Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-02-13 to 2015-07-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study
Remarks:
Sections 7.5.1. and 7.8.2.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
The deviations had no impact on study results.
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Test material form:
solid

Test animals

Species:
rat
Strain:
other: RccHan TM : WIST(SPF)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories, B.V. Kreuzelweg 53, 5961 NM Horst / Netherlands
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) 12 wks
- Weight at study initiation: (P) Males: 268 - 421 g; Females: 193 - 278 g
- Fasting period before study: not specified
- Housing: During acclimatization and pre-pairing in groups of up to three animals by sex inMakrolon type-4 cages, cages with males were interspersed amongst those holding females to promote the development of regular estrus cycles. During pairing females were housed with males (1:1) in Makrolon type-3. Following pairing period males were re-housed in their original prepairing cages and females were individually housed in Makrolon type-3 cages.All cages were provided with wire mesh tops and standard softwood bedding (‘Lignocel’ J. Rettenmaier& Söhne GmbH & CoKG, 73494 Rosenberg / Germany, imported by Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) with paper enrichment (Enviro-dri from Lillico, Biotechnology, Surrey / UK). In addition a wood stick was provided (batch nos. 02105131114, 79 and 132211/1602205).
- Diet (e.g. ad libitum): Microgranulated standard Harlan Teklad 2018C (batch no. 41/13 and 54/13) rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum.
- Water (e.g. ad libitum): Community tap-water from Itingen was available ad libitumin water bottles.
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30-70%
- Air changes (per hr): 10-15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12-hour fluorescent light / 12-hour dark cycle with at least eight hours music during the light period.

IN-LIFE DATES: From: 2014-02-13 To: 2014-04-05

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): Dietary admixtures were prepared at intervals not exceeding 8 days using the test item as supplied by the supplier.
- Mixing appropriate amounts with (Type of food): A portion of Rosin, Maleated, was ground to powder using an electrical grinder, weighed into a tared glass beaker on a suitable precision balance, and mixed with microgranulated feed separately for each dose group. No heat was used during diet preparation
- Storage temperature of food: Stability of test item in feed was at least 8 days upon the results of stability analyses performed within the non-GLP Harlan Laboratories study no. D81027 (Method Implementation and Development). Feed preparations were stored until use in paper bags at room temperature (22 ± 3 °C), protected from light.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: until evidence of copulation was observed
- Proof of pregnancy: vaginal plug / sperm in vaginal smear] referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: not specified
- After successful mating each pregnant female was caged (how): Individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples for analyses of test item, its content and homogeneity in the feed were drawn at the start of the pre-pairing period and at the end of gestation/start of lactation period (i.e. two occasions). Stability of the test item in the feed for 8 days at room temperature was determined at the start of the pre-pairing period (i.e. one occasion). For assessment of content and homogeneity, approximately 100 g sample was collected from each the top, middle and bottom of every dietary admixture of the respective diet preparation (from group 1 admixtures only one sample was drawn from the middle of the preparation). Samples for assessment of content and homogeneity were collected on the day of preparation. For assessment of stability, approximately 100 g sample was drawn from the middle of the dietary admixture on the day of preparation and stored at room temperature for 8 days. After preparation the samples were delivered to the analytical department at ambient temperature and analyzed immediately or stored there frozen (at -20 ± 5 °C) until analysis. The samples were analyzed by HPLC coupled toan ELSD detector following an analytical procedure developed at Harlan Laboratories. The test item was usedas the analytical standard. Duplicates were taken of all samples and were stored at Harlan Laboratories Ltd., Itingen/Switzerland.
Duration of treatment / exposure:
Males: 43 days (during 14 days pre-pairing period, up to 29 days pairing period and postpairing period).
Females: Minimum 5 weeks (14 days pre-pairing period, up to 28 days pairing period, approximately 21 days of gestation period and lactation period to day 4 post partum).
Frequency of treatment:
Continuously (ad libitum)
Doses / concentrationsopen allclose all
Dose / conc.:
0 ppm
Remarks:
Control (Group 1)
Dose / conc.:
1 750 ppm
Remarks:
Low Concentration (Group 2)
Dose / conc.:
3 500 ppm
Remarks:
Intermediate Concentration (Group 3)
Dose / conc.:
7 000 ppm
Remarks:
High Concentration (Group 4)
No. of animals per sex per dose:
12/sex/dose
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: The dose levels were selectedbased on a previous dose range-finding toxicity study in Han Wistar rats, non-GLP Harlan Laboratories study no. D81038, using concentrations infeed of 5000, 10000 and 20000 ppm and resulting in adverse effects on food consumption, body weight loss and pre-termination of animals in high-dose group and dose dependent reduction in food consumption and body weight gain in groups 2 and 3.
- Rationale for animal assignment (if not random): Performed after at least three days of acclimatization using a computer-generatedrandom algorithm. Body weights (recorded on the day of allocation) were taken into consideration in order to ensure similar
mean body weights in all groups.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily cage-side clinical observations (once daily, during acclimatization and up to day of necropsy).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Males: Once prior to the first administration of the test item and weekly thereafter.
Females: Once prior to the first administration of the test item, weekly during the pre-pairing and pairing and on days 0, 6, 13 and 20 post coitum.

BODY WEIGHT: Yes
- Time schedule for examinations:
Males: Once during acclimatization (the data will be used for randomization but will not be reported), together with food consumption during pre-pairing and after pairing periods and at three-day intervals during pairing period.
Females: Once during acclimatization (the data will be used for randomization but will not be reported), together with food consumption during pre-pairing, gestation and lactation periods and at three-day intervals during pairing period.

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
Males: On days 1 - 5, 5 - 8, 8 - 12 and 12 - 14 of pre-pairing and after pairing periods (during after pairing recording of food consumption will be adapted according to the length of this period).
Females: On days 1 - 5, 5 - 8, 8 - 12 and 12 - 14 of pre pairing, on days 0 - 7, 7 - 14 and 14 - 20 post coitum and 1 - 4 post partum. No food consumption was recorded during the pairing period.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Gravimetrical measurement of water consumption was performed daily with exception for acclimatization and pairing periods.
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 anomalies, weight gain, physical or behavioural abnormalities, other: Righting reflex, Animals were observed outside the home cage for the following: changes in skin, fur, eyes, mucous membranes, occurrence of secretionsand excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes or bizarre behavior were also reported.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving males were sacrificed on the day after completion of the treatment for 43 days, when they were no longer necessary for the assessment of reproductive performance
- Maternal animals: All surviving Dams in groups 1, 2 and 3 were sacrificed on day 5 post partum. Because of increased mortality and moribund condition, all remaining females in group 4 were terminated on day 14 of the pre-pairing period. If birth did not occur on the expected date (day 21 post coitum), the dam was sacrificed on day 25 post coitum.

GROSS NECROPSY
- Gross necropsy: All animals surviving to the end of the observation period and all moribund animals were anesthetized by intraperitonealinjection of pentobarbitoneand killed by exsanguinations. All parent animals and pups, except those excessively cannibalized, were examined macroscopically for any structural changes,either at the scheduled necropsy or during the study if death occurred. For the parent animals, special attention was directed at the organs of the reproductive system. The uteri of all dams were placed in a solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites and the number of implantation sites was noted. The number of corpora luteain each ovary was recorded for all pregnant females.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 2 and 3 were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at 4 days of age.
Statistics:
The following statistical methods were used toanalyze food consumption, body weights, water consumption, clinical laboratory data, grip strength, locomotor activity, organ weights, macroscopic findings and reproduction data:
• Means and standard deviationsof various data were calculated and included in the report.
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
• Fisher's exact-test was applied if the variables could be dichotomized without loss of information.
Reproductive indices:
1) Mating Performance and Fertility
2) Corpora Lutea Count
3) Duration of Gestation
4) Implantation Rate and Post-Implantation Loss
5) Litter Size at First Litter Check
6) Postnatal Loss Days 0 - 4 Post Partum
Offspring viability indices:
1) Litter Size at First Litter Check
2) Postnatal Loss Days 0 - 4 Post Partum
3) Sex Ratios
4) Righting Reflex

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
No test item-related findings were recorded in males or females in any group during the weekly detailed observations. The only finding noted was hair loss recorded already during the daily observations for one female in group 3
Mortality:
mortality observed, treatment-related
Description (incidence):
In group 4, four females from two cages (cage numbers 30 and 32) were found dead during prepairing period; one female (no. 89) on day 12, one female (no. 88) on day 13 and two females (nos. 94 and 96) on day 14 of this period. Remaining females in this group showed signs of bad condition like reduced food consumption, body weight loss, hunched posture, stiff gait and ruffled fur, and were therefore terminated for ethical reasons on day 14 of the pre-pairing period. No further unscheduled deaths occurred in any group.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males - Pre-Pairing and Post-Pairing Periods
Treatment with the test item caused a dose-dependent and reversible reduction in body weight gain in groups 3 and 4. Mean body weight gainin order of ascending dose levels was +10.9%, +10.0%, +7.1% and -1.5% during the pre-pairing period and +11.5%, +10.4%, +9.9% and +12.9% during the post-pairing period. Body weights were significantly reduced in group 4 whereas no significant changes were observed in group 3. In group 4, body weight loss up to 4.1% was noted until day 5 of the pre-pairing period followed by statistically significantly reduced body weight gain recorded until the end of this period. During the post-pairing period, body weight gain was slightly, not statistically significantly higher than control values. Consequently, reduced body weights,compared to the control, were recorded from day 5 of the pre-pairing period until the completion of the study. This reduction was statistically significant from day 8 onwards. In group 3, statistically significantly reduced body weight gain was noted from day 5 until the completion of the pre-pairing period. During the post-pairing period, no significant differences in body weight gain were recorded compared tothe control values. Body weights were no
significantly different from these in the control groups during the entire study. In group 2, body weights and body weight gain weresimilar to the respective values in the control group.

Females - Pre-Pairing, Pairing, Gestation and Lactation Periods
Treatment with the test item caused a reduction in body weight gain in groups 3 and 4. During the pre-pairing period mean body weight gain in groups 1, 2, 3 and 4 was +5.5%, +4.8%, +2.1% and -9.5%. During the remaining study period, mean body weight gain in groups 1, 2 and 3 was +49.3%, +48.3% and +44.9% during the gestation period and +6.5%, +6.8% and +5.4% during the lactation period. Body weights were significantly reduced in group 4, and slightly but not statistically significantly reduced in group 3. In group 4, body weight loss up to 9.5% was noted until day 14 of the pre-pairing period. This resulted in a reduction in body weights, which was statistically significant from day 8 until the termination of females in this group on day 14 of the pre-pairing period. In group 3, body weight loss up to 0.7% was noted until day 8 of the pre-pairing period and reduced body weight gain until the end of thisperiod. The reduction in body weight gain was statistically significant until day 12 of the pre-pairing period. During the remaining study period, body weight gain was similar to the values in the control group. Body weights were not significantly different from these in the control groups during the entire study except for day 4 of
the lactation period. In group 2, body weights and body weight gain weresimilar to the respective values in the control group.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Males - Pre- and Post-Pairing Periods
Treatment with the test item caused a dose-dependent, reversible reduction in food consumption in groups 3 and 4. In order of ascending dose levels, mean food consumption was 22.4, 22.6, 21.5 and 16.5 g/animal/day during the pre-pairing period and 23.7, 23.6, 23.4 and 21.3 g/animal/day during the post-pairing period. In group 4, reduction in food consumption was statistically significant from day 1 to 12 of the pre-pairing period and from day 5 to 15 of the post-pairing period. Although food consumption remained lower than in the control group during the post-pairing period, a recovery was observed and from day 15 of this period the differenceswere not longer statistically significant. In group 3, slight but statistically significant reduction in food consumption was recorded from day 1 to 5 of the pre-pairing period. Afterwards, food consumption recovered and was similar to the control during the remaining study period. In group 2, food consumption was similar to thatin the control group during the entire study.

Females - Pre-Pairing, Gestation and Lactation Periods
Treatment with the test item caused adverse reduction in food consumption in group 4 and a reversible reduction in food consumption in group 3. During the pre-pairing period mean food consumption in groups 1, 2, 3 and 4 was 17.4, 16.8, 15.5 and 9.5 g/animal/day. During the remaining study period, mean food consumption in groups 1, 2 and 3 was 23.3, 23.1 and 21.6 g/animal/day during the gestation period and 31.7, 28.8 and 30.4 g/animal/day during the lactation period. In group 4, reduction in food consumption was statistically significant from day 1 to 14 of the pre-pairing period, when females were terminated, and was approximately 50% lower than in the control group. In group 3, a slight but statistically significant reduction in food consumption was observed from day 1 to 5 of pre-pairing period. Afterwards, no statistically significant changes in food consumption were noted in this group. In group 2, food consumption was similar to thatin the control group during the entire study.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Males - Pre- and Post-Pairing Periods
Treatment with the test item caused a dose-dependent, reversiblereduction in food conversion efficiency in groups 3 and 4. In order of ascending dose levels, mean food conversion efficiency was 0.045, 0.043, 0.032 and 0.015 g bw/g food/animal/day during the pre-pairing period and 0.029, 0.027, 0.024 and 0.036 g bw/g food/animal/dayduring the post-pairing period. In group 4, reduction in food conversion efficiency was statistically significant from day 1 to 8 of the pre-pairing period. Afterwards, food conversion efficiency recovered and during the postpairing period was similar than in the control group. In group 3, a statistically significant reduction in food conversion efficiency was recorded from day 1 to 5 of the pre-pairing period. Afterwards, relative food consumption recovered and was similar to the control during the remaining study period. In group 2, slight but statistically significant reduction in food conversion efficiency was recorded from day 1 to 5 of the pre-pairing period. Mean food conversion efficiency during the entire pairing was similar tothe value in the control group. Therefore the difference was considered to be incidental.

Females - Pre-Pairing, Gestation and Lactation Periods
Treatment with the test item caused adverse reduction in food conversion efficiency in group 4 and a reversible reduction in group 3. During the pre-pairing period mean food conversion efficiency in groups 1, 2, 3 and 4 was 0.023, 0.019, 0.020 and 0.003 g bw/g food/animal/day. During the remaining study period, mean foodconsumption in groups 1, 2 and 3 was 0.272, 0.270 and 0.261 g bw/g food/animal/day during the gestation period and 0.193, 0.215 and 0.160 g bw/g food/animal/day during the lactation period. In group 4, mean food conversion efficiency was 0.000 bw/g food/animal/day from day 1 to 5 of the pre-pairing period and after slight increase during the period from day 5 to 8, reduces again to 0.000 bw/g food/animal/day. The reduction was statistically significant from day 1 to 5 of the pre-pairing period but not thereafter. Due to the deaths, lower number of cages was considered for the mean value calculation ingroup 4; 3 cages from day 8 to13 and 2 cages from day 12 to 14 of the pre-pairing period. For this reason the statistical analysis of the differences could be less reliable. In group 3, a statistically significant reduction in food conversion efficiency was observed from day 1 to 5 of pre-pairing period. Afterwards, no statistically significant changes were noted in this group. In group 2, food conversion efficiency was similar to that in the control group during the entire study.
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Males - Treatment with the test item caused a dose dependent reduction in water consumption in groups 3 and 4. Mean water consumption in order of ascending dose levels was 33.0, 29.3, 26.6 and 24.6 g/animal/day during the pre-pairing period and 28.8, 25.4, 22.6 and 23.7 g/animal/day during the post-pairing period. In group 4, reduction in water consumption was statistically significant on pre-pairing period days 4 - 10 and 12 - 14 and on post-pairing days 15 - 17, 19 - 20 and 22 - 24. In group 3, reduction in water consumption was statistically significant on pre-pairing days 5 - 8, 9 - 11 and 12 - 13 and post pairing days 15 - 17, 18 - 20 and 23 - 24. In group 2, a statistically significantly lower water consumption was recorded on two occasions during the pre-pairing period. The differences to the control group were, however, not greater than variability of this parameter within the group on individual days and therefore this observation was considered not to be related to the treatment.

Females - Differences in water consumption between the control and trated groups were observed during the study. A slight and statisticallynot significantly lower water consumption was noted in group 4 during the pre-pairing period and in group 3 during the gestation and lactation periods. During the pre-pairing period mean water consumption in groups 1, 2, 3 and 4 was 27.2, 25.4, 28.5 and 24.4 g/animal/day. During the remaining study period, mean water consumption in groups 1, 2 and 3 was 38.6, 34.0 and 35.0 g/animal/day during the gestation period and 35.7, 31.6 and 30.6 g/animal/day during the lactation period. Because the differences were only minor, with one exception for group 3 on days 14 and 15 of the gestation period, not statistically significant and not clearly correlated with the dose levels, they were considered not to be related to the treatment.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Males - No test item-related effects on hematology parameters were noted in males at any dose level.

The following statistically significant changes were noted: lower concentration of haemoglobin in group 4 (9.8 mmol/L compared to 10.6 mmol/Lin the control group) and higher count of platelets in groups 2 and 4. These differences were only minor and values remained in the range of historical control data, therefore they were considered to be a result of biological variability and not test item-related.

Females - No test item-related effects on hematology parameters were noted in females at any dose level.

The following statistically significant changes were noted: lower haematocrit value in group 2 (38% compared to 41% in the control group), lower relative amount of neutrophiles (0.173 compared to 0.266 in the control group) and higher count of lymphocytes. These differences were only minor and values remained in the range of historical control data, therefore they were considered to be a result of biological variability and not test item-related.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Males - No test item-related effects on clinical biochemistry parameters were notedin males at any dose level.

The following statistically significant changes werenoted: higher concentration of creatinine in group 4 (31.7 µmol/L compared to 26.9 µmol/L inthe control group), lower activity of alanine transferase in groups 3 and 4 (19.1 and 22.8 U/L,respectively, compared to 37.3 U/L in the control group), higher concentration of potassium in group 4 (4.44 mmol/L compared to 4.00 mmol/L in the control group), higher concentration of phosphorus in group 4 (2.01 mmol/L compared to 1.65 mmol/L in the control group), lower protein concentration in group 4 (61.94 g/L compared to 67.80 g/L in the control group), lower concentration of albumin in groups 3 and 4 (39.75 and 38.01 g/L,respectively, compared to 42.19 g/L in the control group) and higher concentration of globulin in group 2 (28.44 g/L compared to 25.60 g/L in the control group). These differences were only minor and values remained in the range of historical control data or were not dose dependent, therefore they were considered to be a result of biological variability and not test item-related.

Females - No test item-related effects on clinical biochemistry parameters were noted in females at any dose level.

The following statistically significant changes were noted: lower concentration of sodium in groups 2 and 3 (139.6 and 139.6 mmol/L, respectively, compared to 142.4 mmol/L in the control group), lower concentration of chloride in groups 2 and 3 (98.9 and 98.3 mmol/L, respectively, compared to 102.2 mmol/L in the control group)and higher concentration of phosphorus in groups 2 and 3 (2.48 and 2.37 mmol/L, respectively, compared to 1.96 mmol/L in the control group). These differences were only minor and values remained in the range of historical control data, therefore they were considered to be a result of biological variability and not test item-related
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No test item-related findings were recorded during functional observational battery in males or females in any group.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Males:
In males, test item related findings were recorded in group 4. Minimal decreased lymphocytes were recorded in the spleen (involving mainlythe marginal zone) of 5/5 males and in the mesenteric lymph node (involving the paracortex) of 3/5 males. This finding was considered to result from non-specific stress. Moderate bilateral seminiferous tubule atrophy in the testes, associated with abnormal content in the epididymides (decreased spermatozoa and increased immature exfoliated germ cells) was noted in 1/12 males at 7000 ppm. This isolated finding, known to occur spontaneously in rat, was considered to be coincidental.

Females:
In females, test item-related findings were observed only in group 4 animals, which were found dead or terminated in extremis; lesions in the kidneys and lymphoid organs were recorded. In the kidneys, there were variable combinationsof pelvis/ureter dilatation, tubular dilatation, tubular degeneration/regeneration, pelvis urothelium erosion/ulceration, pelvis/ureter urothelium hyperplasia and subacute inflammation at the hilus in 11 out of 12 rats. Findings were bilateral (8/12) or unilateral (3/12), the grade severity being higher in animals killed in extremis when compared to animals found dead. In affected animals, the dilated tubules were mainly distal tubules and showed evidences of degeneration/regeneration. Erosion/ulceration of the transitional epithelium lining the pelvis was occasionally associated with hemorrhage. Urothelium hyperplasia was mainlyseen in the pelvis close tothe ureter opening and, when present on sections, in the urothelium lining the ureter. Subacute inflammation was present at the hilus, within the adipose tissue close to the ureter opening. Tubular dilatation and tubular degeneration/regeneration were the histological correlates of the pale discoloration seen at necropsy on the left kidney of one rat.

Minimal to marked decreased lymphocytes, occasionally associated with lymphocytolysis, were present in the thymus, spleen, mammary gland lymph node and/or pancreatic lymph node of 3/4 found dead and 3/8 animals killed in extremis, which was considered to be a result of nonspecific stress. In one female rat, the reddish discoloration ofthe cecum observed at necropsy was correlated histologically with moderate submucosal edema/congestion. All the other microscopic observations were few and were those commonly recorded as spontaneous findings in the Wistar rat. Histopathological examination of reproductive organs including the qualitative examination of the stages of spermatogenesis in the testes (no test item related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle) and the evaluation of the uterus or evaluation of follicles and corpora lutea in the ovaries did not reveal any test itemrelated findings in animals found dead, terminated in extremis or terminated according to the study schedule. Of note, there were no test item related microscopic findings in the males or females suspected of reduced fertility.
Other effects:
no effects observed
Description (incidence and severity):
Locomotor activity was similar inall groups in males and females. The mean low beam counts during 30 minutes of measurement were 1024, 880, 902 and 950 in groups 1, 2, 3 and 4 in males and 819, 790 and 893 in groups 1, 2 and 3 in females.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Corpora Lutea Count: Corpora lutea count was not affected by the treatment with the test item.

Duration of Gestation: Duration of gestation was unaffectedby the exposure to the test item.

Implantation Rate and Post-Implantation Loss: Implantation rate and post-implantation loss were notaffected by the treatment with the test item.

Litter Size at First Litter Check: Litter size at first litter check gave no indicationof a test item-related effect.
Reproductive performance:
no effects observed
Description (incidence and severity):
All paired females in groups 1, 2 and 3 mated within the first pairing period. The median and mean precoital times were unaffected by the treatment with the test item. Percentage mating (number of animals mated as a percentage of animals paired)
was 100% for males and females in all groups. The fertility index (number of females achieving pregnancy as a percentage of females paired) and conception rate (number of females achieving pregnancy as a percentage of females mated) as well as number of males producing offspring as a percentage of males paired/mated were 83.3%, 91.7% and 100.0% in groups 1, 2 and 3, respectively. All pregnant females gave birth to living pups which resulted in gestation index (number of females with living pups as a percentage offemales pregnant) of 100% in all groups.

Females in group 4 were terminated prior to pairing due to significant toxic effects. Therefore no males and females were paired in group 4.

Details on results (P0)

Postnatal Loss Days 0 - 4 Post Partum:
In groups 2 and 3, statistically significantly increased total number of pups lost during lactation and, consequently, statistically significantly reduced viability index were noted. Mean postnatal loss was 0.0, 0.8 and 0.6 whereas the total number of pups lost were 0, 9 (in 2 litters) and 7 (in 3 litters), in group 1, 2 and 3, respectively. In group 3, 5 pups from one litter (no. 80) and two from another litter (no 83) were missing on day 2 post partum, 2 further pups, each from another litter (nos. 73 and 82), were missing on day 3 post partum. In group 2, 8 pups from one litter (no. 71) one further from another litter (no. 62) were missing on day 2 post partum. Viability index (number of pups alive on day 4 post partum as a percentage of pups born alive) was 100.0%, 93.5% and 95.2% in order of ascending dose levels, the reduction in viability index was statistically significant in both treatment groups. The postnatal loss observed in treated groups was higher than the normal background expected in this strain but the increase resulted from the high mortality in only one litter in each group and the effect was not clearly dose dependent. For these reasons, the relation ofthe higher mortality to the treatment with the test item was not clear.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
3 500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Systemic Toxicity
Dose descriptor:
NOAEL
Effect level:
3 500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Reproductive Toxicity

Target system / organ toxicity (P0)

Critical effects observed:
yes
Lowest effective dose / conc.:
7 000 ppm
System:
other: Body weight and body weight gain
Organ:
other: Body weight and body weight gain
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
In one litter in group 2, four pups cold to touch were found at first litter check, one of these pups was missing on day 2 post partum. This finding occurred in one litter only and thereore it was considered not to be related to the treatment.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weights in groups 1, 2 and 3 were respectively 6.87, 5.96 and 5.81 g on day 1 post partum and 10.44, 8.78 and 8.32 g on day 4 post partum. Mean body weight gain from day 1 to 4 post partum was 52.14%, 47.61% and 42.81%.
Treatment with the test item resulted in statistically significantly reduced pup body weights and reduced pup body weight gain in group 3. Mean values of pup body weights recorded in this group on day 1 and day 4 of the lactation period were slightly below the historical control range. Statistically significantly lower body weights were recorded also in group 2 on days 1 and 4 of the lactation period but in the absence of statistically significantdifferences in body weight gain during this period. Mean values in group 2 were within the rangeof historical control groups comprising values of male and female pup weights from 5.9 to 6.6 g on day 1 and from 8.2 to 9.8 g on day 4 post partumwhereas the respective mean values in the control group were higher than the historical range. For this reason, the differences in pup body weights observed between group 2 and the control group were considered to be due to the incidentally higher body weights in the control group and not related tothe treatment with the test item.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related findings were noted at macroscopic examination of F1 pups.

Sore was found in one pup in group 2 and dark red discoloration of lungs was found in one pup in group 3.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No effects on righting reflex were observed in pups at any dose level.

Details on results (F1)

Sex Ratios: Sex ratios at first litter check and on day 4 post partumwere unaffected by exposure to the test item.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 750 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Developmental Toxicity

Target system / organ toxicity (F1)

Critical effects observed:
yes
Lowest effective dose / conc.:
3 500 ppm
System:
other: Body weight
Organ:
other: Body weight and body weight gain
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Overall reproductive toxicity

Reproductive effects observed:
yes
Lowest effective dose / conc.:
7 000 ppm
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:
not specified

Any other information on results incl. tables

Table 4. BODY WEIGHTS (GRAM) OF MALES SUMMARY P GENERATION - PRE-PAIRING PERIOD

 

 

Group 1

0 ppm

Group 2

1750 ppm

Group 3

3500 ppm

Group 4

7000 ppm

 

Day 1

Mean

371

380

382

377

 

ST. Dev

37.7

21.8

22.1

25.6

 

N

12

12

12

12

 

Day 5

Mean

384

391

383

361

 

ST. Dev

39.0

24.6

22.1

25.4

 

N

12

12

12

12

 

Day 8

Mean

395

400

391

362*

 

ST. Dev

389.5

25.7

21.4

28.3

 

N

12

12

12

12

 

Day 12

Mean

406

413

404

369*

 

ST. Dev

40.8

28.7

22.7

30.0

 

N

12

12

12

12

 

Day 14

Mean

411

418

409

371*

 

ST. Dev

41.1

29.7

22.4

33.9

 

N

12

12

12

12

* / **: Dunnett-Test based on pooled variance significant at 5% (*) or 1% (**) level

 

Table 5. BODY WEIGHTS (GRAM) OF MALES SUMMARY P GENERATION - PAIRING

 

 

Group 1

0 ppm

Group 2

1750 ppm

Group 3

3500 ppm

Group 4 7000 ppm

 

Day 1

Mean

410

416

404

373*

 

ST. Dev

41.3

30.3

22.1

35.2

 

N

12

12

12

12

* / ** : Dunnett-Test based on pooled variance significant at 5% (*) or 1% (**) level

Table 6. BODY WEIGHTS (GRAM) OF MALES SUMMARY P GENERATION - POST-PAIRING

 

 

Group 1

0 ppm

Group 2

1750 ppm

Group 3

3500 ppm

Group 4

7000 ppm

 

Day 1

Mean

417

423

411

372*

 

ST. Dev

42.9

30.5

24,5

43.5

 

N

12

12

12

12

 

Day 5

Mean

426

430

419

384*

 

ST. Dev

44.7

33.4

25.8

42.3

 

N

12

12

12

12

 

Day 8

Mean

434

436

426

389*

 

ST. Dev

45.5

33.3

24.5

40.6

 

N

12

12

12

12

 

Day 12

Mean

443

446

436

400*

 

ST. Dev

46.5

35.3

26.1

40.4

 

N

12

12

12

12

 

Day 15

Mean

449

452

439

402*

 

ST. Dev

48.2

35.4

27.3

38.0

 

N

12

12

12

12

 

Day 18

Mean

455

457

443

409*

 

ST. Dev

47.4

35.5

27.3

36.0

 

N

12

12

12

12

 

Day 22

Mean

461

463

450

417*

 

ST. Dev

50.0

36.1

28.1

36.1

 

N

12

12

12

12

 

Day 24

Mean

464

466

452

417*

 

ST. Dev

49.1

35.8

26.6

34.6

 

N

12

12

12

12

* / **: Dunnett-Test based on pooled variance significant at 5% (*) or 1% (**) level

Table 7. BODY WEIGHTS (GRAM) OF FEMALES SUMMARY P GENERATION - PRE-PAIRING PERIOD

 

 

Group 1

0 ppm

Group 2

1750 ppm

Group 3

3500 ppm

Group 4

7000 ppm

 

Day 1

Mean

239

245

241

242

 

ST. Dev

21.1

20.0

15.2

22.3

 

N

12

12

12

12

 

Day 5

Mean

244

250

239

227

 

ST. Dev

22.9

20.8

15.9

21.0

 

N

12

12

12

12

 

Day 8

Mean

248

253

239

228*

 

ST. Dev

25.0

19.2

14.6

21.3

 

N

12

12

12

12

 

Day 12

Mean

250

254

240

218**

 

ST. Dev

24.3

19.8

18.6

23.7

 

N

12

12

12

12

 

Day 14

Mean

253

257

246

219**

 

ST. Dev

24.9

20.0

14.9

21.6

 

N

12

12

12

8

* / **: Dunnett-Test based on pooled variance significant at 5% (*) or 1% (**) level

Table 8. BODY WEIGHTS (GRAM) OF FEMALES SUMMARY P GENERATION - GESTATION

 

 

Group 1

0 ppm

Group 2

1750 ppm

Group 3

3500 ppm

Day 0

Mean

255

258

247

 

ST. Dev

27.5

19.4

12.8

 

N

10

11

12

 

Day 7

Mean

280

283

270

 

ST. Dev

25.8

22.6

13.5

 

N

10

11

12

 

Day 14

Mean

314

315

301

 

ST. Dev

26.4

22.2

11.7

 

N

10

11

12

 

Day 20

Mean

379

381

358

 

ST. Dev

27.6

23.9

16.3

 

N

10

11

12

* / ** : Dunnett-Test based on pooled variance significant at 5% (*) or 1% (**) level

Table 9. BODY WEIGHTS (GRAM) OF FEMALES SUMMARY P GENERATION - LACTATION

 

 

Group 1

0 ppm

Group 2

1750 ppm

Group 3

3500 ppm

Day 1

Mean

290

286

271

 

ST. Dev

29.1

34.1

10.4

 

N

10

11

12

 

Day 4

Mean

308

304

286*

 

ST. Dev

25.3

25.3

10.1

 

N

10

11

12

* / ** : Dunnett-Test based on pooled variance significant at 5% (*) or 1% (**) level

Table 10. BREEDING DATA PER GROUP

 

 

Group 1

0 ppm

Group 2

1750 ppm

Group 3

3500 ppm

Living Pups at First Litter Check

% of Males/Females (#)

52/48

40/60 -

56/44 -

 

Total

111

139

146

 

Mean (+)

11.1

12.6 -

12.2 -

 

ST. Dev

1.7

2.2

2.5

 

N

10

11

12

 

Postnatal Loss Days 0-4 Post Partum

% of Living Pups

0.0

6.5

4.8

 

Litters Affected (#)

0

2 -

3 -

 

Total

0

9 ##

7 #

 

Mean (+)

0.0

0.8 -

0.6 -

 

ST. Dev

0.0

2.4

1,4

 

N

10

11

12

 

Living Pups on Day 4 Post Partum

Total

111

130

139

 

Mean (+)

11.1

11.8 -

11.6 -

 

ST. Dev

1.7

3.2

2.4

 

N

10

11

12

 

Birth Index (#)

 

88.8

94.6 -

88.0 -

 

Viability Index (#)

 

100.0

93.5 ##

95.2 #

Birth index = (pups born alive / number of implantations) * 100

Viability index = (pups alive before culling on day 4 p.p. / pups born alive) * 100

#/##/- : Fisher's Exact Test, significant at 5% (#), 1% (##) or not significant (-)

+/++/- : Steel Test, significant at 5% (+), 1% (++) or not significant (-)

Table 11. Mean Body Weights of Pups per Group On a Litter Basis (G) – Lactation Period

 

 

Group 1

0 ppm

Group 2

1750 ppm

Group 3

3500 ppm

Day 1 - Males

Mean

6.93

6.16**

5.92**

 

ST. Dev

0.56

0.55

0.62

 

N

10

11

12

Day 1 - Females

Mean

6.68

5.81**

5.63**

 

ST. Dev

0.42

0.62

0.68

 

N

10

11

12

Day 1 –

Males + Females

Mean

6.87

5.96**

5.81**

 

ST. Dev

0.33

0.55

0.64

 

N

10

11

12

 

Day 4 - Males

Mean

10.50

8.86**

8.52**

 

ST. Dev

0.94

0.97

1.20

 

N

10

10

12

Day 4 - Females

Mean

10.24

8.63**

8.05**

 

ST. Dev

0.78

1.12

1.18

 

N

10

11

12

Day 4 –

Males + Females

Mean

10.44

8.78**

8.32**

 

ST. Dev

0.75

1.02

1.18

 

N

10

11

12

*/**/- : DUNNETT-Test based on pooled variance sig. at 5% (*), 1% (**) or not sig. (-)

Applicant's summary and conclusion

Conclusions:
Based on the results of this study, a NOAEL (No Observed Adverse Effect Level) for systemic toxicity was established at the dose level of 3500 ppm.
 
The NOAEL and NOEL (No Observed Effect Level) for reproduction were set at the dose level of 3500 ppm whereas the NOEL and NOAEL for developmental toxicity was established at the dose level of 1750 ppm due to the reduced body weights and body weight gain in pups at the dose level of 3500 ppm.
Executive summary:

In a key combined repeated dose, reproductive/developmental toxicity screening study, the test material (Rosin, maleated; CAS# 8050-28-0) was administered daily in dietary mixtures at concentrations of 0, 1750, 3500 or 7000 ppm to male rats for 43 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum.

 

Treatment resulted in slightly higher test item intake in females compared to males during the pre-pairing period. Intake remained fairly constant throughout the entire study, except during the lactation phase when, due to an increase in food consumption, intake in females was almost two-fold higher than during the preceding gestation period.

 

Adverse toxic effects were observed in females in group 4 (7000 ppm) during the pre-pairing period. A severe reduction in food consumption resulting in body weight loss, significantly reduced body weights and significantly reduced food conversion efficiency were recorded. Further, clinical signs indicating worsening of the animal condition like stiff gate, hunched posture and ruffled fur were observed in most females in this group between days 12 and 14 of the pre-pairing period. Four females were found dead between day 12 and 14 and remaining females were terminated for ethical reasons on day 14 of the pre-pairing period. During necropsy, reddish discoloration of the cecum was observed in one female. This finding was histologically correlated with moderate submucosal edema/congestion. In one further female, pale discoloration of kidney and pelvic dilation was observed during the necropsy and histopathological examination confirmed kidney as a target organ in the females in the high concentration group. The pathogenesis of these changes was uncertain, however an acute/subacute impairment of the urinary outflow was suspected. The kidney findings did not appear to be linked to the cause of death as its grade severity was higher in animals killed in extremis when compared to animals found dead. Starvation caused by the aversion to test item containing food possibly contributed to the effects on body weights. However, there was no clear evidence that the food aversion was the immediate reason of the severity of the effects and bad condition of females. Thus, the reason for the mortality of females in the high-dose group remained unclear.

 

Treatment-related reduction in food consumption, food conversion efficiency, water consumption, body weight gain and body weights were also observed in males in the high concentration group (7000 ppm), however, less severe than in females. Although food and water consumption remained also slightly lower during the entire study, they recovered after the significant reduction observed at the beginning of the treatment. A body weight loss was observed at the beginning of the treatment followed by a reduced body weight gain. This resulted in reduced body weights observed until the end of the study.

 

During necropsy of males in the high concentration group, reduced adrenal weights were recorded but no other macroscopic findings. Histopathological examination revealed minimal decreased lymphocytes in the spleen and mesenteric lymph nodes which was considered to be a result of a non-specific stress.

 

In group 3 (3500 ppm), a slight and reversible reduction in food consumption and food conversion efficiency was observed in males and females. This resulted in a slight and reversible reduction in body weights, however without an effect on absolute body weights. A slight reduction in water consumption was recorded in males. During necropsy, reduced adrenal weights were recorded in females. Histopathological examination revealed no test item-related findings in any gender. No further test item-related effects were observed in any group. No test item-related findings were recorded in group 2 (1750 ppm).

 

Due to early termination of females in group 4, reproduction and developmental parameters were not assessed in this group. Mating performance, fertility, duration of gestation, number of corpora lutea, number of implantation sites and post implantation loss, were not affected by the treatment with the test item at any dose level. In group 3 (3500 ppm), reduced pup body weights and reduced pup body weight gain were recorded from day 1 to 4 of the lactation period. The differences of mean values were statistically significant if compared to the respective current control values and the values in group 3 were slightly below the historical control range. Therefore, this effect was considered to be related to the treatment with the test item. The changes in pup body weights were concomitant with slight reduction in maternal weight and weight gain. In group 2, pup body weights and body weight gain were considered not to be affected by the treatment with the test item. Further, an increased postnatal loss was observed in both dose-groups. However, the relation of this observation to the treatment with the test item was not conclusively established.

 

Based on the results of this study, a NOAEL (No Observed Adverse Effect Level) for systemic toxicity was established at the dose level of 3500 ppm.

 

The NOAEL and NOEL (No Observed Effect Level) for reproduction were set at the dose level of 3500 ppm whereas the NOEL and NOAEL for developmental toxicity was established at the dose level of 1750 ppm due to the reduced body weights and body weight gain in pups at the dose level of 3500 ppm.