02 18.39 28.16 26.44 0.57 1.72 12.07 | 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 | 20 1 1 1 5 1 4 6 15 2 9 8 1 4 13 16 16 1 3 11 | 83.33 4.17 4.17 4.17 20.83 4.17 16.67 25.00 62.50 8.33 37.50 33.33 4.17 16.67 56.17 66.67 66.67 4.17 12.50 45.83 |
4 | Forepaw(s) Ribs Ribs Sacral vertebrae Skull Skull Skull Skull Skull Skull Sternebrae Sternebrae Sternebrae Sternebrae Sternebrae Sternebrae Sternebrae Thoracic vertebrae Thoracic vertebrae Thoracic vertebrae | AN AN VA AN AN AN VA VA VA VA AN AN AN VA VA VA VA AN VA VA | Metacarpal(s) no ossification 4th Rudimentary Short 14th Arch(es) incomplete ossification Temporal incomplete ossification Hyoid no ossification Interparietal incomplete ossification Hyoid incomplete ossification Supraoccipital incomplete ossification Parietal incomplete ossification Asymmetrical ossification Bipartite 5th No ossification 6th Incomplete ossification Incomplete ossification 5th No ossification 5th Incomplete ossification 6th Centrum bipartite Centrum incomplete ossification Centrum dumb-bell shaped | 183 183 183 183 183 183 183 183 183 183 183 183 183 183 183 183 183 183 183 183 | 43 6 2 3 1 9 43 12 16 3 3 3 23 1 45 31 53 2 19 2 | 23.50 3.28 1.09 1.64 0.55 4.92 23.50 6.56 8.74 1.64 1.64 1.64 12.57 0.55 24.59 16.94 28.96 1.09 10.38 1.09 | 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 | 16 5 2 2 1 7 17 9 11 3 2 3 9 1 18 17 17 2 11 2 | 66.67 20.83 8.33 8.33 4.17 29.17 70.83 37.50 45.83 12.50 8.33 12.50 37.50 4.17 75.00 70.83 70.83 8.33 45.83 8.33 |
FIXED VISCERAL EXAMINATION OF FOETUSES – GROUP INCIDENCE
Group | Organ | Cat | Observation(s) | No. Foetuses | No. Litters | ||||
Obs | Aff | % | Obs | Aff | % | ||||
1 | Abdomen Brain Heart Heart Heart Heart Kidneys Kidneys Testis Ureter | VA AN AN AN MA VA AN VA AN VA | Umbelical vein left Ventricle enlarged moderate Ventricle enlarged Atrium enlarged Septum incomplete Ventricle enlarged Ectopic Pelvic dilation slight Displaced Enlarged slight | 152 152 152 152 152 152 152 152 152 152 | 1 1 1 1 1 2 2 1 10 7 | 0.66 0.66 0.66 0.66 0.66 1.32 1.32 0.66 6.58 4.64 | 22 22 22 22 22 22 22 22 22 22 | 1 1 3 1 1 3 2 1 9 4 | 4.55 4.55 13.64 4.55 4.55 13.64 9.09 4.55 40.91 18.18 |
2 | Abdomen Abdomen Great vessels Heart Kidneys Kidneys Testis Thoracic cavity Ureter | VA VA VA AN AN VA AN VA VA | Umbelical vein left Haemorrhagic Long innominate artery Atrium enlarged Ectopic Pelvic dilation slight Displaced Haemorrhage Enlarged slight | 162 162 162 162 162 162 162 162 162 | 2 2 1 4 4 1 5 2 5 | 1.23 1.23 0.62 2.47 2.47 0.62 3.09 1.23 3.09 | 23 23 23 23 23 23 23 23 23 | 2 2 1 2 3 1 2 2 5 | 8.70 8.70 4.35 8.70 13.04 4.35 8.70 8.70 21.74 |
3 | Abdomen Abdomen Brain Great vessels Heart Heart Heart Heart Kidneys Kidneys Testis Testis Thoracic cavity Ureter Ureter | VA VA MA VA AN MA MA VA AN VA AN AN VA AN VA | Haemorrhagic Umbelical vein left Ventricles enlarged extreme Long innominate artery Atrium enlarged Septum incomplete Persistant truncus areriosus Pericardium Haemorragic Ectopic Pelvic dilation slight Malpositioned Displaced Haemorrhage Kinked moderate Enlarged slight | 162 162 162 162 162 162 162 162 162 162 162 162 162 162 162 | 3 1 1 1 2 1 1 1 4 2 1 5 5 1 4 | 1.85 0.62 0.62 0.62 1.23 0.62 0.62 0.62 2.47 1.23 0.62 3.09 3.09 0.62 2.47 | 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 | 3 1 1 1 2 1 1 1 3 2 1 5 3 1 4 | 12.50 4.17 4.17 4.17 8.33 4.17 4.17 4.17 12.50 8.33 4.17 20.83 12.50 4.17 16.67 |
4 | Abdomen Great vessels Heart Heart Kidneys Kidneys Testis Testis Thoracic cavity Thoracic cavity Ureter Ureter Ureter | VA VA AN VA AN VA AN AN AN VA AN AN VA | Haemorrhagic Long innominate artery Atrium enlarged Pericardium Haemorragic Ectopic Pelvic dilation slight Hypoplasia Displaced Haemorrhage Haemorrhage Enlarged moderate Kinked moderate Enlarge slight | 173 173 173 173 173 173 173 173 173 173 173 173 173 | 7 3 6 2 1 1 1 10 2 1 1 1 3 | 4.05 1.73 3.47 1.16 0.58 0.58 0.58 5.78 1.16 0.58 0.58 0.58 1.73 | 24 24 24 24 24 24 24 24 24 24 24 24 24 | 5 2 4 1 1 1 1 9 2 1 1 1 3 | 20.83 8.33 16.67 47.17 4.17 4.17 4.17 37.50 8.33 4.17 4.17 4.17 12.50 |
Effect on developmental toxicity: via oral route
- Endpoint conclusion:
- no adverse effect observed
- Dose descriptor:
- NOAEL
- 1 000 mg/kg bw/day
- Study duration:
- subacute
- Species:
- rat
- Quality of whole database:
- K1
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
Prenatal Developmental Toxicity Study
Study design
The effects of 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline were investigated in female Sprague Dawley SD rats during pregnancy and embryo-foetal development according to OECD guideline no. 414 “Prenatal Developmental Toxicity Study”. Females were in-house mated with sexually mature males of the same strain and then assigned to 4 groups of 24 females each as indicated below:
Group Number | Treatment (mg/kg/day)+ | Level | Female Numbers (odd only) |
1 2 3 4 | 0 50 250 1000 | Control Low Medium High | 1-47 49-95 97-143 145-191 |
+: in terms of test item supplied
The test item was suspended in 0.5 % methyl cellulose (0.5% MC) and administered by oral gavage at 50, 250 and 1000 mg/kg/day with the dose volume set at 10 mL/kg body weight.
The animals were treated throughout the gestation period from Day 3 up to Day 19 post coitum. Control females received the vehicle (0.5 % MC) at the same dose volume during the same treatment period.
Parameters assessed included clinical observations, body weight and food consumption during the in-life phase. Blood collection for hormones determination (T3, T4 and TSH) was performed on Day 20 post coitum before dispatch to necropsy.
All females were caesarean-sectioned on Day 20 post coitum and subjected to post mortem examination along with thyroid and brain weights. The ovaries were examined and the corpora lutea counted. Gravid uterus was weighed and the uterine content examined for the number of implantations, intrauterine deaths and live foetuses.
Live foetuses were weighed, sexed and observed for external abnormalities. In addition, the anogenital distance (AGD) was recorded in all live foetuses.
Approximately one half of the foetuses in each litter was examined for fixed-visceral and skeletal abnormalities.
Histopathology of thyroid was performed in all groups.
Fate of females
All females survived to the final sacrifice on gestation Day 20 and were found pregnant with the exception of two controls and one low dose female.
The final number of females with live foetuses on gestation Day 20 was as follows:
– 22 in the control group (0 mg/kg/day)
– 23 in the low dose group (50 mg/kg/day)
– 24 in the mid-dose group (250 mg/kg/day)
– 24 in the high dose group (1000 mg/kg/day)
Clinical signs
No treatment-related findings were recorded.
Maternal body weight and body weight gain
No treatment-related effects were seen in maternal body weight/body weight gain throughout the study.
Food consumption
No treatment-related effects were seen in food consumption throughout the study.
Thyroid hormone determination
Serum levels of T3, T4 and TSH did not differ between treated and control groups.
Terminal body weight, uterus weight and absolute weight gain
Body weight at termination, uterus weight and the absolute weight gain of females were unaffected by treatment.
Organ weight
No effects were seen in thyroid weight between treated and control groups.
Litter data and sex ratios
Litter data and sex ratios were unaffected by treatment.
Anogenital distance
No differences were seen in the anogenital distance between treated and control groups both in male and females foetuses.
Macroscopic and microscopic examinations
At post mortem examination, no treatment-related macroscopic observations were noted in treated females receiving test item at ≥ 50 mg/kg/day, when compared to controls. Any macroscopic observations were within the range of expected spontaneous findings in SD rats of the same age considered incidental and unrelated to the test item.
External examinations of foetuses
No treatment-related effects were seen at external examination of foetuses.
Skeletal examinations of foetuses
No treatment-related effects were seen at external examination of foetuses.
Visceral examination of foetuses
No treatment-related effects were seen at external examination of foetuses.
Conclusion
Based on the results obtained in the study, the NOAEL (No Observed Adverse Effect Level) for maternal and developmental toxicity can be set at 1000 mg/kg/day.
Toxicity to reproduction: other studies
Additional information
No additional information
Justification for classification or non-classification
The above studies has been ranked reliability 1 according to the Klimisch et al system. This ranking was deemed appropriate because the study was conducted to GLP and in accordance with an appropriate OECD Guideline. Sufficient dose ranges and numbers are detailed; hence it is appropriate for use based on reliability and animal welfare grounds.
The above results are considered to trigger no classification under the CLP Regulation (EC No 1272/2008)
Additional information
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