Reaction mass of Fatty acids, montan-wax and Montan wax

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 FEB 2009 to 14 MAR 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD 414)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Remarks:

Deviation: In agreement with the Sponsor, no formulation analyses were performed at RTC for technical reasons (test item not analysable in the vehicle).

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Automatically generated during migration to IUCLID 6, no data available

IUPAC Name:

Automatically generated during migration to IUCLID 6, no data available

Details on test material:

Name of test material (as cited in study report): Licowax E
- Substance type: solid yellowish particulate (stated "powder" in study report)
- Physical state: solid
- Storage condition of test material: room temperature

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Italy S.r.l., San Pietro al Natisone (UD), Italy
- Age at study initiation: females: 9 weeks old; males: 11 weeks old
- Weight at study initiation: females: body weight range of 206.5 ¿ 224.5 g; males: at least 318.6 g
- Housing: in polycarbonte cages, no more than 5 per sex per cage before and after mating; one male, one female during mating
- Diet (ad libitum): commercially available laboratory rodent diet (4 RF 21, Mucedola S.r.l., Via G. Galilei, 4, 20019, Settimo Milanese (MI), Italy)
- Water (ad libitum): drinking water
- Acclimation period: 18 days


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

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: sesame oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The formulation was prepared daily. A double check of the amount of test item suspended in the vehicle was performed at each sample preparation. In addition, formulation samples were constantly stirred from preparation till administration (approximately 4 hours).

VEHICLE
- Amount of vehicle (if gavage): 10 ml/kg bw

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

In agreement with the Sponsor, no formulation analyses were performed at RTC for technical reasons (test item not analysable in the vehicle). A double check of the amount of test item suspended in the vehicle was performed at each sample preparation.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: overnight
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

females from Day 6 through Day 19 post coitum

Frequency of treatment:

once daily

Duration of test:

animals were sacrificed day 20 post coitum and necropsied

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
50 mg/kg bw/day
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
250 mg/kg bw/day
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

24 mated female rats

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: in accordance with the sponsor

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: 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


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: for individual animals from allocation to sacrifice. Once daily during the whole study and, in addition, once daily at approximately 1.5-2 hours after treatment, each animal was observed and any clinical sign was recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: on Days 0, 3, 6, 9, 12, 15 and 20 post coitum

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day: 20
- Organs examined: a detailed post mortem examination was conducted (including examination of the external surface and orifices).


- OTHER: Food consumption was measured on Days 3, 6, 9, 12, 15 and 20 post coitum starting from Day 0 post coitum

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
- Number of early resorptions: Yes
- Number of late resorptions: Yes

- Other: gross evaluation of placentae

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No data

-Other: 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;

Statistics:

For continuous variables the significance of the differences amongst group means were assessed by Dunnett's test or a modified t test, depending on the homogeneity of data.
Statistical analysis of non-continuous variables was carried out by means of the Kruskal-Wallis test and intergroup differences between the control and treated groups assessed by a non-parametric version of the Williams test.

Indices:

Pre-implantation loss was calculated as a percentage from the formula:
(no. of corpora lutea-no. of implantations)x100/no. of corpora lutea

Post-implantation loss was calculated as a percentage from the formula:
(no. of implantations-no. of live young)x100/no. of implantations

Total implantation loss was calculated as a percentage from the formula:
(no. of corpora lutea-no. of live young)x100/no. of corpora lutea

Sex ratios of the foetuses were calculated as the percentage of males per litter.
All derived values (e.g., means, percentages, ratios) first were calculated within the litter and the group values derived as a mean of individual litter values.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:no effects

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
External malformations were recorded in one foetus in the control group and one foetus in the low dose group and were considered to be incidental. Visceral malformations were noted in six foetuses, three in the control group and one in each treated group.Due to their low incidence and to the absence of a dose-relation these malformations and the other findings noted were considered to be incidental.

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, no treatment-related effects were observed up to 1000 mg/kg bw/day, the highest dose tested.

Executive summary:

Groups of 24 mated female Sprague-Dawley rats received the test item at dosages of 50, 250 and 1000 mg/kg/day. The test item was administered orally by gavage at a constant dosing volume of 10 ml/kg from day 6 through day 19 post coitum. A control group of 24 mated female rats received the vehicle (sesame oil) during the same treatment period. All females were sacrificed on gestation day 20 and subjected to post mortem examination.The number of corpora lutea, implantations, early and late resorptions, live and dead foetuses, uterus weight, foetal weight and sex was recorded. All foetuses were examined for external abnormalities. Approximately one half of the foetuses in each litter were examined in all groups for fixed-visceral and skeletal abnormalities.

No mortality occurred during the study. One control female and one low dose female were found not pregnant at necropsy.

The number of females with live foetuses on gestation day 20 was 23 each in the control and low dose group, and 24 each in the mid- and high dose group. No relevant clinical findings nor signs of reaction to treatment were detected in any animal throughout the study. Body weight and body weight gain of treated females were not affected by treatment. No changes were detected in food consumption between treated and control females. No differences were noted in terminal body weight, uterus weight and absolute weight gain within the groups that could be ascribed to treatment. Litter data and sex ratio were not affected by treatment.

Animals did not show macroscopic findings related to the administration of the test item. A total of 14 small foetuses were found at necropsy. They were equally distributed between the groups. External malformations were recorded in one foetus in the control group and one foetus in the low dose group and were considered to be incidental. Visceral malformations were noted in six foetuses, three in the control group and one in each treated group.Due to their low incidence and to the absence of a dose-relation 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 in any group.

On the basis of the results obtained, the dosage of 1000 mg/kg/day could be considered the NOAEL (No Observed Adverse Effect Level) in this study.  Therefore, the test substance has not to be classified for developmental toxicity according to Regulation (EC) 1272/2008 and Council Directive 67/548/EEC.