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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009-10-30 to 2010-04-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
There were minor protocol deviations considered to have not affected the outcome of the study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
minor protocol deviations related to temperature and time of observations. These deviations were considered not to have affected the study integrity.
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
Name of test material: Trimethylolpropanepoly(oxypropylene)triamine
EC no.: 500-105-6
CAS no.: 39423-51-3
Physical state: clear colourless liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 9D416 (source: Sponsor)
- Expiration date of the lot/batch: 27-04-2011

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At ambient temperature and protected from light

OTHER SPECIFICS:
- Composition of test material, percentage of components: Primary amine (>90% of total amine), Total acetylatables (6.5-7.1 meq/g), total amine (6.1-6.6 meq/g), water (0.25 max wt%)

Test animals

Species:
rat
Strain:
other: Crl:CD (SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Ltd., UK
- Age at study initiation: 9-10 weeks
- Weight at study initiation: 314-358 g (males) and 224-268 g (females)
- Fasting period before study: no fasting
- Housing: Individiually and depending upon the phase of the study, animals were housed in P2000 (pre-pairing) or 2154 (post mating) solid-bottomed cages, or RB3 modified cages with stainless steel grid flooring (during mating). Wood based bedding was used, which was sterilised by autoclaving and changed at least twice a week.
- Use of restrainers for preventing ingestion (if dermal): no, after removal of dressing the exposed area was cleaned to reduce the risk of ingestion
- Diet: Standard rodent diet (SDS VRF1 Certified Diet), ad libitum. The diet contained no added antibiotic, or other chemotherapeutic or prophylactic agent
- Water: Potable water taken from the public supply, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 23 °C
- Humidity (%): 40 to 70%
- Photoperiod (hrs dark / hrs light): 12/12 (lights on at 6.00 GMT)

Administration / exposure

Route of administration:
dermal
Vehicle:
water
Details on exposure:
TEST SITE
- Area of exposure: Dorsum between the limb girdles
- % coverage: approx. 10% of total body
- Type of wrap if used: Gauze patch held in place with cotton wool, Tubigrip bandage and surgical tape (semi-occlusive)
- Time intervals for shavings or clipplings: As required

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Exposed area was cleaned with warm tap water and dabbed dry with disposable paper towel/tissues
- Time after start of exposure: not less than six hours (except for females on Day 21 of mating and Day 2 and 3 of lactation when exposure was 3 hours)

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 3 mL/kg
- Concentration (if solution): 3.3, 16.7 and 33.3 mg/mL
- Constant volume or concentration used: yes

USE OF RESTRAINERS FOR PREVENTING INGESTION: no, info on 'Test animals'
Details on mating procedure:
- M/F ratio per cage: one-to-one basis (1:1)
- Length of cohabitation: 14 days
- Proof of pregnancy: Cages were checked daily for ejected copulation plugs and a vaginal smear was examined on the presence of spermatozoa and the stage of the oestrous cycle. The day on which evidence of mating was found was designated Day 0 of gestation.
- After successful mating each pregnant female was caged (how): Individually in 2154 solid-bottomed cages.
- Any other deviations from standard protocol: Not applicable
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of each formulation prepared for administration in the first and last preparations were analysed for achieved concentration of the test substance. Four samples were taken (nominally 1 mL accurately weighed) from all groups; 2 assays from each test group and 1 assay from the control group. The remainder was retained as contingency for analysis if any result required confirmation. The method of analysis was LC-MS and was an adaptation of a method supplied by the Sponsor;
Duration of treatment / exposure:
Males were treated daily 15 days before pairing, throughout pairing until Day 3 after the birth of the F1 generation. Females were treated daily for 15 days before pairing, throughout pairing until Day 22 of gestation with recommencement on Day 2 of lactation until Day 3 after the birth of the F1 generation.
Frequency of treatment:
Daily (7 days per week) at 6 hours per day, except for females on Day 21 of mating and Day 2 and 3 of lactation when exposure was only three hours
Details on study schedule:
Age at mating of the mated animals in the study: 11 to 12 weeks
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
control group
Dose / conc.:
10 mg/kg bw/day (nominal)
Remarks:
low dose group
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
mid dose group
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
high dose group
No. of animals per sex per dose:
10/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the dose levels of 10,50 and 100 mg/kg/day were selected in conjunction with the Sponsor based on findings from previous toxicology studies (dermal LD50 ca. 600 mg/kg; dermal 28-d NOEL of 100 mg/kg bw/d; dermal 90-d NOEL of 16 mg/kg bw/d, systemic 90-d NOEL of >160 mg/kg bw/d).
- Rationale for animal assignment (if not random): The males and females were divided into bodyweight strata (5 g range) and allocated to treatment groups by selecting animals from each bodyweight range in rotation after any grossly atypical animals had been discarded. This procedure ensured that all groups contained populations of rats with similar initial mean and range of bodyweights, and that discarded surplus animals were amongst those with outlying bodyweights.
- Other: The route of administration was chosen to simulate the conditions of human exposure.
Positive control:
Not used

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

DERMAL OBSERVATIONS: daily, before each administration, according to Draize criteria

FOOD CONSUMPTION: mean weekly consumption per animal until mating, mean daily consumption per female animal after mating
Oestrous cyclicity (parental animals):
For 15 days before pairing, daily vaginal smears were taken from all females, using cotton swabs moistened with saline. The smears were subsequently examined to establish the duration and regularity of the oestrous cycle. After pairing with the male, smearing was continued using pipette lavage, until evidence of mating was observed
Sperm parameters (parental animals):
For the assessment of the testes at necropsy, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell- or stage-specificity of testicular findings was noted.
Litter observations:
All litters were examined at approximately 24 hours after birth (Day 1 of age) and then daily thereafter. Clinical signs, Litter size, sex ratioand body weight of individual offsprings were examined.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: After confirmation that a second mating was not required
- Maternal animals: F0 females were killed on Day 4 of lactation. Females that failed to produce a viable litter were killed on Day 25 after mating. Females whose litter died before Day 4 of lactation were killed on the day the last offspring died.

GROSS NECROPSY
- Gross necropsy consisted of a full macroscopic examination of the tissues; a visual examination of all external features and orifices; an in situ examination of neck and associated tissues and the thoracic, abdominal and pelvic cavities and their viscera; examination of size and appearance of organs and tissues. For females the number of implantation sites in each uterine horn was counted.

HISTOPATHOLOGY / ORGAN WEIGHTS
Following organs were weighted: epididymides, ovaries, pituitary, prostate, seminal vesicles, testes and uterus with cervix and oviducts. Following tissues were fixed and histologically examined for all adult animals of Groups 1 (control) and 4 (100 mg/kg/day): application site, epididymides, pituitary, ovaries, seminal vesicles, prostate, testes, uterus and vagina. The reproductive organs (i.e mammary area - caudal) for one Group 2 (10 mg/kg/day) female with a litter death were additionally examined.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring sacrificed at 4 days of age.

GROSS NECROPSY
- Gross necropsy consisted of a full macroscopic examination of the tissues; a visual examination of all external features and orifices; an in situ examination of neck and associated tissues and the thoracic, abdominal and pelvic cavities and their viscera; examination of size and appearance of organs and tissues.
- Premature deaths before weaning were examined similarly as above, if not autolysed or cannibalised. Presence of milk in the stomach was assessed as well.
Statistics:
The following sequence of statistical tests was used for bodyweight and organ weight data: A parametric analysis was performed if Bartlett's test for variance homogeneity was not significant at the 1% level. The F1 approximate test was applied. If the F1 approximate test for monotonicity of dose-response was not significant at the 1% level, Williams' test for a monotonic trend was applied. If the F1 approximate test was significant, suggesting that the dose-response was not monotone, Dunnett's test was performed instead.
A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. The H1 approximate test, the non-parametric equivalent of the F1 test described above, was applied. If the H1 approximate test for monotonicity of dose-response was not significant at the 1% level, Shirley's test for a monotonic trend was applied. If the H1 approximate test was significant, suggesting that the dose-response was not monotone, Steel's test was performed instead.

Significant differences between Control and treated groups were expressed at the 5% (p<0.05) or 1% (p<0.01) level.

For organ weight data, analysis of covariance was performed using terminal bodyweight as covariate
Reproductive indices:
Oestrous cycles
The percentage females showing the following classifications of oestrous cycles before pairing are presented:
- Regular: All observed cycles of 4 or 5 days
- Irregular: At least one cycle of 2, 3 or 6 to 10 days
- Acyclic: At least 10 days without oestrus

Mating performance and fertility
- Percentage mating = (# animals mating/animals paired)*100
- Conception rate (%) = (# animals achieving pregnancy/animals mated)*100
- Fertility index (%) = (# animals achieving pregnancy/animals pairing)*100

Gestation length
- Gestation index (%) = (# live litters born/ # pregnant)*100

Sex ratio in litter
- % males = (# males in litter/ total # offspring in litter)*100
Offspring viability indices:
The following were calculated for each litter:
- Post-implantation survival index (%) = (total # offspring born/ total # uterine implantation sites)*100
Post-implantation survival index was expressed as 100% where the number of offspring exceeded the number of implantation sites recorded.

- Live birth index (%) = (# live offspring on day 1 after littering/ total # offspring born)*100
- Viability index (%) = (# live offspring on day 4 after littering/ #live offspringon day 1 after littering)*100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
no effects observed
Description (incidence and severity):
Dermal reactions at the application site of male animals were limited to very slight erythema and eschar/scab formation, however there was no evidence for either a treatment related or dose related response in either the number of animals or the number of occasions the signs were observed.
For females before pairing, during gestation and lactation dermal signs at the application site and included very slight/well defined eythema, eschar/scab formation, exfoliation and sloughing. The incidence in terms of the number animals affected and the number of occasions observed was marginally higher at 100 mg/kg/day when compared with Controls, but there was no clear evidence of a dose related response at 10 or 50 mg/kg/day.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Overall mean bodyweight gain for males receiving 100 mg/kg/day was low when compared with Controls, approximately 83% of Controls; however this difference did not attain statistical significance. Bodyweight gain for males at 10 or 50 mg/kg/day was unaffected by treatment.
Bodyweight gain for females during the two week treatment period before being paired for mating was unaffected by treatment with the test substance. During gestation mean bodyweight gain for females receiving 100 mg/kg/day was approximately 88% of controls, however statistical significance was not attained. At 10 or 50 mg/kg/day the bodyweight gain of females during gestation was similar to the Controls.
During lactation the bodyweight gain of females was unaffected by treatment.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not specified
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
Mating performance was unaffected by treatment with all paired animals showing positive evidence of mating. One female at 100 mg/kg/day was not pregnant and because of the small group size this lowered the conception and fertility index at 100 mg/kg/day to 90% compared with 100% in the Control group. However a total of 10 Control females and 10, 10 and 9 females at 10, 50 and 100 mg/kg/day, respectively, achieved pregnancy.
The majority of animals commenced parturition within the normal range of 22 to 23.5 days. At 10 mg/kg/day one female the gestation length was 21.5 days however there was no other evidence for a treatment related reduction in gestation length.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
general toxicity
Effect level:
> 100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Key result
Dose descriptor:
NOEL
Remarks:
reproduction and fertility
Effect level:
> 100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Mating performance, fertility and offspring survival and development up to Day4 of age was unaffected by treatment at dose levels up to 100 mg/kg/day.

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Other effects:
not examined

Details on results (F1)

VIABILITY (OFFSPRING)
The mean number of implantations was essentially similar amongst the groups. However at 100 mg/kg/day the mean post-implantation survival and subsequent litter size were low when compared with the Controls; although statistical significance was not attained. Review of the individual litter data revealed that this was attributed to two litters at 100 mg/kg/day, namely 72 and 73. The implantation rate of these animals (nos.72 and 73) was comparable with other animals in the group and although the post-implantation survival rate was low (14 and 35%, respectively), the survival rate in the other seven animals was similar to the Controls ranging between 79 and 100%. Post natal survival for litter nos. 72 and 73 was good and the mean bodyweight of offspring on Day 1 of age was above average, reflecting the low litter size/heavier bodyweight ratio. This heavier weight was maintained to Day 4 of age and there were no significant findings in these dams or offspring at necropsy. It is considered likely that the increased post-implantation loss in these two high dose litters is fortuitous and is exaggerated by the small sample size, and is therefore considered unlikely to be related to treatment. However the potential for higher in-utero loss at 100 mg/kg/day cannot be resolved within the context of the design of this screening study.

Effect levels (F1)

Key result
Dose descriptor:
NOEL
Generation:
F1
Effect level:
> 100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

The mean concentrations in dose formulations analysed during this study were within ± 10% of nominal concentrations, confirming accurate formulation.

Applicant's summary and conclusion

Conclusions:
It was concluded that in this study, in terms of general toxicity, that 100 mg/kg/day was the no observed adverse effect level (NOAEL). Despite the slight bodyweight effects (males and females) and dermal reactions at the application site (females) at this dose level, the general condition of parental animals, food consumption and organ weights showed no adverse effects of treatment, and there were no macroscopic or microscopic lesions that could be attributed to treatment with the test substance. In the absence of any impairment of reproductive function, the high dose of 100 mg/kg/day was considered to be the no observed effect level (NOEL) in terms of reproductive function and fertility.