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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
toxicity to reproduction
Remarks:
other: OECD 421 Reproductive Screeing test
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2007-10-03 till 2008-06-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline-conform study under GLP without deviations

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Milled SLI (76)
- EC name of test material: Fatty acids, C12-18 and C18-unsatd., 2-sulfoethyl esters, sodium salts
- Physical state: Off-white fine powder
- Storage condition of test material: At room temperature (20 ± 5 °C) in the original container away from direct sunlight, with dessicant

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: RCC Ltd, Füllinsdorf / Switzerland
- Age at study initiation: (P) 11 weeks wks
- Weight at study initiation: (P) Males: 291 to 333 g; Females: 172 to 207 g
- Housing: Individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding
- Diet (e.g. ad libitum): Pelleted standard Kliba Nafag 3433 rat / mouse maintenance diet, ad libidum
- Water (e.g. ad libitum): Community tap-water from Füllinsdorf, ad libidum
- Acclimation period: Minimum 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 - 70%
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
For groups 2 and 3: dose formulations were prepared every second week.
Fro group 4: on the first day of the study it was apparent that mixing a batch large enough to cover this length of treatment resulted in the incorporation of a lot of air. From day 2 of the study dose formulation was therefore mixed daily.

VEHICLE
- Concentration in vehicle: no data
- Amount of vehicle (if gavage): 10 mL/kg
- Purity: Milli-Q-water
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: 14 days maximum
- Proof of pregnancy: vaginal plug and sperm in vaginal smear referred to as day 0 post coitum
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- Any other deviations from standard protocol: none
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulations were stored in the refrigerator (2 - 8 °C) in sealed glass containers.

For groups 1, 2 and 3: One mL samples were taken in triplicate from the top, middle and bottom (9 in total) of the treated formulation during stirring.
Five 1 mL samples were taken from the vehicle control group (5 in total). Following 14-days refrigerated storage of the formulations 1 mL samples were taken from the middle of each formulation during stirring and from the vehicle control to confirm achived stability. After dosing on day 1 of each preparation, the residual formulation was stored frozen. From the formulations mixed on each subsequent occasion five 1 mL samples were taken from the middle of each formulation during stirring and from the vehicle control. These samples were used to confirm achieved concentration only.

For group 4: On day 1 of the treatment period, samples were taken from the dose preparation made for 14 days of treatment for homogeneity and concentration. On day 2 of the treatment period, homogeneity and concentration samples were taken when the dose formulation was mixed for the first daily preparation, as described above. Further, on days 7, 10, 13 and 15 of the treatment period and thereafter every two week (to coincide with preparation of dose for groups 2 and 3), samples were taken to confirm the achieved concentration. Five 1 mL samples were taken from the middle (5 in total) of the formulation on each occasion of sampling for achieved concentration.
Duration of treatment / exposure:
Males: 28 days
Females: approx 70 days (approx 10 weeks) (sacrificed on day 22 post partum)
Frequency of treatment:
Males were treated over a 15-day pre-pairing period and during the pairing period up to one day before necropsy.
Females were treated throughout the pre-pairing, pairing, gestation and lactation period up to weaning on day 21 post partum.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
100 mg/kg bw/day
Basis:
no data
Remarks:
Doses / Concentrations:
300 mg/kg bw/day
Basis:
no data
Remarks:
Doses / Concentrations:
1000 mg/kg bw/day
Basis:
no data
No. of animals per sex per dose:
12 males and 12 females per group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Selected by the Sponsor based on previous rat toxicity data.
Positive control:
None

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily


BODY WEIGHT: Yes
- Time schedule for examinations: dailiy /(recorded from treatment start to day of necropsy).


FOOD CONSUMPTION: yes
- Time schedule:
Males: Weekly during pre-pairing and after pairing periods
Females: Pre-pairing period days 1-8 and 8-15; gestation days 0-7, 7-14 and 14-21 post coitum, and lactation days 1-7 and 7-14 post partum
No food consumption was recorded during the pairing period.

OTHER:
Vaginal Smears: Throughout pre-pairing and pairing until the smear was sperm-positive or a copulation plug was observed.
Oestrous cyclicity (parental animals):
Yes
Sperm parameters (parental animals):
Parameters examined in [F1] male parental generations: testis weight, epididymis weight, spermatogenicity, prostata and seminal vesicles (histopathology)
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, body weight, external examination

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 were sacrificed after end of mating period
- Maternal animals: All surviving animals were sacrificed on day 22 post partum (at the end of the waening period)


GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations with special attention at the organs of the reproductive system.


HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated below were prepared for microscopic examination and weighed, respectively:
- gross lesions
- testes
- epididymes
- prostate
- seminal vesicles
- ovaries
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at 21 days of age.
- These animals were subjected to postmortem examinations (macroscopic) as follows:


GROSS NECROPSY
- Gross necropsy consisted of external examinations

HISTOPATHOLOGY / ORGAN WEIGTHS: no
Statistics:
Statistical methods used to analyze food consumption, body weight, macroscopical findings, organ weights and reproduction data:
• Means and standard deviations of various data were calculated and included in this report.
• The Dunnett-test (many to one t-test) based on a pooled variance estimate, applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test), applied instead of the Dunnetttest when the data could not be assumed to follow a normal distribution.
• Fisher's exact-test, applied if the variables could be dichotomized without loss of information
Reproductive indices:
fertility indices, mean precoital time, post-implantation losses
Offspring viability indices:
Mean litter size, pup sex ratios and viability indices.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
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)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
All animals survived until the scheduled necropsy, except one female at 1000 mg/kg, which died after treatment on day 14 of the pre-pairing period. The death was considered that due to a reflux the test item went into the lung (possibly due to a dosing error).
At 1000 mg/kg,one male pushed its head through the bedding and had salivation on days 14 and 15 of the pre-pairing period. Male no. 41 had ruffled fur starting on day 6 of the pairing period and seven days later also a hunched posture until the end of the study. Due to the isolated occurance these findings were considered to be not test item-related.
At 100 mg/kg, one male had a hairless area on its front leg during the pairing and after pairing period. At 1000 mg/kg, one female had a wound on its shoulder on day 2 of the gestation period onwards, which developed a scab and then a hairless area until the end of the study. These findings were considered to be incidental.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Males: Mean body weight gain was slightly reduced during the study at 300 and 1000 mg/kg, and recovered in the after pairing period at 300 mg/kg but not at 1000 mg/kg. At 300 and 1000 mg/kg, mean body weight was statistically significantly reduced from days 13 and 15 of the pairing period, respectively, until the end of the study. No clear dose-dependency was noted.
Mean food consumption was slightly dose-dependently reduced from the second week of the pre-pairing period until the end of the treatment. At 1000 mg/kg in the after pairing period, the difference reached statistical significance.

Females: At 300 and 1000 mg/kg, mean body weight gain was slightly reduced during the lactation period. The difference reached statistical significance from days 10 - 13 at 1000 mg/kg. Mean body weights were not affected by treatment with the test item in all groups.
At 1000 mg/kg, mean food consumption was slightly but not statistically significantly reduced during the treatment period. At 300 mg/kg, mean food consumption was slightly but not statistically significantly reduced during the second week of the lactation period.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
The mean number of days from estrous to estrous was unaffected by treatment with the test item. Mean number of days was 4.6, 4.0, 4.1 and 4.0 days in order of ascending dose level. A shortened cycle was noted in one female at 300 mg/kg and one control female was acyclic.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
The assessment of the integrity of the spermatogenetic cycle did not provide any evidence of impaired spermatogenesis.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
The fertility rate was 100% in all groups. At all dose-levels, there were no treatment-related effects on estrous cycle, precoital time, mean duration of gestation, number of corpora lutea and implantations, post-implantation loss, pup survival or litter size from birth through to scheduled sacrifice on day 21 post partum.

ORGAN WEIGHTS (PARENTAL ANIMALS)
No significant deviations in mean organ weight of testes and epididymides were recorded which could be attributed to the treatment with the test item, since lower mean absolute weights of epididymides were considered to be in relation with lower body weights and no test item-related histopathological findings were present.

GROSS PATHOLOGY AND HISTOPATHOLOGY (PARENTAL ANIMALS)
No test item-related histopathologic findings were observed in the reproductive organs of either sex from the parental generation.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Remarks on result:
other: Generation: P and F1 (migrated information)
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Remarks on result:
other: Generation: P and F1 (migrated information)

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:
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

VIABILITY (OFFSPRING)
Neonatal mortality was generally low and considered to be unaffected by treatment with the test item.
The total numbers of pup loss during the first four days of life were 2, 2, 0 and 1 in order of ascending dose level, corresponding to 1.3, 1.4, 0.0 and 0.7% of living pups. The resulting viability indices were 98.8, 98.6, 100 and 99.3% in order of ascending dose levels.

CLINICAL SIGNS (OFFSPRING)
No findings that were considered to be test item-related were noted at first litter check or during lactation.
Two pups in the control group and at 100 mg/kg, respectively, had no milk in the stomach either at the first litter check or on day 4 post partum and died.

BODY WEIGHT (OFFSPRING)
Mean pup weights were unaffected by treatment with the test item. Mean pup weight development during lactation was unaffected by treatment with the test item.

SEXUAL MATURATION (OFFSPRING)
Not examinated

GROSS PATHOLOGY (OFFSPRING)
No test item-related findings were noted at macroscopic examination of the pups. For one pup at 100 mg/kg, which died spontaneously on day 2 post partum, an advanced austolysis was noted.

Effect levels (F1)

Remarks on result:
not determinable due to absence of adverse toxic effects

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on these results a general NOAEL (No Observed Adverse Effect Level) was considered to be 1000 mg/kg body weight/day.
With respect to reproduction/developmental toxicity a NOEL (No Observed Effect Level) was considered to be 1000 mg/kg/day.
Executive summary:

The purpose of this study was to generate preliminary information concerning the effects of MILLED SLI (76) on male and female reproductive performance such as gonadal function, mating behavior, conception and parturition.

Four groups of 12 males and 12 females were treated by gavage with MILLED SLI (76) once daily. The dose levels were: 0 mg/kg bw/day (control group), 100 mg/kg bw/day, 300 mg/kg bw/day, and 1000 mg/kg bw/day. Males were treated over a 15-day pre-pairing period and during the pairing period up to one day before necropsy. Females were treated throughout the pre-pairing, pairing, gestation and lactation period up to weaning on day 21 post partum. A standard dose volume of 10 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (Milli-Q-water).

All animals survived until scheduled necropsy, except one female at 1000 mg/kg, which died, possibly due to a dosing error.

In males at 300 and 1000 mg/kg, mean food consumption was slightly reduced. Additionally, mean body weights were reduced from the pairing period until the end of the study, whereas no clear dose-dependency was noted. Mean body weight gain was slightly reduced and recovered in the after pairing period at 300 mg/kg but not at 1000 mg/kg.

In females at 1000 mg/kg, mean food consumption was slightly reduced during the treatment period. At 300 mg/kg mean food consumption was only reduced during the second week of the lactation period. As a result, a slight transient reduction of mean body weight gain was noted in the lactation period at both dose levels. Mean body weights were similar in all groups at the end of the study.

The fertility rate was 100% in all groups. At all dose-levels, there were no treatment-related effects on estrous cycle, precoital time, mean duration of gestation, number of corpora lutea and implantations, post-implantation loss, pup survival or litter size from birth through to scheduled sacrifice on day 21 post partum.

No significant deviations in mean organ weight of testes and epididymides were recorded which could be attributed to the treatment with the test item, since lower mean absolute weights of epididymides were considered to be in relation with lower body weights and no test item-related histopathological findings were present.

No test item-related histopathologic findings were observed in the reproductive organs of either sex from the parental generation.

No abnormal findings were noted for pups at first litter check or during the lactation period. Sex ratios at first litter check and on day 21 post partum were unaffected by treatment with the test item. Mean pup weights on day 1 post partum and mean pup weight development during the lactation period were unaffected by treatment with the test item.