Registration Dossier

Administrative data

Endpoint:
repeated dose toxicity: oral
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:
other: see 'Remark'
Remarks:
The study is valid without restriction for the reproductive/developmental endpoints. However, some of the parameters measured in guideline repeated dose studies (hematologies, clinical chemistries and complete organ histopathology) were not performed. Therefore, a rating of (2) is appropriate for the repeated dose toxicity endpoints.
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
Deviations:
not specified
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
Sex:
male/female
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 oral 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 of the nominal concentrations
- Mean analytical concentrations were 129%, 102% and 110% of nominal 3.75, 37.5 and 62.5 mg/mL solutions, with the exception that the low dose formulation had a concentration above the acceptable limit on one occasion.
- The use of this formulation was not considered to influence the outcome of the study.
Duration of treatment / exposure:
19 d (males), 40-41 d (females)
Frequency of treatment:
Once daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
15 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on a range-finding study
- Rationale for animal assignment (if not random): Randomisation procedure based on stratified bw
Positive control:
Not applicable

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS (mortality and moribundity): Yes
- Time schedule: Twice daily, (once daily on weekends)


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 for males and females 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 for males and females during maturation period; in mated females between Days 1-7, 7-14 and 14-20 post coitum and 1-4 post partum
Sacrifice and pathology:
SACRIFICE:
- Males were euthanized after confirmation of successful mating; all surviving adults (including non-fertile animals) and offspring were euthanized on Day 5 postpartum.

EXAMINATIONS:
- All animals were examined macroscopically for internal and external abnormalities.
- Organ weights: The testes and epididymides of all adult males were weighed
- Histopathology: Following list of organs from the high dose and control adult males and females were fixed, processed and examined microscopically: coagulating glands, epididymides, prostate, seminal vesicles, testes, pituitary, ovaries, uterus/cervix and vagina
Other examinations:
REPRODUCTIVE/DEVELOPMENTAL PARAMETERS
- Parameters examined in all male parental generations: Testis weight, epididymis weight, sperm count in testes and sperm count in epididymides
- Number of corpora lutea of all ovaries from pregnant females
- Number of uterine implantation sites

Litter observations: 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
Statistics:
Data were processed to give litter mean values, group mean values and standard deviations. The food conversion ratio (group mean weekly body weight gain/ food consumption) was calculated for the premating period. Adult body weight and food consumption, litter size and weight, individual pup body weight, pinna detachment, reproductive and viability indices and organ weight data were analyzed for homogeneity using Bartlett's' test, followed by a one-way analysis of variance (ANOVA). Data that were not homogeneous were subsequently analyzed using a t-test (assuming unequal variances). Dunnett's multiple comparison method was used to analyze data that were homogenous. Relative organ weights were analyzed using the Kruskal-Wallis non parametric rank sum test. Pairwise comparisons were performed using the Mann-Whitney U-test. Histopathological lesions that occurred at an overall frequency of 1 or greater were analyzed using a chi-squared test. Severity grades were analyzed using a Kruskal-Wallis one-way non-parametric ANOVA. Significant differences were reported at the p < 0.05, p < 0.01 and p < 0.001 level (if present).

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
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
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
Mortality:
- Thirteen animals were found dead or were killed in extremis during the study (four, five and four treated with 15, 150 and 250 mg/kg bw/day, respectively). All mortalities were considered to be related to 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 WEIGHT GAIN
- At 250 mg/kg bw/d: Slight reduction in bodyweight gain in males during weeks 2, 4, 5 and 6; lower body weights in females on Day 0 of gestation and lower body weight gains throughout gestation (which resulted in lower group mean body weights on Days 7, 14 and 20 of gestation). Day 1 and 4 post partum group mean body weights of females treated with 250 mg/kg bw/day were also lower than control.
- At 150 mg/kg bw/d: The only difference observed in weights or weight gains was a slight decrease in Day 1 post partum body weight. The difference was statistically significant (p < 0.05) , but was not considered to represent a significant effect as subsequent weight gain and the Day 4 post partum body weight were not significantly different from control.
- At 15 mg/kg bw/d: There was no effect of treatment on body weight.

FOOD CONSUMPTION
- At 250 mg/kg bw/d: Food consumption of males was significantly lower than control during the first week of dosing and not affected in females during the maturation period. However, it was decreased during gestation days 1-7, 7-14 and 14-20 and lactation days 1 and 4.
- At 150 and 15 mg/kg bw/d: There was no effect of treatment on food consumption.


ORGAN WEIGHTS
- There was no effect of treatment on parental organ weight; both testes and epididymis weights (absolute and relative to body weight) of all treated groups were greater than control. The differences were significant for relative testes (p < 0.001) and epididymis weights (p < 0.05) of high dose animals. This was due to one control male with small testes (testes weight was 0.209% of body weight compared to others in the group being 0.547 - 0.763% of body weight) and epididymides (epididymis weight was 0.108% of body weight compared to others in the group being 0.205 - 0.284% of body weight) and was not considered to be treatment-related.

GROSS PATHOLOGY AND HISTOPATHOLOGY
- All animals found dead or killed in extremis (with the exception of one female at 15 mg/kg bw/d) showed macroscopic changes consistent with dosing trauma, including fluid in the thoracic cavity and fibrous adhesions in thoracic structures. At study termination, there was evidence of gas distension in four high dose males. One mid dose male also had an ulcer in the stomach.
- There were no significant gross abnormalities in treated females or in males treated with 15 mg/kg bw/d.
- No treatment-related histopathological changes were observed in the organs that were examined.

OTHER FINDINGS: Refer to 7.8.1 Reproduction/Developmental toxicity (screening), Knox et al, 2005, KL1

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: clinical signs and effects on body weight and food consumption at 250 mg/kg bw/d
Remarks on result:
other: -
Remarks:
Toxicity potentially linked to irritating properties of the substance

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
250 mg/kg bw/day (actual dose received)
System:
other: irritation type effects
Organ:
other: gastrointestinal tract
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Applicant's summary and conclusion

Conclusions:
Under the study conditions, administration of the test substance to adult male and female rats throughout maturation, mating, gestation and early lactation resulted in significant effects at 250 mg/kg bw/d. Although effects were seen at 150 mg/kg bw/d, they were not considered to be toxicologically significant and this dose was therefore considered to be the NOAEL.
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. On Day 5 post-partum, all surviving animals were killed and examined macroscopically for internal and external abnormalities. All adults and offspring were observed for clinical signs. Male body weights and food consumption per cage were recorded weekly throughout the study. Body weights and food consumption per cage of females were recorded weekly until mating. After mating, body weights of females were recorded on Gestation Days (GD) 0, 7, 14, and 20 and on Lactation Days (LD) 1 and 4, and food consumption of females was determined for GD 1-7, 7-14 and 14-20. Female food consumption also was recorded for the period covering LD 1-4. The litter signs and individual pup bodyweights were recorded on Days 1 and 4 post-partum. 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. There was a total of 13 adult mortalities, distributed across all dose groups. Twelve of these were associated with dosing trauma and the nature of the test substance. The distribution of the mortalities suggested that it was not a consequence of systemic toxicity. At 250 mg/kg bw/day, there were clinical signs of reaction to a potentially irritating substance. These included increased salivation pre- and post-dosing, evidence of a reduction in bodyweight gain throughout the different phases of the study and significant reductions in food consumption. Post-mortem evaluation at termination showed limited evidence of effects on the gastro-intestinal tract. At 150 mg/kg bw/day, there were similar clinical signs of reaction to dosing and the post-mortem evaluation of adults at termination showed isolated incidents of potential gastro-intestinal effects. There were however no significant effects on bodyweight and food consumption during the in-life phase of the study. At 15 mg/kg bw/day, there were similar clinical signs of increased salivation. There were no significant findings at post-mortem macroscopic examination and no effects upon bodyweight or food consumption during the course of the study. There were no significant histopathological changes observed for the reproductive organs of adults at termination in any dose group. Under the study conditions, administration of the test substance to adult male and female rats throughout maturation, mating, gestation and early lactation resulted in significant effects at 250 mg/kg bw/day. Although effects were seen at 150 mg/kg bw/day, they were not considered to be toxicologically significant and this dose was therefore considered to be the NOAEL (Knox, 2005).