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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:
4 August 2004 to 15 February 2005
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

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 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
- Physical state: Brown slightly viscous liquid
- Storage conditions: Room temperature in the dark

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent
- Age at study initiation: approximately 8 weeks
- Weight at study initiation: 201 - 266g (males); 177 - 238g (females)
- Housing: initially in groups of 4, during the mating phase animals were housed in pairs (one of each sex), following mating females where housed individually and males in groups. Cages were polypropylene cages with stainless steel grid floors and tops, suspended over polypropylene trays lined with absorbent paper. Mated females were housed in polypropylene cages with solid floors and stainless steel lids, furnished with softwood flakes (Datesand Ltd. Cheshire, UK).
- Diet (e.g. ad libitum): pelleted diet (Rodent PMI 5002 (Certified) diet, BCM IPS Limited, London, UK), ad libitum
- Acclimation period: Up to 16 days

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
400
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- The appropriate concentrations were prepared in the vehicle weekly and stored at approximately 4 ºC in the dark
- The test material was administered daily by gavage using a plastic catheter attached to a disposable plastic syringe
- Control animals were treated in an identical manner with 4 mL/kg/day of the vehicle
- The volume of test and control material administered to each animal was based on the most recent bodyweight and was adjusted at regular intervals
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples from each preparation were analysed using gas chromatography to verify the concentration, stability and homogeneity of each formulation.

Method:
- Gas chromatography
- Samples and standards were prepared in methanol to give a final concentration of 0.1 mg/mL
- Samples were analysed within two days of preparation
- The method was validated using a range of standard solutions covering the range 0 to 0.1527 mg/mL. The results of which have been considered to be sufficiently accurate for the purpose of this study.

Conditions:
- GC system: Agilent Technologies 5890, incorporating autosampler and workstation.
- Column: DB-1 (30 m x 0.32 mm id x 0.25 µm film).
- Oven temperature program: initial 50 ºC for 0 mins, rate 5 ºC/min, final 200 ºC for 0 mins.
- Injection temperature: 150 ºC
- Flame ionisation detector temperature: 250 ºC
- Injection volume: 1 µL
- Retention time: ~14.7 mins.

Results:
- The results indicate that the prepared formulations were within ± 9 % of the nominal concentration.
- Analytical verification of the stability and homogeneity of the test preparations was also performed and the formulation was found to be stable for at least fourteen days.
Details on mating procedure:
Animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of the oestrous cycle or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating and the males were subsequently returned to their original holding cages. Mated females were housed individually during the period of gestation and lactation.
Duration of treatment / exposure:
54 consecutive days
Frequency of treatment:
Daily
Duration of test:
54 days
No. of animals per sex per dose:
10 males and 10 females per group
Control animals:
yes, concurrent vehicle
Details on study design:
Preliminary Study:
- A fourteen day oral range finding study was conducted to provide information for the selection of dose levels for the definitive study. Animals were dosed and observed for adverse effects resulting from toxicity including; clinical observations, bodyweight and gross pathology.
- Animals were kept under the same conditions as in the definitive study.
- The test material was prepared in the same manner as the definitive study and verified analytically.
- The test material was administered in a limit test daily, for up to 14 consecutive days. Animals were administered 1000 mg/kg/day via oral gavage, a volume of 4mL/kg and a concentration of 250 mg/mL. Control animals were treated in an identical manner with 4 mL/kg/day of polyethylene glycol.

Examinations

Maternal examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: immediately before and after dosing, then at 1 and 5 hours after dosing. The 5 hour observation was omitted at weekends, except for females during parturition where applicable.
- Observations recorded: overt signs of toxicity, ill-health and behavioural changes

BODY WEIGHT: Yes
- Time schedule for examinations: males were weighed on day 0, then weekly until termination. Females were weighed on day 0 and then weekly until evidence of mating was evident. Bodyweights were recorded on day 0, 7, 14 and 20 post coitum, and on day 1 and 4 post partum.

FOOD AND WATER CONSUMPTION: Yes
- Weekly food consumption was recorded for each caged group of adults, which was continued for males after mating. Food consumption for females showing evidence of mating was recorded for the periods covering days 1-7, 7-14 and 14-21. For females with lie litters, consumption was recorded during the lactation period.
- Water consumption was observed daily by visual inspection of water bottles for any overt change.

FUNCTIONAL OBSERVATIONS: YES
- Time schedule: prior to the start of treatment and at weekly intervals
- Observations recorded: signs of functional behavioural toxicity (except for non-pregnant female number 71). Functional performance tests were also performed on five selected males and females per dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.

BEHAVIOURAL ASSESSMENTS: Yes
- Observations recorded: gait, tremors, twitches, convulsions, bizarre/abnormal/stereotypic behaviour, salivation, pilo-erection, exophthalmia, lachrymation, hyper/hypothermia, skin colour, respiration, palpebral closure, urination, defecation, transfer arousal, tail elevation.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on day 14, prior to pairing
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: five males and five females per dosing group
- Parameters examined on blood collected into tubes containing potassium EDTA anti-coagulant: haemoglobin, erythrocyte count, haematocrit, erythrocyte indices, total leucocyte count, differential leucocyte count, platelet count, prothrombin time and activated thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on day 14, prior to pairing.
- Animals fasted: No
- How many animals: five males and five females per dosing group
- Parameters examined on blood collected into tubes containing lithium heparin anti-coagulant: urea, glucose, total protein, albumin, albumin/globulin ratio (by calculation), sodium, pptassium, chloride, calcium, inorganic phosphorus, aspartate aminotransferase, alanine aminotransferase, Alkaline phosphatise, creatinine, total cholesterol and total bilirubin.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Battery of functions tested: sensory activity, forelimb/hind limb grip strength and motor activity

Sensory reactivity: Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. The following parameters were observed: grasp response, vocalisation, toe pinch, tail pinch, finger approach, touch escape, pupil reflex, startle reflex and blink reflex.

Grips strength: An automated grip strength meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. The animal was pulled by the base of the tail until its grip was broken. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal.

Motor activity: Purpose-built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals were randomly allocated to the activity monitors. The tests were performed at approximately the same time each day, under similar laboratory conditions. The evaluation period was thirty minutes for each animal. The percentage of time each animal was active and mobile was recorded for the overall thirty minute period and also during the final 20% of the period (considered to be the asymptotic period).

PREGNANCY AND PARTURITION:
Each pregnant female was observed at approximately 0830, 1230 and 1630 hours and around the period of expected parturition. Observations were carried out at approximately 0830 and 1230 hours at weekends and public holidays. The following was recorded for each female: date of mating, date and time of observed start of parturition, date and time of observed completion of parturition and duration of gestation.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Number of corpora lutea: Yes
- Number of implantations: Yes
- The procedure was enhanced by staining the uteri with a 1 % ammonium polysulphide solution.
Fetal examinations:
No pre-parturition examinations were performed. All offspring, including those dying during the study, were subjected to a full external and internal examination post parturition, and any macroscopic abnormalities were recorded.
Statistics:
Data were processed to give group mean values and standard deviations where appropriate. Haematological, blood chemical, organ weight (absolute and relative to terminal bodyweight), weekly bodyweight gain and quantitative functional performance data were assessed for dose response relationships by linear regression analysis, followed by one way analysis of variance (ANOVA) incorporating Levene’s test for homogeneity of variance. Where variances were shown to be homogenous, pair-wise comparisons were conducted using Dunnett’s test. Where Levene’s test showed unequal variances the data were analysed using non-parametric methods: Kruskal-Wallis ANOVA and Mann-Whitney ‘U’ test.

The haematology variable basophils was not analysed since consistently greater than 30% of the data were recorded as the same value.

Probability values (p) are presented as follows:
p < 0.001 ***
p < 0.01 **
p < 0.05 *
p 20.05 (not significant)
Indices:
MATING PERFORMANEC AND FERTILITY

The following parameters were calculated from the individual data during the mating period of the parental generation.

-Pre-coital interval: Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.

-Fertility Indices, for each group the following were calculated:
Mating Index (%) = [No. of animal mated/No. of animals paired] x 100
Pregnancy Index (%) = [No. of pregnant females/ No. of animals mated] x 100

GESTATION AND PARTURATION DATA

The following parameters were calculated for individual data during the gestation and parturition period of the parental generation.

-Gestation Length: Calculated as the number of days of gestation including the day for observation of mating and the start of parturition. Where the start of parturition occurred overnight, the total was adjusted by subtracting half a day.

-Parturition Index, the following was calculated for each group:
Parturition Index (%) = [No. of females delivering live offspring/No. of pregnant females] x 100

LACTATION DATA

-Live birth and viability indices, the following indices were calculated for each group form individual data:
Live Birth Index (%) = [No. of offspring alive on day 1/No. of offspring born] x 100
Viability Index (%) = [No. of offspring alive on day 4/No. of offspring alive on day 1] x 100

-Sex ratio (% males), group mean values calculated from each litter value on day 1 and 4 using the following formula:
Sex ratio = [No. of male offspring/No. of offspring of determined sex] x 100

-Implantation losses (%), group mean percentile pre-implantation and post implantation loss were calculated as follows:
% pre-implantation loss = [(No. of Corpora Lutea – No. of implantation sites)/No. of corpora lutea] x 100
% post-implantation loss = [(Group no. of implantation sites – No. of offspring)/No. of implantation sites] x 100
Historical control data:
None used.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

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

Details on maternal toxic effects:
Mating:
- No significant intergroup differences were detected for mating, fertility or pregnancy.
- One female treated with 1000 mg/kg/day showed positive evidence of mating after one day of pairing, however, this animal failed to achieve pregnancy. This is a normal low incidence finding on reproductive studies of this type and in isolation, considered unrelated to treatment.
- A reduced parturition index was evident at 1000 mg/kg/day, but this was attributed to the termination of one female due to dystocia.

Uterine examination:
- No treatment-related effects were detected.
- Statistical analysis of corpora lutea and implantation site counts along with percentile pre and post implantation losses did not reveal any significant differences.

Necropsy:
- No treatment-related effects were detected.
- The interim death pregnant female treated with 1000 mg/kg/day showed four placentae and four dead foetuses in the right uterine horn. The left uterine horn was blood filled. This animal was terminated due to difficulties in birthing. Dystocia is a common low incidence finding in reproductive studies of this type and in isolation, this death was considered unrelated to test material toxicity.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:not examined

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks:
maternal dose
Sex:
male/female
Basis for effect level:
other: development toxicity

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no

Any other information on results incl. tables

Offspring Developmental Effects:

Litter size and viability:

-There were no treatment-related intergroup differences in litter sizes, sex ratio, live birth indices or post partum viability.

Growth and development:

- There was no adverse effect of treatment on offspring growth or development.

- There were no significant intergroup differences in mean litter weights or offspring bodyweights. Surface righting or pinna unfolding assessments for treated offspring were comparable to controls.  

Clinical observations:

- No clinically observable signs of toxicity were detected.

- The clinical observations recorded for offspring throughout the dose groups were consistent with normally expected incidence findings in offspring of the age examined and were of no toxicological significance.  

Necropsy:

- No treatment-related macroscopic abnormalities were detected for offspring at terminal kill. The macroscopic findings observed for interim and terminal kill offspring throughout the dose groups, were consistent with normally expected low incidence findings in pups of the age examined and were of no toxicological significance.  

Applicant's summary and conclusion

Conclusions:
There was no evidence of developmental toxicity in this study. One high dose female failed to achieve pregnancy and another female from this dose group was sacrificed at parturition due to dystocia. These are normal low incidental findings observed on reproductive studies and considered unrelated to treatment. No adverse effects were recorded in offspring growth or development.

No effect of treatment was detected in offspring development, therefore the ‘No Observed Adverse Effect Level’ (NOAEL) for reproductive toxicity was considered to be 1000 mg/kg/day.
Executive summary:

In a GLP compliant study the developmental toxicity of the test material was determined in a repeat dose toxicity study, performed according to the OECD guideline 422.

Sprague-Dawley rats (10 per sex per dose) were given 54 consecutive doses of the test material, receiving 100, 300 or 1000 mg/kg/day. Both the adult and the F1 generation were observed during 54 day study and subjected to necropsy at termination. All animals, including those dying during the study, were subjected to a full external and internal examination post parturition, and any macroscopic abnormalities were recorded.

No adverse effects or systemic signs of toxicity were recorded as a result of treatment during this study. One high dose female failed to achieve pregnancy and another female from this dose group was sacrificed at parturition due to dystocia. These are normal low incidental findings observed on reproductive studies and considered unrelated to treatment. No treatment related effects were detected in offspring growth or development, therefore the ‘No Observed Adverse Effect Level’ (NOAEL) for developmental toxicity was considered to be 1000 mg/kg/day.