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Key value for chemical safety assessment

Effects on fertility

Description of key information

Testing proposed: Extended One-Generation Reproductive Toxicity Study (EOGRTS, OECD 443), basic test design (cohorts 1A and 1B without extension to include a F2 generation), rat, oral administration

Effect on fertility: via oral route
Endpoint conclusion:
no study available
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

A testing proposal for an Extended One-Generation Reproductive Toxicity Study (EOGRTS, basic test design (cohorts 1A and 1B without extension to include a F2 generation), rat, oral administration), according to OECD guideline 443 is provided to fulfil the standard information requirement of Annex X, Item 8.7.3.,of Regulation (EC) No. 1907/2006. As soon as the final results of the study are available, the technical dossier will be updated.

Effects on developmental toxicity

Description of key information

Developmental toxicity / teratogenicity (OECD 414, rat): NOAEL(maternal toxicity) ≥ 1000 mg/kg bw/day; NOAEL(development) ≥ 1000 mg/kg bw/day

Testing proposed: Prenatal Developmental Toxicity Study (PNDTS, OECD 414), rabbit, oral administration

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
Adopted in 2001
Deviations:
no
GLP compliance:
yes
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, Italy
- Age at study initiation: 9 weeks (females) and 11 weeks (males)
- Weight at study initiation: 191 - 221 g (females) and at least 316 g (males)
- Housing: Before and after pairing, the animals were housed no more than 5 of one sex to a cage, in clear polycarbonate cages measuring 59 x 38.5 x 20 cm with a stainless steel mesh lid and floor (Techniplast Gazzada S.a.r.l., Buguggiate, Varese, Italy). 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 43 x 27 x 18 cm with a stainless steel mesh lid and floor (Techniplast Gazzada S.a.r.l., Buguggiate, Varese, Italy). Each cage tray held absorbent material which was inspected and changed daily.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): 15 - 25
- Photoperiod (hrs dark / hrs light): 12 / 12
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
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.

VEHICLE
- 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:
yes
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:
daily, 7 days/week
Duration of test:
Days 6 - 19 of gestation
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
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:
CAGE SIDE OBSERVATIONS: Yes
- 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.

DETAILED CLINICAL OBSERVATIONS: Yes
- 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 h after treatment, each animal was observed and any clinical signs were recorded.

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

FOOD CONSUMPTION: Yes
- 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.
- Head examinations: Yes, part of skeletal examination

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.
Statistics:
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
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.
Key result
Dose descriptor:
NOAEL
Remarks:
maternal toxicity
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: no effects observed
Key result
Abnormalities:
no effects observed
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.
Key result
Dose descriptor:
NOAEL
Remarks:
development
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects observed
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Executive summary:

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.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
June - Nov 2008
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
Adopted January 2001
Deviations:
yes
Remarks:
Range-finder with reduced foetus analysis, reduced number of animals, no formulation analysis
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Italy
- Age at study initiation: 12 weeks
- Weight at study initiation: approx. 230 g
- Housing: up to 5/cage
- Diet : Mucedola S.r.l, 4RF 21, ad libitum
- Water : tap water, ad libitum
- Acclimation period: 5 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): 15 to 25
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The required amount of the test item was suspended in the vehicle; the formulations were prepared daily and concentrations were calculated and expressed in terms of test item as supplied.

VEHICLE
- Justification for use and choice of vehicle (if other than water): solubility
- Concentration in vehicle: 10, 30, 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw
Analytical verification of doses or concentrations:
no
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 and/or sperm in vaginal smear, referred to as day 0 of pregnancy
Duration of treatment / exposure:
14 days (from Day 6 to Day 19 of gestation)
Frequency of treatment:
once daily, 7 days/week
Duration of test:
approximately 1 month
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
8 mated females/group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: selected by sponsor
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: All clinical signs were recorded for individual animals. Each animal was observed daily before the start of treatment, at least once daily during the treatment period and on the day of necropsy. Any clinical signs were recorded from allocation until sacrifice.

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

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: Uterus and Ovaries (macrosocopic observation); furthermore, external surfaces and orifices; abnormalities were preserved in 10% buffered formol saline
Ovaries and uterine content:
•Gravid uterine weight
•number of corpora lutea
•number of implantation sites
•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; Intra-uterine deaths were classified as early resorptions (only placental remnants visible) or late resorptions (placental and foetal remnants visible)
•gross evaluation of placentae
Other: Uteri or individual uterine horns without visible implantations were immersed in a 20% solution of ammonium sulphide to reveal evidence of embryonic death at very early stages of implantation.

Litter data: Pre-implantation loss, post-implantation loss, total implantation loss, sex ratios
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No

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.
Statistics:
For continuous variables the significance of the differences amongst group means was 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.
Details on maternal toxic effects:
Maternal toxic effects: no effects

Details on maternal toxic effects:
- No mortality occurred during the study.
- All females were found pregnant at necropsy.
- No signs of toxicological significance were noted in any treated female.
- No signs of reaction to treatment were recorded at the daily post-dose observations performed 1 h after administration.
- Soft faeces on the cage tray were occasionally observed in the high dose group.
- Body weight and body weight gain in treated females were comparable to controls through the study.
- No differences were noted in litter data parameters between control and treated females.
- No relevant findings were reported at the macroscopic observation of females.
- Staining on different areas of body surface noted in some animals was not considered treatment-related.
Dose descriptor:
NOAEL
Remarks:
maternal toxicity
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: no effects observed
Abnormalities:
no effects observed
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects: no effects

Details on embryotoxic / teratogenic effects:
- No abnormalities were detected at the external examination of foetuses.
Dose descriptor:
NOAEL
Remarks:
development
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effect observed
Abnormalities:
no effects observed
Developmental effects observed:
no
Executive summary:

The results indicate that Licowax C is not maternally toxic or embryotoxic at the tested dose levels and the high dosage of 1000 mg/kg bw/day could be used for a subsequent main study.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The available information comprises adequate, reliable (Klimisch score 1 and 2) and consistent studies, and is thus sufficient to fulfil the standard information requirements set out in Annexes IX and X, Item 8.7.2., of Regulation (EC) No. 1907/2006.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

There is data available from a prenatal developmental toxicity study (Liberati, 2009), which was chosen as the key study, and the corresponding preliminary test which served as a range finder (RTC, 2008).

In the key study doses of 100, 300 and 1000 mg/kg bw/day, which had been analytically confirmed, were administered by oral gavage. No maternal toxic effects were observed up to the highest dose of 1000 mg/kg bw/day, there were no mortality or clinical signs observed. Food consumption and body weight development were not affected by treatment with the test substance, either. The number of dams with live fetuses was comparable among all dose groups. Litter data and sex ratio were not affected by the treatment, and no visceral or skeletal abnormalities were noted within the foetuses which could be attributed to the test substance.

This view was supported by the corresponding range-finder study (RTC, 2008), which was conducted with a reduced animal number, and no analysis of the administered dose. No mortality occurred during the study, either. All females were found pregnant at necropsy. No signs of toxicological significance were noted in any treated female. No signs of reaction to treatment were recorded at the daily post-dose observations performed 1 h after administration. Soft faeces on the cage tray were occasionally observed in the high dose group. Body weight and body weight gain in treated females were comparable to controls through the study. No differences were noted in litter data parameters between control and treated females. No relevant findings were reported at the macroscopic observation of females. Staining on different areas of body surface noted in some animals was not considered treatment-related. In this range-finder study the foetuses were only examined externally, and no abnormalities were detected.

In order to investigate the developmental toxicity in a second (non-rodent) species, a testing proposal for a Prenatal Developmental Toxicity Study (PNDTS, OECD 414), rabbit, oral administration, is provided to fulfil the standard information requirement of Annex X, Item 8.7.2., of Regulation (EC) No.. 1907/2006. As soon as the final results of the study are available, the technical dossier will be updated.

Justification for classification or non-classification

The available data on developmental toxicity of the test substance in the rat do not meet the criteria for classification according to Regulation (EC) No. 1272/2008. However, as no studies investigating the reproductive toxicity and the developmental toxicity in a second (non-rodent) species are available, no final hazard conclusion, incl. classification, can be made. Since there is no indication of any adverse systemic toxic effect in any of the available acute and subchronic studies, the registered substance is not classified for reproductive toxicity based on 'data lacking'. As soon as the reproductive and developmental toxicity studies proposed are finalised, the hazard assessment and the 'justification for classification or non-classification' will be updated.

Additional information