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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From Feb. 03, 2003 to Feb. 16, 2005
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Cross-reference
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD Guideline 421
Principles of method if other than guideline:
A study was conducted to identify the potential adverse effects of test material on reproduction including embryo/foetal development in rats, on oral exposure to 0, 15, 150 and 250 mg/kg bw/d test concentartions, throughout maturation, mating, gestation and up to Day 4 post partum.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): TMXDI® (META) (CT -759-02)
- Physical state: Extremely pale yellow liquid; characteristic odor
- Analytical purity: > 98 %
- Lot/batch No.: UC3081102
- Storage condition of test material: Room temperature in the dark
- Stability under test conditions: Test material was stable in vehicle (arachis oil) for at least 14 d at 4 °C in the dark


Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Manston Road, Margate, Kent
- Weight at study initiation: Males: 347-387 g; Females: 212-241 g
- Housing: 5/cage; polypropylene cages with solid floors and stainless steel tops
- Diet: Certified Rodent Diet PMI 5002; ad libitum
- Water: Mains water; ad libitum
- Acclimation period: 7 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2 °C
- Humidity (%): 55 ± 15 %
- Air changes (per hr): 15/h
- Photoperiod (hrs dark / hrs light): 12 h dark/12 h light

IN-LIFE DATES: From: Feb. 03, 2004 To: Mar. 19, 2004

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Preparation of the test material/vehicle formulations were performed weekly. Analysis showed test material to be stable in Arachis oil for at least 14 d at ambient temperature and humidity in the dark.

VEHICLE
- Concentration in vehicle: 0, 3.75, 37.5 and 62.5 % mg/mL
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Samples of each formulation were taken on three occasions throughout the study (representing the start, middle and end of the dosing period) and analysed for achieved concentration
- Results showed that the majority of preparations to be within acceptable limits
Details on mating procedure:
- M/F ratio per cage: One male and one female per cage
- Length of cohabitation: 14 d
- Proof of pregnancy: Vaginal plug and sperm in the vagina referred to as Day 1 of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility
- Further matings after two unsuccessful attempts: No
- After successful mating each pregnant female was caged: Housed individually during the period of gestation and lactation



Duration of treatment / exposure:
- Male and female rats were dosed during maturation (14 d), mating (14 d), gestation and up to Day 4 post partum
- Both male and female animals were dosed for 14 d at their appointed dose levels, prior to pairing

Frequency of treatment:
Once daily
Duration of test:
14 days prior to mating to Lactation Day 5
No. of animals per sex per dose:
Ten
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on a range-finding study
- Rationale for animal assignment: Randomisation procedure based on stratified bw



Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily before and after dosing, 1 and 5 h post-dosing


BODY WEIGHT: Yes
- Time schedule for examinations: Weekly (during maturation and mating period); in mated females on Day 0, 7, 14 and 20 post coitum and Days 1 and 4 post partum


FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule: Weekly (during maturation period); in mated females between Days 1-7, 7-14 and 14-20 post coitum and 1-4 post partum

POST-MORTEM EXAMINATIONS:
SACRIFICE
- Male animals: All adult males and females that died or were killed in extremis
- Maternal animals: On Day 5 post partum all surviving females, including non-fertile animals


GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations


HISTOPATHOLOGY / ORGAN WEIGHTS
Following list of organs were preserved in buffered 10 % formalin (testes and epididymides preserved in Bouin's fluid) and examined at histopathology for the control and high dose levels: coagulating glands, epididymides, prostate, seminal vesicles, testes, pituitary, ovaries, uterus/cervix and vagina


Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes (using the technique proposed by Salewski)
- Number of early resorptions: No
- Number of late resorptions: No
- Other: The uteri of apparently non-pregnant females were also examined.
Fetal examinations:
For each litter the following was recorded:
- Number of pups born
- Number and sex of pups alive recorded daily and reported on Day 1 and 4 post partum
- Clinical condition of pups from birth to Day 4 post partum
- Individual litter weights on Day 1 and 4 post partum
- Macroscopical examinations for internal and external examinations (Alive offspring on Day 5 were killed by intracardiac overdose of sodium pentobarbitone)
Statistics:
The following parameters were analysed statistically, where appropriate using the test methods outlined as follows:
- Adult male bodyweight and female bodyweight during the maturation, gestation and lactation periods, adult male food consumption, female food consumption during maturation, gestation and lactation, litter size, litter weight, individual offspring bodyweight and adult absolute organ weights. Values were analysed to establish homogeneity of group variances using Bartletts test followed by one-way analysis of variance. If the variances were unequal subsequent comparisons between control and treated groups were performed using t-test assuming unequal variances. If variances were equal subsequent comparisons between control and treated groups were performed using Dunnett's Multiple Comparison Method.
- Adult pre-coital intervals, female gestation lengths, offspring reflexological responses, landmarks of physical development, reproductive and viability indices and litter sex ratios, relative organ weights. Individual values were compared using Kruskal-Wallis non-parametric rank sum test. Where significant differences were seen, pairwise comparison of control values against treated group values was performed using Mann-Whitney "U" test.
Indices:
Fertility Indices
For each group the following were calculated:
Mating index (%) = (Number of animals mated)/ (Number of animals paired) X 100
Pregnancy Index (%) = (Number of pregnant females)/ (Number of animals mated) X100
Gestation Length: Number of days of gestation including the day for observation of mating and the start of parturition
Parturition Index (%): (Number of females delivering live pups)/ (Number of pregnant females) X 100
Live Birth and Viability Indices:
Live Birth Index (%) = (Number of pups alive on Day 1)/ (Number of pups born) X 100
Viability Index (%) = (Number of pups alive on Day 4)/ (Number of pups alive on Day 1) X 100
Sex Ratio = (Number of male pups)/ (Number of pups of determined sex) X 100
Offspring Physical Development: Calculated as the day of appearance and completion of physical landmarks of development


Historical control data:
No data

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS):
Mortality:
- Thirteen mortalities occured during the study; one female dosed at 250 mg/kg bw/d had a total litter loss on Day 2 post partum and was subsequently terminated prior to Day 5 post partum.
- Mortalities were seen across all dose groups which suggests that these findings were not a result of systemic toxicity but a consequence of mal-administration of the test material

Clinical signs:
- At 250 mg/kg bw/d: Increased salivation pre dose and up to 1 h post dose for various durations. Isolated incidents of increased salivation 5 h post dose. Diarrhoea, diuresis, pilo-erection, hunched posture and tiptoe gait were seen sporadically.
- At 150 mg/kg bw/d: Increased salivation pre dose and up to 1 h post dose for various durations. Isolated incidents of increased salivation 5 h post dose. Pilo-erection, hunched posture, tiptoe gait were seen sporadically.
- At 15 mg/kg bw/d: Isolated incidents of increased salivation both pre and post dose. Occasional clinical signs including tiptoe gait were observed in isolated individuals during the study.


BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
- Slight reduction in bodyweight gain in males when compared with controls during the maturation phase which resulted in a slightly lower group mean bodyweight
- Group mean food efficiency was also decreased for this period


REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
- No treatment-related effects on gestation length or parturition


REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- No treatment-related effects on fertility or mating performance
- No significant differences in the number of corpora lutea or implantation sites between groups


ORGAN WEIGHTS (PARENTAL ANIMALS)
- No significant treatment-related effects were observed


HISTOPATHOLOGY (PARENTAL ANIMALS)
- No treatment-related changes were observed

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Maternal abnormalities

Key result
Abnormalities:
not specified

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
VIABILITY (OFFSPRING): No treatment related effects on litter size. No significant effects on offspring viability between Day 1 and 4 of lactation

CLINICAL SIGNS (OFFSPRING): No treatment related effects

BODY WEIGHT (OFFSPRING)
- At 250 mg/kg bw/d there was a lower mean pup weight on Day 1 and 4 of lactation compared to controls.
- No treatment related effects on offspring bodyweight at other doses

SEXUAL MATURATION (OFFSPRING)
- No treatment related effects on offspring development

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
Under the test conditions, administration of the test material to adult male and female rats throughout maturation, mating, gestation and early lactation resulted in significant effects on adults at 250 mg/kg bw/d. However, there were no significant effects on reproductive or developmental parameters, so that the NOAEL for these endpoints was considered to be 250 mg/kg bw/day. 
Executive summary:

A repeated dose reproductive/developmental toxicity study was conducted to screen potential adverse effect of test substance on systemic toxicity and reproduction, including embryo/foetal development in the rat according to OECD Guideline 421, in compliance with GLP. The test substance was administered by gavage at concentrations of 0, 15, 150 and 250 mg/kg bw/day to groups of ten rats of either sex for a period of 19 d in males and 40-41 d in females. Following 14 days of dosing, male and female rats were paired within their dose groups to produce litters. On Day 5 post-partum, all surviving animals were killed and examined macroscopically. Parental animals were observed for clinical signs. Bodyweights and food consumption were recorded during the maturation phase which was continued for males after the mating phase. Mated females were weighed on Days 0, 7, 14 and 20 post-coitum and Days 1 and 4 post-partum and food consumptions recorded between Days 1 to 7, 7 to 14 and 14 to 20 post-coitum and 1 to 4 post-partum. The offspring were observed daily for clinical signs. The litter signs and individual pup bodyweights were recorded on Days 1 and 4 post-partum. During the lactation period the offspring were observed for intra-litter onset and duration of landmarks of physical development. On specific days of lactation, reflexological assessment of offspring was performed. Post mortem macroscopic examinations were performed on all adults and offspring including decedents. Histopathology was carried out on reproductive organs from control and high dose group parental animals at termination. The effects observed in adult animals are detailed in the chapter on repeated dose toxicity (Section 5.6.1). The NOAEL for adults was established at 150 mg/kg bw/day. There were no treatment related effects on fertility, mating performance gestation length at any dose level, and no significant histopathological changes were observed for the reproductive organs of adults at termination. There were no treatment related effects upon litter sizes at birth or on subsequent offspring survival throughout lactation. There were no effects on offspring reflexological responses and no effect on the intra-litter sex ratios. At 250 mg/kg bw/day group mean bodyweight of offspring at Day 1 and 4 of lactation were lower than controls. However, study control weight values were generally higher than historical controls; this was due to one control litter with higher than normal mean offspring weights. Under the test conditions, administration of the test material to adult male and female rats throughout maturation, mating, gestation and early lactation resulted in significant effects on adults at 250 mg/kg bw/day. However, there were no significant effects on reproductive or developmental parameters, so that the NOAEL for these endpoints was considered to be 250 mg/kg bw/day (Knox, 2005).