Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
yes
Remarks:
During the study several minor deviations from protocol occurred. All deviations were considered to not have adversely affected the study integrity.
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
Crl:WI(Han) (outbred, SPF-quality), nulliparous and nonpregnant females and untreated animals were used at initiation of the study.
- Age at study initiation: Approximately 5-6 weeks.
- Housing:
Pre-mating: Animals were housed in groups of 4 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 4 animals/cage. Females were individually housed in Macrolon cages. Pups were kept with the dam until termination
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%, at least 10 room air changes/hour, and a 12-hour light/12-hour dark cycle. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.

IN-LIFE DATES
From: June 2014 to April 2015

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): once per week
- Mixing appropriate amounts with (Type of food): the test substance was mixed without the use of a vehicle with some powder feed (premix) and subsequently mixed with the bulk of the diet. Water (approx. 15%) was added to aid pelleting. The pellets were dried for approx. 24 hours at 35°C before storage.
- Storage temperature of food: room temperature
Details on mating procedure:
- M/F ratio per cage: 1 to 1
- Length of cohabitation: maximum of 15 days; if no evidence of copulation was obtained after 10 days, the female was placed with another male (for max. 5 days) of the same treatment group who had successfully mated before.
- Proof of pregnancy: sperm in vaginal lavage or appearance of copulatory plug was referred to as day 0 post-coitum
- After successful mating each pregnant female was caged individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentrations and homogeneity of test substance in feed were controlled analytically in weeks 1, 11, 14, 15, 22, and 23. A validated method using UPLC with UV-detection and a C18 column was used. For extraction of the test substance the pellets were powdered and then extracted with methanol. The filtered extract was diluted before being analysed by UPLC chromatography.
The concentrations analysed in the diets of groups 2, 3 and 4 were in agreement with the target concentrations (i.e. mean accuracies between 80% and 120%) except for Week 14 Group 2 samples of 500 ppm and 667 ppm where 70% and 130%, respectively, of the nominal concentration was determined.
The diets were homogeneous (i.e. coefficient of variation < 10%). At two occasions analysis of Group 2 samples showed a coefficient of variation of 11% and 12% (week 15 and 22, respectively). This was considered to have no effect on the integrity of the study.
Duration of treatment / exposure:
F0 generation: a minimum of 70 days prior to mating and continuing until euthanasia
F1 generation (F0 pups): the F1 generation was potentially exposed to the test substance in utero, through nursing during lactation and directly when they begin eating solid food. After weaning, pups were treated for a minimum of 70 days prior to mating and continuing until euthanasia..
F2 generation:(F1 pups): the F2 generation was potentially exposed to the test substance in utero, through nursing during lactation.
Frequency of treatment:
The test substance was included in the diet; thus treatment was ad libitum.
Details on study schedule:
- Selection of parents from F1 generation when pups were 21 days of age.
- Age at mating of the mated animals in the study: F0 animals approx. 16 weeks, F1 animals approx. 13 weeks
Doses / concentrationsopen allclose all
Dose / conc.:
1 000 mg/kg diet
Remarks:
concentration for F0 generation
Dose / conc.:
3 000 mg/kg diet
Remarks:
concentration for F0 generation
Dose / conc.:
10 000 mg/kg diet
Remarks:
concentration for F0 generation
Dose / conc.:
1 000 mg/kg diet
Remarks:
concentration for F1 generation
Dose / conc.:
3 000 mg/kg diet
Remarks:
concentration for F1 generation
No. of animals per sex per dose:
24
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: results from OECD 421 study plus dose range finding study for palatability
- Rationale for animal assignment (if not random): animals of F0 generation selected randomly according to body weight. Selection of animals for the F1 generation: a minimum of one male and one female per litter were selected when litters of the F0 generation attained an age of 21 days. Only pups not expected to survive due to physical limitations were not available for selection.
- The amount of test substance incorporated in the diet was kept at a constant level in terms of ppm, throughout the study period. As during the lactation phase the relative food consumption is markedly higher than compared to the remainder of the study period based on historical control data, the concentrations (ppm) were reduced accordingly during the lactation phase. After termination, the actual substance intake for each period (pre-mating, mating, post-mating, post-coitum and lactation) was estimated based on the body weight and food consumption values.

Examinations

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- 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, 4, 7, 14, and 21.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
Oestrous cyclicity (parental animals):
Daily vaginal lavage was performed to determine the stage of estrous beginning 21 days prior to initiation of the mating period and until evidence of copulation was observed. Vaginal lavage continued for those females with no evidence of copulation until termination of the mating period.
Sperm parameters (parental animals):
Parameters examined in P and F1 male parental generations:
testis weight, epididymis weight, sperm count in testes, sperm count in epididymides, sperm motility, sperm morphology
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: 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 / F2 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals as soon as possible after delivery of the litters in each generation.
- Maternal animals: All surviving animals on Lactation Day 21-24

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in the following were prepared for microscopic examination and weighed, respectively.

(adrenal glands), (brain), (caecum), cervix, clitoral gland, coagulation gland, (colon), (duodenum), epididymidis, female mammary gland area, (ileum), (jejunum), (kidneys), liver, ovaries, (pituitary gland), preputial gland, prostate gland, (rectum), seminal vesicles, (spleen), (stomach), testis, (thyroid incl. parathyroid), uterus, vagina, all gross lesions. Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals (and not already separated during culling on Day 4) and all F2 offspring not already separated during culling on Day 4 were sacrificed at 21 days of age.
- These animals were subjected to postmortem examinations as follows: external examination of the cranium, macroscopic examination of the thoracic and abdominal tissues and organs. Gross lesions and brain, spleen, thymus and liver were collected and placed in 10% buffered formalin.

HISTOPATHOLOGY / ORGAN WEIGTHS
Brain, spleen, thymus and liver of one pup/sex/litter were weighed. The spleen of the first ten F2 pups/sex/group were processed for histopathology examination.
Statistics:
The following statistical methods were used to analyze 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.
- The Kruskal-Wallis nonparametric ANOVA test was applied to motor/activity data to determine intergroup differences.

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. Test statistics were calculated on the basis of exact values for means and pooled variances.
Reproductive indices:
For each group, the following calculations were performed:
- Mating index males: Number of males mated/Number of males paired x 100
- Mating index females: Number of females mated/Number of females paired x 100
- Fertility index males: Number of pregnant females/Number of males paired x 100
- Fertility index females: 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
- Percentage of breeding loss Days 5 until weaning: Number of dead pups between Days 5 and 21 of lactation/Number of live pups on Day 4 of lactation x 100
- Percentage of live males at weaning: Number of live male pups on Day 21 of lactation/Number of live pups on Day 21 of lactation x 100
- Percentage of live females at weaning: Number of live female pups on Day 21 of lactation/Number of live pups on Day 21 of lactation x 100
- Viability index: Number of live pups on Day 4 of lactation / Number of pups born alive x 100
- Weaning index: Number of live pups on Day 21 of lactation / Number of live pups on Day 4 of lactation x 100

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Commencing from the last week of premating hunched posture, piloerection and/or lean appearance in 5/24 males and 23/24 females of the 10000 ppm group. No effects at 1000 and 3000 ppm.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In the 10000 ppm group body weight gain in males and females was significantly lower during the premating period, reduced by 18% and 15%, respectively. No effects in animals of the 1000 and 3000 ppm groups.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food intake of males and females at 10,000 ppm and of males at 3000 ppm was lower over Days 1-4 of the premating period. On subsequent days of the premating period, food intake was slightly higher than controls. Over Days 8-15 of the premating period, this increase was most pronounced. At other time points of the premating and mating phase, the statistically significantly higher food intake noted at 3000 and 10,000 ppm on various occasions showed no clear dose-related trend. When corrected for body weight, a dose related increase in relative food intake was noted over the dose groups throughout the premating, mating, post mating and post coitum phase, especially for males and being most prominent over Days 8-15 of the premating phase.

At 3000 ppm, absolute and relative food intake of females was also higher during the post coitum period (statistically significant on most occasions), and during the last week of the lactation period.

The lower absolute food intake and higher relative food intake of females at 10,000 ppm during the post-coitum period was ascribed to the fact that these females did not deliver offspring.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Mean feed conversion ratio was statistically significantly lower than controls for males at 3000 and 10,000 ppm on most occasions during the premating, mating and post-mating period. For males at 10,000 ppm, the difference in mean over means to control levels was approximately 27%, 80% and 90% at the end of the premating, mating and post-mating period, respectively. For males at 3000 ppm, the difference in mean over means to control levels was approximately 11%, 27% and 40% at the end of the premating, mating and post-mating period, respectively.

Females at 10,000 ppm also showed a statistically significantly lower mean feed conversion ratio than controls on some occasions during the premating phase and on all occasions during the post-coitum phase.For females at 10,000 ppm, the difference in mean over means to control levels was approximately 28% and 92% at the end of the premating and post-coitum period, respectively.

For females at 1000 and 3000 ppm, feed conversion ratio was also lower than controls from Day 4 post-coitum onwards, achieving a level of statistical significance on a few occasions For females at 1000 and 3000 ppm, the difference in mean over means to control levels was approximately 16% and 22% at the end of the post-coitum period, respectively.

A summary table of food efficiency in animals of the F0 generation is provided as attachment (Table 1.14) under “attached background material”
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The following (statistically significant) changes in clinical biochemistry parameters distinguished treated from control animals at the end of premating:
− Higher alanine aminotransferase activity (ALAT) in males and females at 10,000 ppm.
− Higher aspartate aminotransferase activity (ASAT) in males at 3000 and 10,000 ppm.
− Higher alkaline phosphatase activity (ALP) levels in males and females at 10,000 ppm, and in males also at 3000 ppm (no clear dose-related trend in males).
− Lower total protein in females at 10,000 ppm.
− Higher total bilirubin in females at 10,000 ppm.
− Higher albumin in males at 10,000 ppm, and lower albumin in females at 3000 and 10,000 ppm (no clear dose-related trend for females).
− Higher urea in males and females at 10,000 ppm, and in females also at 3000 ppm.
− Lower glucose in males at 10,000 ppm.
− Higher cholesterol in females at 3000 and 10,000 ppm (no clear dose-related trend).
− Higher potassium in males and females at 10,000 ppm.
− Higher chloride in males at 10,000 ppm, and lower chloride in females at 3000 and 10,000 ppm (no clear dose-related trend in females).
− Higher inorganic phosphate levels in males and females at 3000 and 10,000 ppm (no clear doserelated trend in males).
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
At 10000 ppm a lower absolute and relative prostate weight was recorded. All other organ weight differences observed were considered unrelated to treatment.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Test item-related macroscopic findings consisted of:
- General observation, emaciated in 1/24 males and 1/24 females of the 10,000 ppm group
- Mesenteric lymph node, dark red or reddish discoloration at an increased incidence in 9/24 males of the 10,000 ppm group, compared to 1/24 females of the 1000 ppm group and 1/24 males and 3/24 females of the 3000 ppm group. The histologic correlate consisted of sinus erythrophagocytosis / sinus erythrocytes.
- Cecum, enlarged was noted in 2/24 females of the 1000 ppm group and in 14/24 females of the 3000 ppm group. This finding was absent in females treated at 10,000 ppm and occurred without histologic correlates.

The remainder of the recorded macroscopic findings were considered to be spontaneous in nature and did not distinguish treated animals from the controls or were related to differences in physiologic status of females (absence of thickened duodenum in non-pregnant females at 10,000 ppm).
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related microscopic observations consisted of:
− Increased incidence and/or severity of sinus erythrophagocytosis/sinus erythrocytes in males at 10,000 ppm (11/11 males: 3 minimal, 7 slight, 1 moderate) and in females at 3000 ppm (4/6 females: 1 minimal, 2 slight, 1 moderate), compared to 3/4 females (2 minimal, 1 slight) of the 1000 ppm group, 1/2 males (1 slight) at 3000 ppm and 1/5 females (1 minimal) of the 10,000 ppm
group.
− Increased incidence of reduced contents of the prostate gland in males at 10,000 ppm (5/24 males: 4 minimal, 1 slight) compared to 1/15 males of the 3000 ppm group (1 slight).
− Increased incidence of reduced contents of the preputial glands in males at 10,000 ppm (15/24 males: 11 minimal, 4 slight), compared to 1/4 males of the 1000 ppm group (minimal) and 3/15 males of the 3000 ppm group.

Reproductive performance
At 10,000 ppm, there were some findings of note present in the reproductive system of the females. These findings consisted of the presence of ovarian luteal cyst(s) (in 2/24) and follicular cyst(s) (in 1/24) at minimal degrees, or absence of corpora lutea (1/24, recorded as immature/abnormal) and atrophy/inactivity of the vaginal epithelium in 3/24 females. Furthermore, there were 16 females with implantation sites and 5 without, within the group of 21 females with evidence of mating. Only a few females did show glandular development of the mammary gland, although reduced compared to the other groups.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, treatment-related
Description (incidence and severity):
At 10,000 ppm, regularity and duration of the estrous cycle was affected by treatment. A total of 11/24 of the females had an irregular cycle, 4 of which also had extended di-estrus. Another 5/24 females were acyclic, two of which also had extended di-estrus. One female had an extended estrus and extended di-estrus. The remainder of the females (29.2% (7/24)) had a ‘regular’ cycle of 4-5 days.

At 1000 and 3000 ppm, regularity and duration of the estrous cycle were unaffected by treatment. The percentage of females per group classified as having a ‘regular’ cycle of 4-5 days at 0, 1000, and 3000 ppm were 87.5, 100 and 95.8%, respectively. In the control group, 3/24 females had an irregular cycle. One female at 3000 ppm had an extended estrus lasting throughout the measurement interval. Two control females and two females at 3000 ppm also had extended di-estrus, next to regular cycles.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Sperm motility, concentration and morphology were considered to have been unaffected by treatment up to and including males of the 10000 ppm group.
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
At 10,000 ppm, 3/24 females did not mate resulting in a lower mating index (88% vs 100% in the control group). Out of the 21 mated females, 18 females were found pregnant based on implantation sites only with a consequent lower fertility index of 75% (vs 88% in the control group). None of the mated females produced offspring, resulting in a gestation index of 0% (vs. 100% in the control group). Precoital time was also increased at 10,000 ppm at an average of 3.8 days vs. 2.3 days for the other dose groups including control. In addition, the number of implantations was reduced at 10,000 ppm (8.3, vs. 12.0 in the control and 3000 ppm group and 10.2 at 1000 ppm).

Most of the mated F0 females of the 10000 ppm group had implantation sites only. Microscopically, implantation sites of females at 10,000 ppm generally seemed to be further in regression compared to female rats that had a normal pregnancy. Only in a few of these mated females glandular development of the mammary gland was noted microscopically, although reduced compared to other dose levels. Overall, these findings were indicative for (early) embryonal loss.

Conception index at 10,000 ppm was similar to control levels.

At 1000 and 3000 ppm, mating, fertility and conception indices, precoital time, gestation index or duration and number of implantation sites were unaffected by treatment. All females at these dose levels showed evidence of mating. A total of 3, 2 and 3 females in the control, 1000 and 3000 ppm group were not pregnant. All pregnant females at these dose groups delivered live pups. There were 21, 22, and 21 litters available for evaluation in the control, 1000 and 3000 ppm groups, respectively.

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 and no deficiencies in maternal care were observed for other animals.

A summary of reproductive indices and offspring viability of F0 and F1 generation is provided as attachment (Table 1.19) under “attached background material”.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 3 000 - < 10 000 mg/kg diet
Based on:
test mat.
Remarks:
When corrected for mean test substance intake, the NOAEL of 3000 ppm corresponds to 184-261 mg and 230-286 mg test substance per kg body weight per day for males and females, respectively.
Sex:
male/female
Basis for effect level:
clinical signs
body weight and weight gain
food consumption and compound intake
food efficiency
clinical biochemistry
reproductive function (oestrous cycle)
reproductive performance
other: food efficiency was strongly depressed in high dose animals; disruption of the gut microbiota by the antimicrobial activity of the test substance is very likely and could have caused the observed (secondary) effects.

Target system / organ toxicity (P0)

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
10 000 mg/kg diet
System:
other: food efficiency was strongly depressed in high dose animals; disruption of the gut microbiota by the antimicrobial activity of the test substance is very likely and could have caused the observed (secondary) effects.
Organ:
mesenteric lymph node
preputial gland
other: food efficiency was strongly depressed in high dose animals; disruption of the gut microbiota by the antimicrobial activity of the test substance is very likely and could have caused the observed (secondary) effects.
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 3000 ppm, female body weight gain appeared slightly higher than controls during the premating phase and at commencement of the mating phase (statistically non-significant). Absolute body weight of these females achieved statistical significance on Day 64 of the premating phase and on Day 1 of the mating phase. Absolute body weight of these females was also statistically significantly higher on most occasions during the post coitum and lactation phase, while weight gain was statistically significantly higher on a few occasions only, i.e. on Day 4 of the post-coitum phase and on Days 7 and 21 of the lactation phase.

The statistically significantly lower body weight gain of females at 1000 ppm on Day 14 of the lactation phase occurred in the absence of a dose-related trend and was therefore considered to be unrelated
to treatment.

Body weights and body weight gain of males appeared unaffected by treatment up to 3000 ppm.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 3000 ppm, absolute and relative food consumption was higher in both sexes throughout the treatment period, achieving a level of statistical significance on most occasions.

At 1000 ppm, absolute and relative food consumption was higher in males during the mating period (statistically significantly higher on Days 1-8 and 15-22) and post mating period. Relative food consumption was also higher only on Days 1-8 of the premating phase for males, while absolute food intake was similar to controls throughout the premating phase. Females at 1000 ppm showed a higher absolute food intake during the premating period (statistically significant on several occasions) and on a single occasion (Days 7-11) during the post coitum period. Relative food intake of these females appeared similar to controls throughout the treatment period.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Females at 3000 ppm showed a lower mean feed conversion ratio than controls from Day 4 of the post-coitum phase onwards, achieving a level of statistical significance on a few occasions. The difference in mean over means to control levels was approximately 15%.

Mean feed conversion ratio for males up to 3000 ppm and for females at 1000 ppm appeared unaffected by treatment; any statistically significant variations in mean feed conversion ratio between these animals and control animals were not consistently seen over the treatment duration and/or occurred in the absence of a dose-related trend.

A summary table of food efficiency in animals of the F1 generation is provided as attachment (Table 3.14) under “attached background material”
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At 3000 ppm, higher cholesterol was recorded for males and females (an apparent dose-related trend was noted for both sexes).

Any other (statistically significant) changes in clinical biochemistry parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend and/or remained within the range of values expected for rats of the same strain and of similar age.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
At 3000 ppm, reduced grip strength was recorded for fore- and hindlimbs of males. At 1000 ppm, forelimb grip strength was reduced for males.

Hearing ability, pupillary reflex, static righting reflex and foot pain response were normal in all examined animals. The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with very high activity in the first interval that decreased over the duration of the test period.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Adrenal gland weights were statistically significantly lower in females of the 1000 ppm and 3000 ppm groups (absolute and relative to body weight). Relative weight was 11 and 19% lower than controls at 1000 and 3000 ppm, respectively.

All other organ weight differences observed, including those that reached statistical significance, were considered incidental and/or occurred in the absence of a dose-related trend and were therefore considered unrelated to the administration of ADK STAB NA-11.
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Description (incidence and severity):
A summary of reproductive indices and offspring viability of F1 and F2 generation is provided as attachment (Table 3.19) under “attached background material”.

Effect levels (P1)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 3 000 mg/kg diet
Based on:
test mat.
Remarks:
When corrected for mean test substance intake, the NOAEL of 3000 ppm corresponds to 184-261 mg and 230-286 mg test substance per kg body weight per day for males and females, respectively.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed at the highest dose level

Target system / organ toxicity (P1)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
Incidental clinical signs of pups surviving until scheduled necropsy remained within the range considered normal for pups of this age, and were therefore not considered to be toxicologically relevant.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related of toxicologically relevant effects on pup mortality seen up to 3000 ppm. None of the pup deaths between first letter check and Day 21 of lactation were considered toxicologically relevant as all remained within the range seen for animals used in this type of study and no dose response effect was seen.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
At 3000 ppm, pup body weights (both sexes) were statistically significantly lower on Day 14 of lactation. Body weights of these pups were similar to control levels towards the end of the lactation period.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
see information provided for P1 generation.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
see information provided for P1 generation.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
see information provided for P1 generation.
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Description (incidence and severity):
The day of vaginal opening and balanopreputial separation was similar for control and treated pups.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
see information provided for P1 generation.
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
Other effects:
no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):
no effects observed

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 3 000 mg/kg diet
Based on:
test mat.
Remarks:
When corrected for mean test substance intake, the NOAEL of 3000 ppm corresponds to 184-261 mg and 230-286 mg test substance per kg body weight per day for males and females, respectively.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed at the highest dose level

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Results: F2 generation

General toxicity (F2)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related or toxicologically relevant effects on pup mortality seen up to 3000 ppm. None of the pup deaths between first litter check and Day 21 of lactation were considered toxicologically relevant as all remained within the range seen for animals used in this type of study and no dose response effect was seen.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
A summary of pup body weights development of the F2 generation is provided as attachment (Table 3.23) under “attached background material”.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Absolute and relative spleen weight of male and female pups at 3000 ppm was higher than controls (not statistically significant for relative spleen weight of male pups). Absolute spleen weight of male pups at 1000 ppm was lower than controls.

There were no test material related histopathological findings observed in the spleen of selected pups.

Other pup organ weights were similar to control levels.
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
Description (incidence and severity):
There were no test item-related findings observed in the spleen of the selected pups of the F2-generation.
Other effects:
no effects observed

Developmental neurotoxicity (F2)

Behaviour (functional findings):
no effects observed

Developmental immunotoxicity (F2)

Developmental immunotoxicity:
not examined

Effect levels (F2)

Key result
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
>= 3 000 mg/kg diet
Based on:
other: test material contained in diet of lactating dams of F1 generation.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed at the highest dose level

Target system / organ toxicity (F2)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
10 000 mg/kg diet
Treatment related:
yes
Relation to other toxic effects:
reproductive effects as a secondary non-specific consequence of other toxic effects
Dose response relationship:
yes
Relevant for humans:
no

Applicant's summary and conclusion

Conclusions:
The NOAEL was derived at 3000 ppm test substance in the diet. When corrected for mean test substance intake, the NOAEL of 3000 ppm corresponds to 184-261 mg and 230-286 mg test substance per kg body weight per day for males and females, respectively.

The effect observed on reproduction in high dose animals (10000 ppm) is considered a secondary effect and not primary reproduction toxicity of the test substance ADK STAB NA-11. This effect is not relevant for humans.

In the meantime additional studies were performed confirming the secondary effect:

The test substance has strong antimicrobial activity leading to disruption of the gut microbiome of orally dosed rats. Subsequently, animals of high dose groups suffered from disruption of nutritional homoeostasis and imbalanced nutrition leading secondarily to adverse effects on reproduction and developmental parameters (see the review and evaluation of effects observed in reproduction toxicity studies attached to the endpoint summary).