Registration Dossier

Administrative data

Description of key information

In a dietary reproscreen study according to OECD TG 421 several systemic parameters were scored in addition to what is required in the reproscreen in view of the systemic effects seen in the 28 -day oral gavage study. The doses were 13 and 16, 28 and 32 and 70 -85 mg/kg bw for males and females, respectively (using the actual intake via food being 134, 284 and 770 ppm (200, 400 and 1000 nominal ppm Salicynalva in feed, respectively).
Body weight and clinical signs were similar to control values. For haematology parameters no treatment related findings were seen up to the highest dose. Macroscopic and microscopic observations did not show treatment related effects. Relative liver weights were increased at 70 -80 mg/kg bw in both sexes, circa 10% for males and 20% for females. No organ weight effect were seen in: kidney, spleen, testes and epididymides.
Considering systemic effects the NOAEL is considered to be 70 mg/kg bw for both males and females, using the lower value of the males on the intake of the substance. Though the liver weights at this dose are increased in the absence of any and macroscopic and microscopic findings this increase in liver weight is considered to be a non-adverse adaptive change.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
repeated dose toxicity: oral
Remarks:
other: reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 December 2011 - 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.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Reference:
Composition 0
Qualifier:
according to
Guideline:
other: OECD Guidelines for Testing of Chemicals, Guideline 421, Reproduction/Developmental Toxicity Screening Test, July 1995.
Deviations:
yes
Remarks:
Additional liver and heamatology parameters were evaluated.
GLP compliance:
yes (incl. certificate)
Limit test:
no
Test material information:
Composition 1
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
Route of administration:
oral: feed
Details on oral 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 diets: 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.
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.
Frequency of treatment:
Ad libitum.
Remarks:
Doses / Concentrations:
15, 30 and 70 mg/kg bw (nominal doses were 0, 200, 400, 1000 ppm i n the diet)
Basis:
actual ingested
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 (See attached results).
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.
Positive control:
No.
Observations and examinations performed and frequency:
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

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.

Sacrifice and pathology:
GROSS PATHOLOGY:
- The animals were not deprived of food. Animals surviving to scheduled necropsy and all moribund animals were deeply anaesthetised and subsequently exsanguinated.
- According to test guidelines

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

HISTOPATHOLOGY:
- According to test guidelines
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 (Dunnett, 1955) (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 (Miller, 1981) (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test (Fisher, 1950) 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.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
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
Histopathological findings: neoplastic:
no effects observed
Details on results:
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.

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.
Dose descriptor:
NOAEL
Remarks:
Parental generation
Effect level:
>= 70 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Treatment with Salicynalva by dietary administration in male and female Wistar Han rats at dose levels of circa 15, 30 and 70 mg/kg bw (200, 400 and 1.000 ppm nominal, respectively) revealed no parental toxicity for treatment up to 70 mg/kg bw.
Critical effects observed:
not specified
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 mg/kg bw (200, 400 and 1.000 ppm nominal, respectively) revealed no parental, reproduction and developmental toxicity for treatment up to 70 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:

In a dietary reproscreen study according to OECD TG 421 several systemic parameters were scored in addition to what is required in the reproscreen in view of the systemic effects seen in the 28-day oral gavage study. The doses were 13 and 16, 28 and 32 and 70 -85 mg/kg bw for males and females, respectively (using the actual intake via food being 134, 284 and 770 ppm (200, 400 and 1000 nominal ppm Salicynalva in feed, respectively).

Body weight and clinical signs were similar to control values. For haematology parameters no treatment related findings were seen up to the highest dose. Macroscopic and microscopic observations did not show treatment related effects. Relative liver weights were increased at 70-80 mg/kg bw in both sexes, circa 10% for males and 20% for females. No organ weight effect were seen in: kidney, spleen, testes and epidymides.Considering systemic effects the NOAEL is considered to be 70 mg/kg bw for both males and females, using the lower value of the males on the intake of the substance. Though the liver weights at this dose are increased in the absence of any and macroscopic and microscopic findings this increase in liver weight is considered to be a non-adverse adaptive change.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
70 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The information on repeated dose toxicity from the 28-day study and the reproscreem are both of adequate quality to derive information on the systemic toxicity of the substance

Mode of Action Analysis / Human Relevance Framework

Additional information

Two sub-acute repeated dose toxicity studies are available of at least 28 days of exposure. In addition, there is a 14 days dermal repeated dose toxicity test. These studies are described in more detail below.

The reproscreen study is selected as the key study because in the 28-day repeated dose toxicity study via gavage effects were seen at the high dose of 250 mg/kg bw, which are considered to be partly due to peak dosing when dosing via gavage. This type of peak exposures are being less relevant for the hazard and risk assessment than compared to a more gradual dosing as is simulated via the diet to which the animals were exposed in the reproscreen study. In both studies the liver is the target organ. In addition, the NOAEL of the reproscreen study of 70 mg/kg bw is well below the effect dose of 250 mg/kg bw in the 28-day gavage study and above the NOAEL in the latter, which was the next lower dose: 15 mg/kg bw.

Summary of the repeated dose toxicity: 28 -day oral gavage study: This study was performed to assess the systemic toxicity of Salicynalva to the rat. The method followed was that outlined in: OECD Guideline for Testing of Chemicals No. 407, "Repeated Dose Oral Toxicity - Rodent 28-day or 14-day study". Adopted: 12 May 1981 (as well as Joint directive of the Japanese Environmental Protection Agency and the Ministries of Health and Welfare and International Trade and Industry, 5 December 1986 and EEC Methods for the determination of toxicity, Annex to Directive 92/69/EEC (01 No. L383A, 29.12.92), Part B, Method B7. Subacute toxicity (oral).

Salicynalva was administered by oral gavage, once daily, to three groups of rats for a minimum of twenty eight consecutive days, at dosage levels of 1, 15 or 250 mg/kg/day. The test material was prepared as suspensions in corn oil at a maximum dosage volume of 5 ml/kg/day. Control animals received the vehicle (corn oil) alone at the same dose volume (5 ml/kg/day).

All rats of Groups 2 and 3 (1 and 15 mg/kg/day respectively) and five males and five females from each of Groups 1 and 4 (Control and 250 mg/kg/day respectively) were killed following the four-week treatment period. The remaining animals ( five males and five females from Groups 1 and 4) were retained for a two-week recovery period, following which, they were also killed. Bodyweights, food and water consumption and clinical observations were recorded during the study. Blood and urine samples were taken from all rats shortly prior to termination following the four-week treatment and two-week recovery periods. All animals were killed and subsequently examined macroscopically, specified tissues were then prepared for histopathological examination. Treatment resulted in:

Mortality: A male rat from the high dosage group was killed for humane reasons on Day 5. Clinical signs prior to sacrifice included hunched posture, piloerection, gasping and an unwillingness to move. Gastric ulceration, seen macroscopically and microscopically, was considered to be the factor contributory to death. Also one high dosage group male rat was killed for humane reasons on Day 7. Prior to death this animal was lethargic, unwilling to move, had partially closed eyelids, piloerection, hunched posture and had a thin ungroomed appearance. A post mortem examination revealed haemorrhagic depressions in the duodenum and microscopically, mucosal ulceration in the duodenum was noted . Duodenal ulceration was considered to be the factor contributory to death. There were no further mortalities that were considered to be related to treatment. Clinical signs: Ungroomed appearance was noted for high dosage group rats frequently throughout the study from Day 6 (males) or Day 14 (females). This sign persisted for approximately one week into the recovery period. Bodyweights: Bodyweight gains were statistically significantly lower than control for high dosage group male rats during the treatment period. During the recovery period bodyweight gains for these rats were higher than controls. This may be due to lower food consumption because lower than control food consumption was noted for male rats receiving 250 mg/kg/day. Food consumption was similar to control during the recovery period. Water consumption: Higher than control water was noted for high dosage group male and female rats during Week 3 . Water consumption was similar to control during the recovery period.

Haematology: Lower than control white blood cell counts, haemoglobin concentration and hence lower mean corpuscular haemoglobin concentration and mean corpuscular haemoglobin levels were recorded for high dosage group male rats at the end of the treatment period. Following the recovery period the haemoglobin concentration and lower mean corpuscular haemoglobin concentration and mean corpuscular haemoglobin levels remained lower than control. Biochemistry: Following the treatment period statistically significantly higher than control alkaline phosphatase, glutamic pyruvic transaminase, glutamic oxaloacetic transaminase and urea nitrogen levels were recorded for male rats receiving 250 mg/kg/day. For rats receiving 250 mg/kg/day, high triglyceride and bilirubin levels (males and females), high cholesterol levels (females) and low glucose levels (males) were recorded. For high dosage group rats higher than control albumin (males and females), globulin (females) and hence total protein (males and females) were recorded. Disturbances in electrolyte levels recorded for high dosage group male rats included higher than control sodium, chloride and inorganic phosphorus levels and lower than control potassium ion levels. There were no differences from control following the two-week recovery period.

Macroscopy and histopathology: Treatment-related changes detected in rats receiving 250 mg/kg/day included the following: Generalised swollen hepatocytes with ground glass cytoplasm in the liver of male and female rats at the end of treatment. Minimal centrilobular hepatocyte necrosis in one male and one female decedent. Reduced colloid in the prostate of one male rats during and at the end of treatment with associated atrophic acini in one decedent male and of doubtful relevance. Minimal epithelial hyperplasia with associated minimal subepithelial inflammation and oedema in the non-glandular region of the stomach in male rats was seen. Gastric ulceration in one decedent male and duodenal ulceration in another decedent male rat were observed both indicating irritation by the substance.

In summary: The mortality and treatment-related findings, in particular in the liver were considered to be systemic adverse effects. The effects seen in the stomach and duodenum of rats from the high dosage group were considered to be local adverse effects. No treatment-related findings were seen following the 2-week recovery period, indicating reversibility of treatment-related effects, resulting in a NOAEL of 15 mg/kg bw.

A 14-day dermal range finding study was performed to assess the systemic toxic potential of Salicynalva, to the rat, in order to assess the suitability of the dermal route of administration. The protocol of the OECD TG 410 was used. The test substance was administered dermally (occlusive dressing over the treatment area for 6 hours per day) to groups of five male and five female rats at 50, 140, 400 or 1000 mg/kg/day (at a dosage volume of 2 ml/kg/day) in corn oil. A further group of five male and five female rats received the vehicle (corn oil) alone, at the same dose volume as control. The study started initially for a period of seven days, though this was extended to fourteen days by the Sponsor, since the initial seven days of treatment did not reveal any clear adverse effects of treatment. All animals were killed on Day 15. Clinical signs including examination of the dermal test site of each animal, bodyweight, food consumption, liver and prostate weights, macroscopic and microscopic examinations were recorded for the study. The dermal route of administration was well tolerated at dosages up to 1000 mg/kg/day, with no treatment-related effects at the site of administration. There were no treatment-related effects on bodyweight gain, food consumption, liver and prostate weights or macroscopic findings at any dosage. Males receiving 1000 mg/kg/day showed slight centrilobular hepatocyte hypertrophy (indicating that dermal absorption occurred) although a similar effect was not seen in females and no associated increase in liver weight was noted in males. The No Observed Adverse Effect Level (NOAEL) was 1000 mg/kg/day for males and females.

For the overall assessment: The local effects seen in the 28-oral gavage study at the highest dose of 250 mg/kg bw are considered irritant effects due to high peak exposures and are not considered for systemic effects. The moderate liver effects and heamatology effects seen at this highest dose are considered to be a LOAEL. Only slight and non-adverse liver effects were seen in the reproscreen study and no effects on haematology at 70 mg/kg bw. Therefore the highest dose in this study is considered to be an overall NOAEL for the repeated dose toxicity study. The 14-day dermal toxicity study with slight non-adverse liver effects at 1000 mg/kg bw further supports that the NOAEL of 70 mg/kg bw is sufficiently conservative.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The reproscreen study is selected for the key repeated dose toxicity because the NOAEL from this dietary study reflects the repeated dose toxicity better than the 28-day gavage study. In the latter study the animals are more gradually exposed compared to peak exposure via gavage in which a.o. irritation effects are observed. The reproscreen study is sufficiently adequate for covering this endpoint because additional systemic effect parameters were included in this study.

Justification for classification or non-classification

Based on the NOAEL of 70 mg/kg bw/day observed in the dietary repeated dose / reproscreen study the substance does not need to be classified for (oral) repeated dose toxicity when considering the criteria outlined in Annex I of 1272/2008/EC (CLP) and Annex VI of 67/548/EEC (DSD). Though this NOAEL is just below 100 mg/kg bw classification and labelling is not warranted because the systemic effects found at 250 mg/kg bw in the 28 -repeated dose toxicity study do not indicate severe organ damage also because these effects were not seen after the recovery period. The mortalities at this 250 mg/kg bw are due to local irritant effects likely due to gavage dosing.