Registration Dossier

Administrative data

Endpoint:
developmental toxicity
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-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other 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:
other: OECD Guidelines for Testing of Chemicals, Guideline 421, Reproduction/Developmental Toxicity Screening Test, July 1995.
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
- Purity: 99.3%
- 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)
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).
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.
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).
- Age at mating: Approximately 14 weeks
- 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 the appearance of an intravaginal copulatory plug and/or by determination of the estrous stage. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated.
- After successful mating each pregnant female was caged individually in Macrolon cages (MIII type, height 18 cm).
Duration of treatment / exposure:
Ad libitum.

Pups were not treated directly, but were potentially exposed to the test substance in utero and through lactational transfer.
Frequency of treatment:
Ad libitum.
Duration of test:
Males: 32 days
Females: 43-56 days
Doses / concentrations
Remarks:
Doses / Concentrations:
circa 15, 30 and 80 mg/kg bw
Basis:
actual ingested
Based on analysis of 200, 400 and 1000 ppm nominal concentrations in the diet
No. of animals per sex per dose:
10
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale:
- 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.

Examinations

Maternal 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

NEUROBEHAVIOURAL EXAMINATION: No

GENERAL REPRODUCTION DATA
- Male number paired with, mating date, confirmation of pregnancy, and delivery day was recorded.
- Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of these females was examined to detect signs of abortion or premature birth.
- Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.

GROSS PATHOLOGY
- The animals were not deprived of food. Animals surviving to scheduled necropsy were deeply anaesthetised and subsequently exsanguinated.
- The number of former implantation sites and corpora lutea were recorded for all paired females.
- According to test guidelines

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

HISTOPATHOLOGY
According to test guidelines

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.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No
Fetal examinations:
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.
Statistics:
- 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.
Indices:
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 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: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
MORTALITY
No mortality occurred during the study period.

CLINICAL SIGNS
No treatment related clinical signs were noted up to 80 mg/kg bw.

One female at 15 mg/kg bw 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 80 mg/kg bw, 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 30 mg/kg bw 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 80 mg/kg bw.

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 30 mg/kg bw 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.


HAEMATOLOGY
For haematology parameters, no treatment related findings were noted up to 80 mg/kg bw ppm for parental females.

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 80 mg/kg bw for parental females. This was statistically significant for absolute liver weight for the parental females
No treatment related effects were noted for the weights of the kidneys, spleen, testes and epididymides up to 80 mg/kg bw for parental females.

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 correlation to the statistically significant increased group mean liver weights of 80 mg/kg bw for parental females.

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 30 mg/kg bw 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 (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
>= 80 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
> 80 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects. Remark: Only early postnatal pup development parameters were examined including body weight, post-natal loss, sex ratio, clinical signs, body weight and external macroscopy.

Details on embryotoxic / teratogenic effects:
There was no evidence of teratogenic effects based on the absence of relevant clinical signs and external macroscopic findings.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental 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 80 mg/kg bw, respectively. The average values for the low and mid dose for males and females are presented in the text and for the high dose the dose of 80 mg/kg bw is used for parental females. The analysis showed that the intake for females varied between 79 and 124 mg/kg bw.

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.

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 15 mg/kg bw and one pup at 30 mg/kg bw 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 the highest dose tested.

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

Applicant's summary and conclusion

Conclusions:
In conclusion, treatment with Salicynalva by dietary administration in male and female Wistar Han rats at actual dose levels of circa 15, 30 and 80 mg/kg bw (nomina dose levels of 200, 400 and 1.000 ppm in the diet) revealed no maternal and developmental toxicity for treatment up to 80 mg/kg bw.

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:

The developmental toxicity of Salicynalva was tested in a developmental / reproscreen study according to OECD TG 421 using dietary exposure. The exposure resulted in doses of circa 15, 30 and 80 mg/kg bw for dams. The dams showed no toxicologically significant effects up to the highest dose level tested. The increased liver weights at the highest dose was not accompanied with macroscopic or microscopic changes and were therefore considered to be an adaptive response to the test substance. No developmental toxicity was observed up to the highest dose tested. Treatment with Salicynalva by dietary administration in female Wistar Han rats at dose levels of circa 15, 30 and 80 mg/kg bw did not reveal maternal and developmental toxicity for treatment up to the highest dose. The No Observed Adverse Effect Level (NOAEL) of at least 80 mg/kg bw was established based on the actual test substance intake 79 to 124 mg substance/kg body weight/day for the females.