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Toxicological information

Developmental toxicity / teratogenicity

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developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study.

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guideline
according to
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:

Test material

Test material form:
solid: particulate/powder
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Licowax C
- Physical state: yellowish powder (supplied as yellowish solid granules)
- Analytical purity: 99.9 % (w/w)
- Lot/batch No.: DEF2004602
- Expiration date of the lot/batch: 11 February 2012
- Stability under test conditions: stability for 24 hours at room temperature confirmed
- Storage condition of test material: room temperature
- Other: Source: Clariant Produkte (Deutschland) GmbH

Test animals

Details on test animals and environmental conditions:
- Source: Harlan Italy S.r.l., San Pietro al Natisone, Italy
- Age at study initiation: 9 weeks (female) and 11 weeks (male)
- Weight at study initiation: 191 - 221 g (female) and at least 316 g (male)
- Housing: Before and after pairing, the animals were housed no more than 5 of one sex to a cage, in clear polycarbonate cages measuring 59x38.5x20 cm with a stainless steel mesh lid and floor (Techniplast Gazzada S.a.r.l., Buguggiate, Varese). Each cage tray held absorbent paper which was inspected and changed at least 3 times per week. During the mating period, the rats were housed on the basis of 1 male to 1 female in clear polycarbonate cages measuring 43x27x18 cm with a stainless steel mesh lid and floor (Techniplast Gazzada S.a.r.l., Buguggiate, Varese). Each cage tray held absorbent material which was inspected and changed daily.
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: 2 weeks

- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): 15 - 25
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: gavage
corn oil
Details on exposure:
The required amount of test substance was suspended in the vehicle; the formulatiuons were prepaired daily and concentrations were calculated and expressed in terms of test item as supplied.

- Justification for use and choice of vehicle (if other than water): Solubility
- Concentration in vehicle: 10, 30 and 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw/day
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
By chemical analysis (concentration, homogeneity and stability), all parameters in range limits
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1 in the home cage of the male
- Length of cohabitation: left overnight
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
All animals were dosed from Day 6 through Day 19 post coitum.
Frequency of treatment:
Once a day
Duration of test:
20 days post coitum
Doses / concentrations
Doses / Concentrations:
100, 300 and 1000 mg/kg bw/day
nominal conc.
No. of animals per sex per dose:
24 mated females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on preliminary study


Maternal examinations:
- Time schedule: Throughout the study, all animals were checked early in each working day and again in the afternoon. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day.

- Time schedule: All clinical signs were recorded for individual animals from allocation to sacrifice.
Once daily before commencement of treatment and once daily at approximately 1-1.5 hours after treatment, each animal was observed and any clinical signs were recorded.

- Time schedule for examinations: All animals were weighed on Days 0, 3, 6, 9, 12, 15 and 20 post coitum.

- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, On Days 3, 6, 9, 12, 15 and 20 post coitum starting from Day 0 post coitum.

POST-MORTEM EXAMINATIONS: Yes, The animals were killed with carbon dioxide on Day 20 post coitum and necropsied , all foetuses were sacrificed by hypothermia. Necropsy: The clinical history of the animal was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices) and changes were noted.
- Sacrifice on gestation day 20
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
Fetal examinations:
- External examinations: Yes: all per litter, Number, sex and weight of all live foetuses; Number and sex of dead foetuses (foetuses at term without spontaneous movements and breathing); Number of intra-uterine deaths classified as: Early resorptions: only placental remnants visible, Late resorptions: placental and foetal remnants visible.
- Soft tissue examinations: Yes: half per litter, fixed-visceral examination of all groups, gross evaluation of placentae.
- Skeletal examinations: Yes: half per litter, skeletal examinations were performed in all groups. Structural deviations were classified as follows: Malformations: major abnormalities that are rare and/or affect the survival or health of the species under investigation; Anomalies: minor abnormalities that are detected relatively frequently; Variants: a change that occurs within the normal population under investigation and is unlikely to adversely affect survival or health. This might include a delay in growth or morphogenesis that has otherwise followed a normal pattern of development.
- Head examinations: Yes, part of skeletal examination
Continuous variables: Dunnett's test or a modified t test
Non-continuous variables: Kruskal-Wallis test
Intergroup differences between the control and treated groups: Non-parametric version of the Williams test

Results and discussion

Results: maternal animals

Maternal developmental toxicity

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

Details on maternal toxic effects:
- No mortality occurred during the study.
- One female in the low dose group was found not pregnant at necropsy.
- Unilateral implantation was detected in one low dose female.
- The number of females with live foetuses on gestation Day 20 was 24 each in the control, mid- and high dose groups and 23 in the low dose group.
- No clinical sign was detected in any animal during the whole study and no sign of reaction to treatment was noted.
- In all treated females body weight and body weight gain were comparable to controls throughout the study.
- No changes were detected in food consumption between treated and control females.
- No significant differences were noted in terminal body weight, uterus weight and absolute weight gain within the groups.
- No findings were detected in treated females that could be considered treatment-related.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

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

Details on embryotoxic / teratogenic effects:
- Litter data and sex ratio were not affected by treatment.
- Two small foetuses (weight less than 2.7 g) were found in the control group and one in each of the treated groups.
- No other abnormalities were detected at the external examination of all foetuses.
- Malformations were noted in three foetuses, one in the control group, one in the mid-dose group and one in the high dose group. These malformations and the other findings noted were considered to be incidental.
- No changes that could be considered treatment-related were noted at skeletal examination of the foetuses between control and treated groups.

Fetal abnormalities

not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Neither clinical signs nor signs of reaction to treatment were noted in treated females. No significant differences were noted in body weight, food consumption, gravid uterus weight, litter data and macroscopic observation of treated females when compared to controls. No treatment-related changes were seen at the external, visceral and skeletal examination of foetuses from all groups. On the basis of the results obtained in this study, the dosage of 1000 mg/kg bw/day is considered as the NOAEL.

Applicant's summary and conclusion