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Repeated dose toxicity: oral

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Administrative data

Endpoint:
repeated dose toxicity: oral, other
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 May - 08 Jul 2011
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD Guideline 421
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Inc., Portage, Michigan, USA
- Age at study initiation: (P) approximately 8 weeks
- Weight at study initiation: (P) Males: 255.2 ± 4.9-256.6 ± 6.2 g; Females: 190.6 ± 8.6- 194.6 ± 8.5 g (mean ± SD)
- Fasting period before study: no
- Housing: animals were housed individually in stainless steel cages, except during breeding and during the littering phases; during breeding, one male and one female were housed per cage; during littering, dams and their litters were housed in plastic cages provided with ground corncob nesting material from approximately GD 19 until termination. Cages had wire mesh floors and were suspended above catch pans. Non-woven gauze was placed in the cages to provide a cushion from the flooring for rodent feet and also provided environmental enrichment. In order to better visualize copulation and plugs, gauze was not placed in cages during the breeding phase.
- Diet: LabDiet Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, Missouri) in meal form, ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 1 (maximum permissible excursion of ± 3 °C)
- Humidity (%): 40-70
- Air changes (per hr): 12-15, on average
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 17 May 2011 To: 15 Jun 2011 (males); 30 Jun and 08 Jul 2011 (females)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test material was mixed with deionised water to reach a dose volume of 4 mL/kg bw; calculated according to the latest body weight, and the solution was adjusted to pH 9. Dose solutions were prepared periodically during the study period based on stability data.

VEHICLE
- Concentration in vehicle: 25, 75 and 250 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses by HPLC/ELSD were performed to determine the concentration of the test material of all dosing solutions from the first mix of the main study prior to the start of dosing. The low- and high-dose solutions from the first mix of the main study were analyzed to confirm homogeneous distribution of the test material concurrent with dose confirmation. The stability was measured on Day 0, 1, 6, 15 and 22. The stability results were 93.4-112.9% of target concentration with RDS up to 18.1% on Day 0; and 81.7-109.4% of target concentration with RDS up to 13.9% on Day 22. The concentration and homogeneity of the dose solutions was 91.4-109.1% of target concentration, with RSD of up to 12.5% (N = 6). Quantitation was performed using external standard calibration; the accuracy of the standards was 94.6-110% (mean) of the nominal values. The large RSDs were explained by the use of an external standard calibration and the ELSD, which both tend to give a larger RSD.
Duration of treatment / exposure:
(P) Males: 29 days (14 days prior to mating, 14 days during mating, up to and including Day 29)
(P) Females: up to 54 days (14 days prior to mating, up to 14 days during mating, approximately 22 days during gestation, 4 days during lactation)
Frequency of treatment:
Daily, 7 days/week
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 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: a range-finding study was performed in which 3 rats/sex/dose were administered 0, 250, 500, 750 or 1000 mg/kg bw/day via gavage for 14 days. All the animals survived to scheduled termination and there were no treatment-related clinical signs. The body weights and feed consumption were comparable between the control and treatment groups, and no effects were noted on organ weights or during gross necropsy. As no effects were observed at the highest dose level of 1000 mg/kg bw/day and this is the limit dose defined in the EPA OPPTS 870.3550 guideline, dose levels of 100, 300 and 1000 mg/kg bw/d were selected for the main study.
- Other: the pH of the test solution was adjusted to 9 prior to dosing

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily during the study period for all animals, including dams and their litters
- Cage side observations included, but were not limited to: morbidity, mortality, decreased/increased activity, repetitive behaviour, vocalization, incoordination/limping, injury, neuromuscular function (convulsion, fasciculation, tremor, twitches), altered respiration, blue/pale skin and mucous membranes, severe eye injury (rupture), alterations in fecal consistency, and fecal/urinary quantity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily. Animals were observed for abnormalities in the eyes, urine, feces, gastrointestinal tract, extremities, movement, posture, reproductive system, respiration, skin/hair-coat, and mucous membranes, as well as an assessment of general behaviour, injuries, or palpable mass/swellings. Females were observed for signs of parturition from around gestation day 20.

BODY WEIGHT: Yes
- Time schedule for examinations: for all animals, at least once during the pre-exposure period and on the first day of dosing (Day 0). Males were also weighed weekly during the study period. Females were weighed weekly during the pre-mating and mating period; on gestation day (GD) 0, 7, 14, 17 and 20; females that delivered litters were weighed on lactation day 1 and 4; females that failed to mate or deliver a litter were weighed at least weekly until termination.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes. Feed consumed was determined weekly during the two week pre-breeding period for males and females by weighing feed crocks at the start and end of a measurement cycle. Feed consumption was not measured for males or females due to co-housing during breeding. Following breeding, feed consumption was not measured for males. For females during gestation, feed consumption was measured on GD 0, 7, 14, and 20. After parturition, feed consumption was measured on lactation day 1 and 4. Feed consumption was not recorded for females that failed to mate or deliver a litter.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No
Sacrifice and pathology:
SACRIFICE
- Male animals: all surviving animals were sacrificed after 29 days of exposure, in a fasted state
- Maternal animals: all surviving animals were sacrificed between lactation day 5 and 8, or at least 24 days after the end of the mating period for females not producing a litter, in a fasted state

GROSS NECROPSY
- Gross necropsy consisted of external examination of tissues and orifices, in situ examination of the eyes, and internal examinations including the cervical, thoracic, and abdominal viscera.

Samples of the following tissues/organs were collected from all the animals and preserved in neutral, phosphate-buffered 10% formalin: kidneys, liver, pituitary, gross lesions, cervix, coagulating glands, mammary gland (females), ovaries, oviducts, prostate, seminal vehicles, uterus, vagina. The testes and epididymides were fixed in Bouin’s. Transponders were removed and placed in jars with the tissues.
The uteri of all females in control and treatment groups were stained with an aqueous solution of 10% sodium sulfide stain (Kopf et al., 1964) for approximately one minute and were examined for the presence and number of implantation sites, prior to preservation.

HISTOPATHOLOGY / ORGAN WEIGHTS
The weights of the epididymides, kidneys, liver, and testes were recorded for all the animals in the control and treatment groups.
Histological examinations were performed on the preserved tissues/organs from the animals in the control and high-dose groups. A qualitative assessment of the stages of spermatogenesis was made of the testes in control and high-dose males by examining the relationship between spermatogonia, spermatocytes, spermatids, and spermatozoa (cycle of spermatogenesis) as seen in cross sections of the seminiferous tubules by microscopic evaluation; and by examination of sections of both testes for the presence of degenerative changes (e.g., vacuolation of the germinal epithelium, a preponderance of Sertoli cells, sperm stasis, inflammatory changes, mineralization, and fibrosis)
Other examinations:
Repro Screening related axaminations
Statistics:
Parental body weights and gestation and lactation body weight gains, litter mean body weights, feed consumption, and organ weights (absolute and relative) was first evaluated by Bartlett's test (alpha = 0.01; Winer, 1971) for equality of variances. Based upon the outcome of Bartlett's test, either a parametric (Steel and Torrie, 1960) or non-parametric (Hollander and Wolfe, 1973) analysis of variance (ANOVA) was performed. If the ANOVA was significant at alpha = 0.05, a Dunnett's test (alpha = 0.05; Winer, 1971) or the Wilcoxon Rank-Sum (alpha = 0.05; Hollander and Wolfe, 1973) test with Bonferroni's correction (Miller, 1966) was performed. Feed consumption values were excluded from analysis if the feed is spilled or scratched. Gestation length, average time to mating, and litter size were analyzed using a nonparametric ANOVA. If the ANOVA was significant, the Wilcoxon Rank-Sum test with Bonferroni's correction was performed. Statistical outliers (alpha = 0.02) were identified by the sequential method of Grubbs (1969) and only excluded from analysis for documented, scientifically sound reasons. The mating, conception, fertility and gestation indices were analyzed by the Fisher exact probability test (alpha = 0.05; Siegel, 1956) with Bonferroni's correction. Evaluation of the neonatal sex ratio on postnatal day 1 was performed by the binomial distribution test (alpha = 0.05; Steel and Torrie, 1960). Gender of pups found dead on postnatal day 0 was included in sex ratio calculations. Survival indices, post-implantation loss, and other incidence data among neonates was analyzed using the litter as the experimental unit by the censored Wilcoxon test (alpha = 0.05; Hollander and Wolfe, 1973) as modified by Haseman and Hoel (1974) with Bonferroni’s correction. Non-pregnant females, females with resorptions only, or females found to be pregnant after staining of their uteri were excluded from the appropriate analyses.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No treatment-related clinical signs were observed during the study period.
Mortality:
no mortality observed
Description (incidence):
There was no mortality during the study period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No significant differences in body weight or body weight gain were observed between the male control and treatment groups. In females, no significant differences in body weight gain were noted between the control and treatment groups. A statistically significant increase in body weight gain was observed in the female mid-dose group during lactation day 1-4, compared to the control group. This effect is considered to be incidental, as no effect was noted in the female high-dose group.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no significant differences in feed consumption between the control and treatment groups during the study period.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There was no significant difference in organ weights between the control and treatment groups.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
10/10 females and 10/10 males in the high-dose groups had a thickened limiting ridge of the stomach (see Table 1). The limiting ridge is a thin protrusion from the forestomach that demarcates the forestomach from the glandular stomach in rats. The effects are considered to be caused by a local irritation effect of the test substance and are therefore treatment-related. However, as humans do not have a forestomach and limiting ridge, the findings are of limited relevance to humans. The gross pathology examinations did not reveal any treatment-related findings in the reproductive organs.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Very slight to slight hyperplasia of the stratified squamous epithelium was observed at the limiting ridge of the stomach of 1, 0, 4 and 10 male, and in 2, 0, 2 and 10 female rats in the control, low-, mid- and high-dose group, respectively (see Table 1). In addition, subacute to chronic inflammation of the submucosa of the glandular stomach was noted in at least 1 animal in all groups; classified as very slight or slight. A clear treatment-related effect was seen only in the cases classified as slight; where 0, 0, 3 and 9 males, and 0, 0, 1 and 4 females in the control, low-, mid- and high-dose group, respectively, were affected. This is considered to be a local irritating effect of the test substance, which was administered in a relatively large volume. As humans do not have a limiting ridge and forestomach, the effect is considerd to have limited toxicological relevance to humans. There were no treatment-related histopathological findings in the reproductive organs.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
systemic effects
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Dose descriptor:
NOAEL
Remarks:
local effects
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
gross pathology
histopathology: non-neoplastic

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (actual dose received)
System:
gastrointestinal tract
Organ:
stomach
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no

Any other information on results incl. tables

Table 1: Results of histopathology

Sex

Males

Females

Dose (mg/kg bw/day)

0

100

300

1000

0

100

300

1000

Stomach, No. examined

10

10

10

10

10

10

10

10

Hyperplasia, with inflammation, epithelium, limiting ridge

 

 

 

 

 

 

 

 

- Very slight

 

1

0

4

3

2

0

2

5

- Slight

0

0

0

7

0

0

0

5

Subacute to chronic inflammation, glandular submucosa, multifocal

 

 

 

 

 

 

 

 

- Very slight

3

2

3

1

2

1

6

5

- Slight

0

0

3

9

0

0

1

4

 

Applicant's summary and conclusion

Conclusions:
In this study there was no systemic toxicity observed in the parent animals exposed up to 1000 mg/kg bw/d Trometamol. Therefore this stuudy shows a systemic NOAEL of >1000 mg/kg bw.