Registration Dossier

Administrative data

Endpoint:
toxicity to reproduction
Remarks:
other: reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 December 2011 to 26 April 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: The United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, July 2000.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): Salicynalva
- Substance type: Clear colourless to pale yellow liquid
- Physical state: liquid
- Expiration date of the lot/batch: 31 October 2013
- Storage condition of test material: At room temperature in the dark
- Volatile: Not indicated
- Relative density: 0.9460
- Stability at higher temperatures: Not indicated
- Vapour pressure: 6.4 Pa

Test animals

Species:
rat
Strain:
other: Crl:WI(Han).
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
Nulliparous and nonpregnant females and untreated animals were used at initiation of the study.
- Age at study initiation: Approximately 12 weeks.
- Weight at study initiation: mean weight at start of treatment was 321 - 325 gr (males) or 203 - 204 gr (females).
- Fasting period before study: no
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon cages.
Mating: Females were caged together with males on a one-to-one-basis in Macrolon cages.
Post-mating: Males were housed in their home cage with a maximum of 5 animals/cage. Females were individually housed in Macrolon cages.
General: Sterilised sawdust as bedding material and paper as cage enrichment were supplied.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, approximately 15 room air changes/hour, and a 12-hour light/12-hour dark cycle.

Temporary deviations from the minimum level of relative humidity occurred.
Evaluation: Laboratory historical data do not indicate an effect of the deviations.

IN-LIFE DATES: From: 27 December 2011 to 26 April 2012

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
- Method of formulation: Oral, by inclusion in the diet.
- Dietary Inclusion Levels: The amount of test substance incorporated into the diet was kept at a constant level in terms of ppm, throughout the study period. After termination, the actual test substance intake was estimated based on the body weight and food consumption values.
- Storage conditions of formulations: In the freezer until day of use. Twice weekly the pellets were defrosted and offered to the animals for a maximum of four days.
- Stability of pelleted diet: At least 14 days stable in the freezer and 4 days at room temperature (concentration range of 200 to 4.500 ppm; determined during NOTOX Project 498293).
Details on mating procedure:
- M/F ratio per cage:1/1 (one female was cohabitated with one male of the same treatment group, avoiding sibling mating (Charles River supplied non-litter mates).
- Length of cohabitation: A maximum of 14 days was allowed for mating.
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage, by staging of the estrous cycle and/or or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged individually in Macrolon cages (MIII type, height 18 cm).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on a single occasion during the treatment phase (07 March 2012), according to a validated method (NOTOX Project 498293). Dietary samples were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations).

In addition, random samples were taken and stored at ≤-15°C for possible future analysis. Any remaining samples will be discarded after approval by the sponsor, or at finalization of the study report.

The accuracy of diet preparations was considered acceptable if the mean measured concentrations were 80-120% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%.

The concentrations analysed in the diets of Group 2, Group 3 and Group 4 were below target concentrations (i.e. mean accuracies between 67% and 77%). This was probably due to loss of test substance during preparation of the pellets (volatilized).

A small response at the retention time of the test substance was observed in the chromatograms of the Group 1 diet. The maximum contribution to the samples based on area was 0.6%.

The diets of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

For the diets of Groups 2, 3 and 4 a mean accuracy of 67%, 71% and 77%, respectively, was obtained, which was below the criterion range 80-120%.
Evaluation: As no treatment related findings were noted up to the highest dose level tested (1.000 ppm) and as this level was just below the criterion range (77% instead of 80%), it was decided this slight deviation was negligible.
Duration of treatment / exposure:
Males were exposed for 32 days, i.e. 2 weeks prior to mating, during mating until scheduled necropsy. Females were exposed for 43-56 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 5 days of lactation.

Pups were not treated directly, but were potentially exposed to the test substance in utero and through lactational transfer.
Frequency of treatment:
Ad libitum.
Details on study schedule:
- Age at mating: Approximately 14 weeks
Doses / concentrations
Remarks:
Doses / Concentrations:
circa 15, 30 and 70 mg/kg bw
Basis:
actual ingested
Based on measured concentrations in the diet which were nominally 200, 400 and 1000 ppm
No. of animals per sex per dose:
10
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Dose levels were based on results of a 14-Day dose range finding study.
Based on the results of this dose range finding study, dose levels for the main study were selected by the sponsor and determined to be 200, 400 and 1.000 ppm.
- Parturition: The females were allowed to litter normally. Day 1 of lactation was defined as the day when a litter was found completed (i.e. membranes, placentas cleaned up, nest built up and/or feeding of pups started). Females that were littering were left undisturbed.
- Identification of pups: On Day 1 of lactation, all pups were individually identified by means of subcutaneous injection of Indian ink.
Positive control:
no

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS:
- Time schedule: At least twice daily.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: Daily, detailed clinical observations were made in all animals. The time of onset, grade and duration of any observed sign was recorded.

BODY WEIGHT:
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum, and during lactation on Days 1 and 4.

FOOD CONSUMPTION:
- Twice weekly, except for males and females which were housed together for mating and for females without evidence of mating.
Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.

FOOD EFFICIENCY:
- (average food consumption [per animal per day]/average body weight per cage)x1000

WATER CONSUMPTION : No.
Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY:
- Time schedule for collection of blood: between 7.00 and 10.30 a.m.
- Anaesthetic used for blood collection: Yes (iso-flurane)
- Animals fasted: No.
- How many animals: 10 animals/sex/group
- Parameters checked were: According to test guidelines

CLINICAL CHEMISTRY: No

URINALYSIS: No

On Day 1 of lactation, an unrealistic high value for body weight was collected for one female from the control group; this value was excluded from the tables.
Evaluation: Sufficient information available for evaluation.

Clinical signs observations were not performed for one female from the control group on Day 3 of lactation.
Evaluation: Sufficient information available for evaluation.

Observations of pups were not recorded online on the following occasions:
Day 4 of lactation for one litter from the control group, two litters from Group 3, and on Day 5 of lactation for one pup of one litter from Group 2 and one pup of one litter from Group 3.
Evaluation: Body weight of these pups was determined on Day 4 and therefore these pups were also observed at that moment. In addition, no symptoms were recorded the day before and the day following this omission. Sufficient data is available for a thorough evaluation.

One pup of one litter from the control group was not observed and weighed on Day 1 of lactation. It was not recorded if one pup of one litter from Group 2 went into necropsy. In addition, for 11 pups of one litter from the control group external examination at necropsy was not recorded.
Evaluation: No findings were noted on the other days. Sufficient information available for evaluation.


NEUROBEHAVIOURAL EXAMINATION: No
Estrous cyclicity (parental animals):
Not determined.
Sperm parameters (parental animals):
Slides of the testes were prepared for histopathological staging of spermatogenesis (males of the control and high dose group and animals suspected to be infertile).

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

- Mortality: The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated.
- Clinical signs: At least once daily, detailed clinical observations were made in all animals.
- Body weights: Live pups were weighed on Days 1 and 4 of lactation.
- Sex: Sex was determined for all pups on Days 1 and 4 of lactation (by assessment of the ano-genital distance).

GROSS EXAMINATION OF DEAD PUPS
Yes, if possible, defects or cause of death were evaluated.
Postmortem examinations (parental animals):
GROSS PATHOLOGY
- The animals were not deprived of food. Animals surviving to scheduled necropsy were deeply anaesthetised and subsequently exsanguinated.
- According to test guidelines.
- The number of former implantation sites and corpora lutea were recorded for all paired females.

ORGAN WEIGHTS
- Epididymides, Kidneys, Liver, Spleen and Testes.

HISTOPATHOLOGY
According to test guidelines
Postmortem examinations (offspring):
SACRIFICE
Pups surviving to planned termination were killed by decapitation on lactation Days 5-7.

GROSS NECROPSY
All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated. Any abnormal pup, organ or tissue was preserved in 10% buffered formalin for possible further examination.

HISTOPATHOLOGY / ORGAN WEIGHTS
No.
Statistics:
The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables.
Reproductive indices:
For each group, the following calculations were performed:
- Mating index: Number of females mated/Number of females paired x 100
- Fertility index: Number of pregnant females/Number of females paired x 100
- Conception index: Number of pregnant females/Number of females mated x 100
- Gestation index: Number of females bearing live pups/Number of pregnant females x 100
- Duration of gestation: Number of days between confirmation of mating and the beginning of parturition
Offspring viability indices:
- Percentage live males at First Litter Check: Number of live male pups at First Litter Check/Number of live pups at First Litter Check x 100
- Percentage live females at First Litter Check: Number of live female pups at First Litter Check/Number of live pups at First Litter Check x 100
- Percentage of postnatal loss Days 0-4 of lactation: Number of dead pups on Day 4 of lactation/Number of live pups at First Litter Check x 100
- Viability index: Number of live pups on Day 4 of lactation / Number of pups born alive x 100

Results and discussion

Results: P0 (first parental animals)

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
Description (incidence and severity):
As no microscopic correlate was noted for the increased liver weights at 1.000 ppm, it was not considered toxicologically significant.
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: estrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

MORTALITY
No mortality occurred during the study period.

CLINICAL SIGNS
No treatment related clinical signs were noted up to 1.000 ppm.

One female at 200 ppm showed swelling of the right foreleg for several days and one female at 400 ppm showed piloerection for one day. At this low incidence and as it was not noted at 1.000 ppm, it was not considered toxicologically significant.

BODY WEIGHTS
For all dose levels, body weights and body weight gain remained in the same range as controls over the treatment period.

One female at 400 ppm showed a body weight loss of 9% over Days 1 to 4 of lactation. At this single occurrence at the mid dose level, it was not considered toxicologically relevant.

FOOD CONSUMPTION
No toxicologically relevant findings were noted for food consumption before or after allowance for body weight for animals treated up to 1.000 ppm.

The statistical significant changes noted for treated females (all dose levels) during post-coitum and/or lactation were not considered toxicologically relevant as all values were well within normal limits of which the concurrent control group at the lower end.

The female at 1000 ppm that showed a severe body weight loss during lactation also showed a severely reduced food consumption during that period. This was not considered toxicologically relevant, as it was only noted for one animal at the mid dose.


Test article intake
The mean test article intake values (mg substance/kg body weight/day) are summarized in the following two tables.

Mean test article intake when corrected for nominal dose level:

Nominal dose level Group 2 Group 3 Group 4
200 ppm 400 ppm 1.000 ppm

MALES
Premating 14 29 73
Mating 13 28 70

FEMALES
Premating 16 33 79
Post-coitum 16 32 85
Lactation 23 44 124

Mean test article intake when corrected for mean value of recovery:

Mean value of accuracy Group 2 Group 3 Group 4
134 ppm 284 ppm 770 ppm

MALES
Premating 9 21 56
Postmating 9 20 54

FEMALES
Premating 11 23 61
Postcoitum 11 23 65
Lactation 15 31 95

HAEMATOLOGY
For haematology parameters, no treatment related findings were noted up to 1.000 ppm for both sexes.

Two females of the control group showed a high percentage of neutrophils and eosinophils with concurrent low percentage of lymphocytes. All other parameters determined for these females were within normal limits, and it was therefore not considered toxicologically significant. Moreover, as it concerned two control animals it was not treatment related.

MACROSCOPIC EXAMINATION
Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment.

Incidental findings consisted of pelvic dilation of the kidneys, nodule at the epididymides, discolouration of the preputial or clitoral glands, enlarged liver or spleen, fluid in the uterus, and spleen reduced in size. These findings were within the background range of findings that are encountered among rats of this age and strain and did not show a dose-related incidence trend, and were therefore not considered to be of any toxicological relevance.

ORGAN WEIGHTS
Increased liver weights were noted at 1.000 ppm for both sexes. This was statistically significant for absolute liver weight for the females and for absolute and relative liver weights for the males.

No treatment related effects were noted for the weights of the kidneys, spleen, testes and epididymides up to 1.000 ppm.

MICROSCOPIC EXAMINATION
There were no treatment related microscopic observations. Recorded findings were considered to be within the normal range of background pathology encountered in Wistar (Han) rats of this age and strain. There was no correlate to the statistically significant increased group mean liver weights of 1.000 ppm animals.

No abnormalities were seen in the reproductive organs of the pair of Group 3 animals that failed to conceive. There was evidence that the female was cycling. Staging of spermatogenesis did not provide any evidence of test article related impairment to the spermatogenetic cycle.

REPRODUCTIVE DATA
No toxicologically relevant effects on reproductive parameters were noted.

Of the control group one female mated within 14 days. All other females mated within 4 days of pairing. One female (no. 61) at 400 ppm was not pregnant.

Mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment.

GESTATION
Gestation index and duration of gestation were within normal limits for all groups.

PARTURITION/MATERNAL CARE
No signs of difficult or prolonged parturition were noted among the pregnant females.
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
(parental)
Effect level:
>= 70 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No parental toxicity up to the highest dose tested
Dose descriptor:
NOAEL
Remarks:
for reproduction
Effect level:
> 70 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No toxicity to reproduction up to highest dose tested.

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

Details on results (F1)

EARLY POSTNATAL PUP DEVELOPMENT
Number of dead and living pups at first litter check, postnatal loss, viability index and sex ratio were unaffected by treatment, and clinical signs, body weight and external macroscopy did not reveal treatment-related findings.

MORTALITY PUPS
Two pups of the control group, one pup at 200 ppm and one pup at 400 ppm were found dead or missing during the first days of lactation. Pups missing were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

CLINICAL SIGNS PUPS
Pale and/or lean appearance was noted for two pups (one at the control and one at the mid dose group). At this low incidence, it was not considered treatment related.

BODY WEIGHT PUPS
Body weights of pups were considered to have been unaffected by treatment up to 1.000 ppm.

MACROSCOPY PUPS
Pale and lean appearance was noted for one pup at 400 ppm. At this single occurrence it was considered to be of no toxicological relevance.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

The dietary concentrations were analysed and for the 200, 400 and 1000 ppm the doses for males were 13, 28 and 70 mg Salicynalva, respectively and for the females this was, 16, 32 and 85 mg/kg bw, respectively. The average values for the low and mid dose are presented in the text and for the high dose the lower dose of 70 mg/kg bw from the males is also used for females being the lower value.

Applicant's summary and conclusion

Conclusions:
In conclusion, treatment with Salicynalva by dietary administration in male and female Wistar Han rats at dose levels of circa 15, 30 and 70-85 mg/kg bw revealed no parental, reproduction and developmental toxicity for treatment up to the highest dose.

A parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 1.000 ppm was established.

The actual test substance intake (based on body weight and food consumption) at 1.000 ppm was 70 to 73 mg substance/kg body weight/day for the males and 79 to 124 mg substance/kg body weight/day for the females.
Executive summary:

A reproscreen study according to OECD TG 421 has been conducted for the substance. Nominal values of 200, 400 and 1000 ppm have been administered via the diet. Chemical analysis showed mean accuracies of 67%, 71% and 77% for the diets of Groups 2, 3 and 4, respectively, which was below the criterion range 80-120%. Therefore the analysed concentrations were used for determining the doses resulted in circa 15, 30 and 70 to 73 mg substance/kg body weight/day for the males and 79 to 124 mg substance/kg body weight/day for the females.

Parental systemic effects: No toxicologically significant parental toxicity was observed up to the highest dose level tested. In the males liver weights were slightly increased but without microscopic changes. Therefore the liver effects were considered to be of an adaptive nature to administration of the test substance.

Fertility: No fertility toxicity was observed up to 70 mg/kg bw. Therefore a parental and fertility No Observed Adverse Effect Level (NOAEL) of at least 70 mg/kg bw was established.