Reaction mass of fatty acids, montan-wax, 1-methyl-1,3- propanediyl esters and fatty acids, montan-wax, ethylene esters and fatty

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

28 AUG 2008 to 15 NOV 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD 421), for justification of read-across see Chapter 1 of CSR

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

: treatment of females only until day 3 post partum

GLP compliance:

yes

Remarks:

deviation: no analysis of the formulated test item was performed (technical reasons, test item in vehicle not analysable; in agreement to the sponsor)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Montan waxes, Type E

IUPAC Name:

Montan waxes, Type E

Details on test material:

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

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Italy, San Pietro al Natisone, Italy
- Age at study initiation: (P): 6-7 wks
- Weight at study initiation: (P) Males: 183-206 g; Females: 148-177 g
- Housing: 5 per cage during the pre-mating period, mating one male/one female per cage
- Diet (ad libitum): 4RF21 rodent diet, Mucedola S.r.L., Settimo Milanese, Italy
- Water (ad libitum)
- Acclimation period: approx. 2 weeks

ENVIRONMENTAL CONDITIONS
- 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

Vehicle:

other: sesame oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was suspended in sesame oil, the formulation was prepared daily in concentrations of 1, 10 and 100 mg/ml

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

Details on mating procedure:

- M/F ratio per cage: 1/1
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): individually

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

No analysis of the formulated test item was performed (technical reasons, test item in vehicle not analysable; in agreement with the sponsor). A double check of the amount of test item to be suspended in the vehicle was performed at each sample preparation

Duration of treatment / exposure:

males: 49-52 days (2 consecutive weeks prior to mating, until necropsy)
females: 2 consecutive weeks prior to mating, throughout gestation until day 3 post partum

Frequency of treatment:

once daily

Details on study schedule:

pups were sacrificed at Day 4 post partum

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
10 mg/kg bw/day
Basis:
nominal conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
100 mg/kg bw/day
Basis:
nominal conc.

Doses / concentrations 3

Remarks:

Doses / Concentrations:
1000 mg/kg bw/day
Basis:
nominal conc.

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily


BODY WEIGHT: Yes
- Time schedule for examinations: on the day of allocation to treatment groups, the day after exposure start, weekly thereafter and just prior to necropsy (females also on days 0, 7, 14 and 20 post coitum and days 1 and 4 post partum)

OTHER: determination of mean food consumption (mean value of each cage weekly until mating, for female animals also on days 7, 14 and 20 post coitum and day 4 post partum

Oestrous cyclicity (parental animals):

Vaginal smears were taken daily in the morning from the first day of treatment until a positive identification of mating is made. The vaginal smear data were examined to determine the following:
a) anomalies of the oestrous cycle;
b) the pre-coital interval (i.e., the number of nights paired prior to the detection of mating).

Sperm parameters (parental animals):

During the necropsy procedure, one cauda from one epididymis of each animal in the high dose group and control group was taken for sperm count and evaluation of motility and viability.

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
sex and number of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight, physical or behavioural abnormalities


GROSS EXAMINATION OF DEAD PUPS: yes, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals
- Maternal animals: All surviving animals


GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations


HISTOPATHOLOGY / ORGAN WEIGHTS

Organs / Tissues Weight Fixation Microscopic
Preservation Examination
Abnormalities ¿ ¿
Adrenals ¿ ¿
Coagulating glands ¿
Epididymides ¿ ¿ ¿
Kidneys ¿ ¿
Liver ¿ ¿ ¿
Ovaries ¿ ¿ ¿
Prostate gland ¿ ¿
One Epididymal cauda a) ¿
Seminal vesicles ¿ ¿
Spleen ¿ ¿
Testes ¿ ¿b) ¿b)
Thyroid ¿ ¿
Uterus - cervix ¿
Vagina ¿

a: only in control and high dose group animals
b: one testis in control and high dose group animals

Postmortem examinations (offspring):

All pups found dead in the cage or sacrificed for human reasons were necropsied. All live pups were killed on Day 4 post partum and examined for the following:
a) external and internal abnormalities;
b) sex confirmation by gonadal inspection.

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.
Statistical analysis of histopathological findings was carried out by means of the non-parametric Kolmogorov-Smirnov test.

Reproductive indices:

Copulatory Index (%): No. of animals mated/No. of animals paired x 100
Fertility Index (%): No. of animals fertile/No. of animals paired x 100

Offspring viability indices:

Pre-birth loss was calculated as a percentage from the formula:
(No. of visible implantations - total litter size) /No. of visible implantations x 100

Pup loss at birth was calculated as a percentage from the formula:
(Total litter size - live litter size)/Total litter size x 100

Cumulative pup loss on Day 4 post partum was calculated as a percentage from the formula:
(Total litter size at birth - live litter size at Day 4)/Total litter size at birth x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
A slight increase in pre-coital interval and a slight reduction in copulation plugs were noted in all treated animals compared to control. The differences did not show any dose-relation and were comparable with our historical control data. Therefore, they were not considered to be of toxicological relevance.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: no treatment-related effects were observed

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: no treatment-related effects were observed

Overall reproductive toxicity

Reproductive 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 10 males and 10 females Sprague-Dawley rats received the test item by gavage at dosages of 10, 100 and 1000 mg/kg/day. A similarly constituted control group of animals received the vehicle (sesame oil). All doses were administered at a constant volume of 10 ml/kg bodyweight.

Males were treated for 2 weeks prior to pairing and during pairing of all females until the day before necropsy, for a total of approximately 7 weeks. Females were treated for 2 weeks prior to pairing, during pairing and throughout thegestation and lactation periods until Day 3 post partum. The following investigations were performed in all groups: body weight, clinical signs, food consumption, oestrous cycle, mating performance, litter data, macroscopic observations,organ weights and histopathological examination of abnormalities.

The following investigations were performed only on control and high dose groups: spermanalysis, testicular sperm head count and histopathological examination of testes,epididymides, liver and ovaries. Additionally, histopathological examination was performed also on the liver.

No mortality occurred in females. Animals did not show any sign of toxicity. No treatment related changes were seen in either sex. No effects on food consumption were observed in either males or females. Oestrus cycle, copulatory index and fertility index did not show intergroup differences. A slight increase in pre-coital interval and a slight reduction in copulation plugs were notedin all treated animals compared to control. These changes were not considered to be clearly treatment-related.

No significant differences were observed for reproductive parameters between the treated groups and the controls. No differences in total and live litter size were noted between groups at birth and on Day 4 post partum. In addition, litter weight, mean pup weight and sex ratio were comparable between groups on Days 1 and 4 post partum. Pre-weaning clinical signs did not show treatment-related effects.
Necropsy findings in decedent pups, human killed pups and pups at Day 4 post partum did not reveal any treatment-related effect.

Terminal body weight and organ weights were unaffected by treatment in both sexes.
Sperm analysis performed in control and high dose males was unaffected by treatment. Daily sperm production measured by testicular sperm head counts, did not show any differences in the high dose group compared to the control group.

No macroscopic observation was reported in treated parental animals that could be considered treatment-related. The histopathological examination of testes, epididymides, liver and ovaries did not reveal any evident differences between the findings observed in high dose and control animals that could be considered treatment-related. In addition, there were no treatment related findings at the histopathological examination performed on the liver. On the basis of the results obtained the NOAEL (No Observed Adverse Effect Level) could be considered 1000 mg/kg/day (dose for parental animals).